Structure of the O‑Specific Polysaccharide from The

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Structure of the O‑Speciﬁc Polysaccharide from the Lipopolysaccharide of Psychrobacter cryohalolentis K5T Containing a 2,3,4-Triacetamido-2,3,4-trideoxy‑L‑arabinose Moiety † ‡ † § # Anna N. Kondakova,*, Kseniya A. Novototskaya-Vlasova, Nikolay P. Arbatsky, Marina S. Drutskaya, , ⊥ † ‡ § # Victoria A. Shcherbakova, Alexander S. Shashkov, David A. Gilichinsky, Sergei A. Nedospasov, , † and Yuriy A. Knirel † Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia ‡ Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, 142290 Pushchino, Russia § Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia # Faculty of Biology, Moscow State University, 119991 Moscow, Russia ⊥ Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290, Pushchino, Russia

ABSTRACT: A novel constituent of bacterial polysaccharides, 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose, was found in the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5T and identified by 1D and 2D 1H and 13C NMR studies of the polysaccharide and a disaccharide obtained by solvolysis of the polysaccharide with triflic acid. The following structure of the branched polysaccharide was established by sugar analysis, triflic acid solvolysis, Smith degradation, and 2D NMR spectroscopy.

Psychrobacter is a genus within the family Moraxellaceae of arabinose, which has not been hitherto reported in natural gamma-proteobacteria, which comprises psychrophilic to carbohydrates. mesophilic, halotolerant, aerobic, nonmotile, Gram-negative The lipopolysaccharide was obtained from dried bacterial coccobacilli.1 These bacteria live in extremely cold habitats, cells by the phenol−water procedure8 and degraded under mild such as Antarctic ice, soil, and sediments, as well as in deep sea acidic conditions. The resultant high molecular mass O-specific − environments.2 4 Studies on Psychrobacter structures and polysaccharide was isolated by gel-permeation chromatography metabolic pathways may aid in research on potential on Sephadex G-50. Sugar analysis by GLC of the acetylated extraterrestrial cryo-dwelling organisms and provide better alditols and (S)-2-octyl glycosides9 derived after full acid insight into the evolution of microbes. hydrolysis of the polysaccharide revealed the presence of D- Lipopolysaccharide structures of Psychrobacter have not been galactose (D-Gal) and L-rhamnose (L-Rha). Three additional studied intensively. Recently, we have established the structures monosaccharide components, 2,4-diacetamido-2,4,6-trideoxy-D- of the O-specific polysaccharide chains of the lipopolysacchar- glucopyranose (D-Qui2,4NAc), 2,3,4-triacetamido-2,3,4-tri- ides of Psychrobacter muricolla 2pST isolated from overcooled deoxy-L-arabinose (L-Ara2,3,4NAc), and 2,3-diacetamido-2,3- water brines within permafrost5 and Psychrobacter maritimus dideoxy-D-glucuronic acid (D-Glc2,3NAcA), were not detected 3pS from the same habitat.6 The polysaccharides contain in the sugar analysis but were identified in further studies of the unusual components, such as an amide of 2-acetamido-2-deoxy- polysaccharide by NMR spectroscopy (see below). 5 13 L-guluronic acid with glycine and a 2-acetyl-4-[(S)-3- The C NMR spectrum of the polysaccharide (Figure 1) δ − hydroxybutanoyl] derivative of 2,4-diamino-2,4,6-trideoxy-D- showed signals for six anomeric carbons at 98.1 103.9, seven glucopyranose (bacillosamine).6 In this paper, we report on the nitrogen-bearing carbons of amino sugars at δ 48.4−58.1, three structure of the O-specific polysaccharide of Psychrobacter methyl groups (C-6 of 6-deoxyhexoses) at δ 18.1−18.8, two cryohalolentis K5T isolated from the lens of overcooled (−9 °C), oxymethylene groups (C-5 of a pentose and C-6 of a hexose) at highly saline (13%) water brine within a permanently frozen δ 62.0 and 63.5, other oxygen-bearing sugar carbons at δ 69.3− marine layer that was deposited beneath shallow lagoons at 81.7, one carboxylic group (C-6 carbonyl of a hexuronic acid) δ δ − temperatures slightly above 0 °C and frozen subaerially as the at 173.7, and N-acetyl groups at 23.2 24.0 (CH3) and Polar Ocean regressed 110 000 to 112 000 years ago.7 The polysaccharide was found to include several diamino and Received: July 11, 2012 triamino sugars, including 2,3,4-triacetamido-2,3,4-trideoxy-L- Published: November 29, 2012

Figure 1. 13C NMR spectrum of the O-speciﬁc polysaccharide from Psychrobacter cryohalolentis K5T. Numerals refer to carbons in sugar residues denoted as follows: A, Ara2,3,4NAc; G, Gal; GA, Glc2,3NAcAN; Q, Qui2,4NAc; RI, RhaI; RII, RhaII.

− 1 175.2 175.9 (CO). The H NMR spectrum showed inter alia Glc2,3NAcA were recognized by relatively large J2,3, J3,4, and J4,5 signals for six anomeric protons at δ 4.61−5.23, three methyl values (8−10 Hz) typical of the glycopyranosyl configuration groups (H-6 of 6-deoxyhexoses) at δ 1.21−1.37, sugar ring and correlations of protons at the nitrogen-bearing carbons to protons at δ 3.45−4.29, and N-acetyl groups at δ 1.90−2.06. the corresponding carbons (C-2 and C-4 for Qui2,4NAc; C-2 The NMR spectra of the polysaccharide were assigned using and C-3 for Glc2,3NAcA) in the 1H,13C HSQC spectrum. That 2D 1H,1H COSY, TOCSY, ROESY (Figure 2), and 1H,13C the latter monosaccharide is a hexuronic acid followed from the absence of C-6 protons. The remaining spin system was assigned to Ara2,3,4NAc based on correlations of H-2, H-3, and H-4 to the nitrogen- bearing C-2, C-3, and C-4 at δ 48.4−49.2 in the 1H,13C HSQC ∼ spectrum. A relatively large J2,3 value of 10 Hz and relatively small J3,4 and J4,5ax values of <4 Hz were estimated for this monosaccharide from the 2D NMR spectra (more exactly the coupling constants tabulated in Table 1 were measured in a better-resolved 1H NMR spectrum of disaccharide 1; see below). These data showed that Ara2,3,4NAc occurs in the pyranose form, H-2 and H-3 are axial, and H-4 is equatorial; hence, this monosaccharide has the arabinopyranosyl configuration. In the 1H NMR spectrum of the polysaccharide measured in a 9:1 H2O/D2O mixture, in addition to signals of NH protons of amino sugars (Table 1), there were two signals at δ 7.54 and 8.20, which showed no correlations with other protons in the TOCSY spectrum. In the ROESY spectrum, they correlated to each other and, in addition, that at δ 7.54 showed a correlation with H-5 of Glc2,3NAcA at δ 4.14. Therefore, it was suggested that the two signals belong to an NH2 group at C-6 of Figure 2. Part of a 2D ROESY spectrum of the O-specific Glc2,3NAcA, which is present in the amide form designated as polysaccharide from Psychrobacter cryohalolentis K5T. The correspond- Glc2,3NAcAN. This conclusion was confirmed by the mass ing parts of the 1H NMR spectrum are displayed along the axes. spectrum of disaccharide 1; see below. Numerals refer to protons in sugar residues denoted as follows: A, A relatively small J1,2 value of 4 Hz for Ara2,3,4NAc indicated Ara2,3,4NAc; G, Gal; GA, Glc2,3NAcAN; Q, Qui2,4NAc; RI, RhaI; the equatorial orientation of H-1, i.e., the β configuration of this RII, RhaII. ∼ monosaccharide. A relatively large J1,2 value of 8 Hz and the presence in the 2D ROESY spectrum of H-1,H-3 and H-1,H-5 HSQC experiments (Table 1), and spin systems for six sugar correlations (Figure 2) indicated that Glc2,3NAcAN is β- pyranosides were identified. Those of 6-deoxyhexoses (one linked. Only a H-1,H-2 correlation was observed for Gal and Qui2,4NAc and two Rha residues, RhaI and RhaII) were showed its α-linkage. The α configuration of RhaI and RhaII and distinguished by coupling of C-5 protons to C-5 methyl groups the β configuration of Qui2,4NAc were determined by a in the COSY spectrum. The spin systems of Rha and Gal were comparison of the C-5 chemical shifts (Table 1) with published identified by relatively small J or J values (∼3 Hz), data for the α-andβ-anomers of the corresponding 2,3 3,4 − respectively, determined from the 2D spectra. Qui2,4NAc and monosaccharides.10 12

2237 dx.doi.org/10.1021/np300484m | J. Nat. Prod. 2012, 75, 2236−2240 Journal of Natural Products Note

a Table 1. 1H and 13C NMR Data (δ, ppm; J, Hz)

C-1 C-2 C-3 C-4 C-5 C-6 b sugar residue H-1 H-2 H-3 H-4 H-5/5a, 5b H-6/6a, 6b NH (NH2) Polysaccharide →3)-β-D-Quip2,4NAc-(1→ 103.9 56.5 77.8 58.1 72.6 18.1 4.61 3.93 3.83 3.83 3.53 1.21 7.94, 8.14 →3,4)-α-D-Galp-(1→ 102.0 69.3 80.2 79.7 72.6 62.0 5.23 3.75 3.74 4.13 3.89 3.71, 3.68 I →2)-α-L-Rhap -(1→ 100.3 80.2 71.2 73.8 71.1 18.3 5.11 4.04 3.85 3.45 3.75 1.30 II →3)-α-L-Rhap -(1→ 102.6 71.6 81.7 72.1 70.6 18.8 5.14 4.21 3.86 3.55 3.80 1.37 →4)-β-D-Glcp2,3NAcAN-(1→ 103.5 55.9 56.1 74.1 76.8 173.7 4.97 3.86 4.25 3.99 4.14 8.07, 8.28 (7.54, 8.20) α-D-Arap2,3,4NAc-(1→ 98.1 48.9 48.4 49.2 63.5 5.23 4.12 4.22 4.29 3.51, 4.00 7.93, 7.99, 8.39 Disaccharide α-D-Arap2,3,4NAc 97.8 48.6 48.1 48.9 63.2 5.25c 4.12 4.24 4.30 3.53, 4.02

J1,2 3.8 J2,3 12.3 J3,4 3.9 J4,5a 1.5 J4,5b 2.3 J5a,5b 12.6 →4)-α-D-Glcp2,3NAcAN-(1→ 91.9 53.6 53.2 73.9 71.7 n.d. 5.22 4.06 4.35 3.99 4.49

J1,2 3.5 J2,3 11.3 J3,4 9.8 J4,5 9.7 →4)-β-D-Glcp2,3NAcAN-(1→ 96.2 56.6 56.1 73.8 76.9 n.d. 4.93 3.75 4.22 3.99 4.14

J1,2 8.5 J2,3 11.1 J3,4 10.2 J4,5 9.5 a δ − δ − − b Chemical shifts for the NAc groups are H 1.90 2.06 and C 23.0 23.2 (CH3), 175.4 175.7 (CO). n.d., not determined. Measured in a 9:1 H2O/ c α δ β D2O mixture. When linked to -Glc2,3NAcAN; 5.24 when linked to -Glc2,3NAcAN.

A significant downfield shift to δ 74.1−81.7 of the signals for RhaI-(1→4)-Gal disaccharide to a pseudodisaccharide with the C-2 of one of the Rha residues (RhaI), C-3 of Qui2,4NAc and destroyed RhaI residue in an acyclic form. The former is the other Rha residue (RhaII), C-4 of Glc2,3NAcAN, and C-3 characterized by a number of specific interactions between and C-4 of Gal, as compared with their positions in the spectra protons of the linked monosaccharide residues, which influence − of the corresponding nonsubstituted monosaccharides,10 12 chemical shifts of both protons and linked carbons, whereas the demonstrated the glycosylation pattern in the polysaccharide. latter, being much less hindered sterically, showed significantly In accordance with the branched character of the poly- different interresidue proton-to-proton interactions. saccharide and the absence in Ara2,3,4NAc of any hydroxy Solvolysis of the polysaccharide with triflic acid14 cleaved group, this monosaccharide occupies the nonreducing end of glycosidic linkages of all constituent monosaccharides but the side chain. Ara2,3,4NAc. As a result, the α-Arap2,3,4NAc-(1→4)- The following interresidue cross-peaks showing the spatial Glc2,3NAcAN disaccharide (1) was isolated by gel-permeation proximity of the transglycosidic protons were observed in the chromatography on fractogel TSK HW-40. The molecular mass 2D ROESY spectrum of the polysaccharide (Figure 2): of 1 determined by negative ion electrospray ionization mass Ara2,3,4NAc H-1/Glc2,3NAcAN H-4, Glc2,3NAcAN H-1/ spectrometry (530.2329 amu; calculated molecular mass RhaII H-3, RhaII H-1/RhaI H-2, RhaI H-1/Gal H-4, Gal H-1/ 530.2336 amu) confirmed both triacetamidopentose and Qui2,4NAc H-3, and Qui2,4NAc H-1/Gal H-3. These data diacetamidohexuronamide moieties. The full structure of 1 confirmed the positions of glycosylation of the monosaccharide was established by 2D NMR spectroscopy as described for the residues and revealed their sequence in the repeating unit. polysaccharide (for assigned 1H and 13C NMR chemical shifts In an attempt to confirm the monosaccharide sequence, the and 3J values see Table 1; for the 1H,13C HSQC spectrum see polysaccharide was subjected to Smith degradation,13 which, as Figure 3). expected, resulted in selective destruction of 2-substituted RhaI. As indicated above, the absolute configurations of Gal and However, the subsequent mild acid hydrolysis did not cleave Rha were established as D and L, respectively. The absolute the side chain, most likely due to steric hindrance. A configurations of the other monosaccharides were determined comparison of the NMR spectra of the initial and degraded by α- and β-effects of glycosylation on 13C NMR chemical polysaccharides revealed dramatic changes in chemical shifts of shifts, which are calculated as differences between the chemical the destroyed RhaI residue and, in addition, the neighboring shifts of the linked and adjacent carbons, respectively, in an Gal residue, thus corroborating the attachment of RhaI to Gal. oligosaccharide or a polysaccharide and the corresponding Particularly, the 1H,13C HSQC spectrum showed significant nonsubstituted monosaccharide. In many cases, these effects displacements upon Smith degradation of the following signals depend significantly on the absolute configurations of the for Gal: H-3/C-3 from δ 3.74/80.2 to 3.81/80.0, H-4/C-4 from linked monosaccharides and enable discrimination between δ 4.13/79.7 to 4.32/75.0, and H-5/C-5 from δ 3.89/72.6 to disaccharides (or disaccharide fragments) composed of 3.85/70.8. These displacements reflected conversion of the monosaccharides having the same or different absolute

2238 dx.doi.org/10.1021/np300484m | J. Nat. Prod. 2012, 75, 2236−2240 Journal of Natural Products Note

bacterial cell surface and is believed to be important for bacterial survival and niche adaptation. ■ EXPERIMENTAL SECTION General Experimental Procedures. NMR spectra were recorded at 50 °C on a Bruker Avance II 600 spectrometer using a 5 mm broadband inverse probehead for solutions in 99.95% D2O (after deuterium exchange by freeze-drying sample solutions in 99.9% D2O) or in 9:1 H2O/D2O mixture. Sodium 3-(trimethylsilyl)propanoate- δ δ 2,2,3,3-d4 ( H 0.0) and acetone ( C 31.45) were used as internal calibration standards for 1Hand13C NMR chemical shifts, respectively. 2D NMR spectra were obtained using standard Bruker software, and the Bruker Topspin 2.1 program was used to acquire and process the NMR data. The 1D and 2D TOCSY and 2D ROESY spectra were recorded with a 150 ms duration of MLEV-17 spin-lock and a 100 ms mixing time, respectively. ESIMS was performed in the negative mode using a micrOTOF II instrument (Bruker Daltonics). A sample (∼50 ng μL−1) was dissolved fl μ in a 1:1 (v/v) H2O/MeCN mixture and sprayed at a ow rate of 3 L min−1. Capillary entrance voltage was set to 4.5 kV and exit voltage to −150 V; the drying gas temperature was 180 °C. GLC experiments were done on an Agilent 7820A GC system using a temperature − Figure 3. 2D 1H,13C HSQC spectrum of disaccharide 1. Numerals program from 160 (1 min) to 290 °Cat7°C min 1. Gel-permeation refer to H/C pairs in sugar residues denoted as follows: A, chromatography was done on a column (56 × 2.5 cm) of Sephadex G- Ara2,3,4NAc; αGA, α-Glc2,3NAcAN; βGA, β-Glc2,3NAcAN. Struc- 50 Superfine (Amersham Biosciences) using 0.05 M pyridinium ture of 1 is shown in the inset. acetate pH 4.5 as eluent or a column (80 × 1.6 cm) of TSK HW-40 (S) (Merck) in 1% HOAc/H2O; monitoring was performed using a 15 ff configurations. Thus, in the β-Glcp2,3NAcAN-(1→3)-α-L- di erential refractometer (Knauer). RhapI fragment of the polysaccharide, the α-effect on C-1 of the Growth of Bacteria and Isolation of the Lipopolysaccharide and O-Specific Polysaccharide. Psychrobacter cryohalolentis K5T was glycosylating Glc2,3NAcAN residue is relatively large (7.1 16 provided by the All-Russian Collection of Microorganisms (B-2378 ppm ) and indicative of different absolute configurations of ° fi type strain). Bacteria were grown to late log phase at 24 C in a pH 7.6 the constituent monosaccharides, i.e., the D con guration of medium containing (per 1 L of distilled) 4 g of yeast extract; 1.12 g of fi Glc2,3NAcAN (in the case of the same absolute con guration, Na2HPO4; 0.4 g of KH2PO4; 19.5 g of NaCl; 2 g of NH4Cl; 4 g of α ff 15 β ff · · the -e ect on C-1 would be <5 ppm ). A small -e ect of MgSO4 7H2O; 0.01g of CaCl2; 0.005 g of FeSO4 7H2O; and 10 mL of glycosylation on C-3 of Glc2,3NAcAN (<0.3 ppm) showed SL-10 micronutrient solution.18 Bacterial cells were harvested, washed, 18 different absolute configurations of the monosaccharides in the and dried as described. − 8 β-Arap2,3,4NAc-(1→4)-D-Glcp2,3NAcA disaccharide (a higher Cells were extracted using the phenol H2O procedure, and the β ff ∼ isolated crude material was purified by precipitation of nucleic acids -e ect on C-3 of 1 ppm would be observed in the case of the 19 fi 15 and proteins by treatment with TCA to yield a lipopolysaccharide same absolute con guration ). Therefore, Ara2,3,4NAc has the preparation in a yield of 6% of dried cell mass. The O-specific L configuration. Finally, in the β-Quip2,4NAc-(1→3)-α-D-Galp α ff polysaccharide was obtained by degradation of the lipopolysaccharide disaccharide, the -e ect on C-1 of the glycosylating with 2% HOAc/H O for 1.5 h at 100 °C; after centrifugation at 11 2 Qui2,4NAc residue is relatively large (8.1 ppm ) and indicated 13000g the supernatant was fractionated by gel-permeation the same absolute configurations of the monosaccharides, i.e., chromatography on Sephadex G-50, yielding 11% of the purified the D configuration of Qui2,4NAc (in the case of different material. absolute configurations, the α-effect on C-1 would be <4 Smith Degradation. A polysaccharide sample (20 mg) was 15 ° ppm ). oxidized with 0.1 M NaIO4 (1.0 mL) in the dark at 20 C for 72 h. On the basis of the data obtained, it was concluded that the After reduction with an excess of NaBH4 and desalting on a fractogel TSK HW-40 column, the product was hydrolyzed with 1% HOAc/ polysaccharide of P. cryohalolentis has the structure shown in ° fi H2O at 100 C for 2 h, and the modi ed polysaccharide was isolated Figure 4. The polysaccharide is neutral, as the constituent acidic by gel-permeation chromatography on TSK HW-40. Triflic Acid Solvolysis. A polysaccharide sample (7 mg) was treated with 0.5 mL of triflic acid for 4 h at 20 °C; after neutralization with 12% NH3/H2O the products were fractionated by gel-permeation chromatography on fractogel TSK HW-40 to give disaccharide 1 (2 mg) and a mixture of monosaccharides. Figure 4. Structure of the O-specific polysaccharide from Psychrobacter T ■ AUTHOR INFORMATION cryohalolentis K5 . Corresponding Author * monosaccharide, Glc2,3NAcA, occurs in the amide form. Tel: +7(499) 137-6148. Fax: +7(499) 135-5328. E-mail: Another peculiar feature of the polysaccharide is the presence [email protected]. of a novel unique triamino sugar, 2,3,4-triacetamido-2,3,4- Notes fi trideoxy-L-arabinose, which occupies the terminal position in a The authors declare no competing nancial interest. rather long, tetrasaccharide side chain. The terminal monosaccharides of carbohydrate antigens are most accessible to ■ ACKNOWLEDGMENTS environmental factors, such as immune system and bacter- The authors thank D. N. Platonov for help with GLC-MS iophages; their diversity provides most specificity to the analysis and Bruker Moscow Ltd for providing access to a

2239 dx.doi.org/10.1021/np300484m | J. Nat. Prod. 2012, 75, 2236−2240 Journal of Natural Products Note micrOTOF II instrument. This work was supported by the Russian Foundation for Basic Research (Projects Nos. 10-04- 00590 and 10-04-01470), the Fundamental Research Program “Molecular and Cellular Biology” of the Presidium of the Russian Academy of Sciences, and the Federal Targeted Program for Research and Development in Priority Areas of Russia’s Science and Technology Complex for 2007−2013 (state contract no. 16.552.11.7050). ■ REFERENCES (1) Bowman, J. P. In The Prokaryotes: an Evolving Electronic Resource for the Microbiological Community, 3rd ed.; Dworkin, M., Ed.; Springer: New York, 2005; Release 3.19. (2) Bowman, J. P.; Nichols, D. S.; McMeekin, T. A. Syst. Appl. Microbiol. 1997, 20, 209−215. (3) Maruyama, A.; Honda, D.; Yamamoto, H.; Kitamura, K.; Higashihara, T. Int. J. Syst. Evol. Microbiol. 2000, 50, 835−846. (4) Romanenko, L. A.; Schumann, P.; Rohde, M.; Lysenko, A. M.; Mikhailov, V. V.; Stackebrandt, E. Int. J. Syst. Evol. Microbiol. 2002, 52, 1291−1297. (5) Kondakova, A. N.; Novototskaya-Vlasova, K. A.; Drutskaya, M. S.; Senchenkova, S. N.; Shcherbakova, V. A.; Shashkov, A. S.; Gilichinsky, D. A.; Nedospasov, S. A.; Knirel, Y. A. Carbohydr. Res. 2012, 349,78−81. (6) Kondakova, A. N.; Novototskaya-Vlasova, K. A.; Shashkov, A. S.; Drutskaya, M. S.; Senchenkova,S.N.;Shcherbakova,V.A.; Gilichinsky, D. A.; Nedospasov, S. A.; Knirel, Y. A. Carbohydr. Res. 2012, 359,7−10. (7) Bakermans, C.; Ayala-del-Rio, H. L.; Ponder, M. A.; Vishnivetskaya, T.; Gilichinsky, D.; Thomashov, M. F.; Tiedje, J. M. Int. J. Syst. Evol. Microbiol. 2006, 56, 1285−1291. (8) Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5,83− 91. (9) Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87−89. (10) Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175,59−75. (11) MacLean, L. L.; Vinogradov, E.; Crump, E. M.; Perry, M. B.; Kay, W. W. Eur. J. Biochem. 2001, 268, 2710−2716. (12) Dmitriev, B. A.; Kocharova, N. A.; Knirel, Y. A.; Shashkov, A. S.; Kochetkov, N. K.; Stanislavsky, E. S.; Mashilova., G. M. Eur. J. Biochem. 1982, 125, 229−237. (13) Goldstein, I. J.; Hay, G. W.; Lewis, B. A.; Smith., F. Methods Carbohydr. Chem. 1965, 5, 361−370. (14) Knirel, Y. A.; Perepelov, A. V. Aust. J. Chem. 2002, 55,69−72. (15) Shashkov, A. S.; Lipkind, G. M.; Knirel, Y. A.; Kochetkov, N. K. Magn. Reson. Chem. 1988, 26, 735−747. (16) Knirel, Y. A.; Vinogradov, E. V.; Kocharova, N. A.; Shashkov, A. S.; Dmitriev, B. A.; Kochetkov, N. K. Carbohydr. Res. 1983, 122, 181− 188. (17) DSMZ Catalogue of Strains, 7th ed.; Deutsche Sammlung von Mikroorganismen und Zellkulture: Braunschweig, 2001. (18) Robbins, P. W.; Uchida, T. Biochemistry 1962, 1, 323−335. (19) Knirel, Y. A.; Kaca, W.; Paramonov, N. A.; Cedzynski, M.; Vinogradov, E. V.; Ziolkowski, A.; Shashkov, A. S.; Rozalski, A. Eur. J. Biochem. 1997, 247, 951−954.

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