Carbohydrate Research 372 (2013) 69–72

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Carbohydrate Research

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Structure of the O-specific polysaccharide from a marine bacteriumCellulophaga pacifica containing rarely occurred sugars, Fuc4NAcand ManNAcA

0008-6215/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.carres.2013.02.011

⇑ Corresponding author. Fax: +7 423 231 40 50.E-mail address: tomsh@piboc.dvo.ru (S.V. Tomshich).

Andrei V. Perepelov a, Alexander S. Shashkov a, Svetlana V. Tomshich b,⇑, Nadezhda A. Komandrova b,Ol’ga I. Nedashkovskaya b

a N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federationb G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, 690022 Vladivostok, Russian Federation

a r t i c l e i n f o

Article history:Received 26 December 2012Received in revised form 25 February 2013Accepted 26 February 2013Available online 6 March 2013

Keywords:O-specific polysaccharideCellulophaga pacificaBacterial polysaccharide structureMarine bacterium4-Acetamido-4,6-dideoxy-D-galactose2-Acetamido-2-deoxy-D-mannuronic acid

a b s t r a c t

The O-polysaccharide was isolated from the lipopolysaccharide of Cellulophaga pacifica and studiedby chemical methods including Smith degradation as well as 1H and 13C NMR spectroscopy. Thefollowing structure of the O-polysaccharide of Cellulophaga pacifica containing N-acetyl-D-glucosamine(GlcNAc), 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc) and two residues of 2-acetamido-2-deoxy-D-mannuronic acid (ManNAcA) was established.

α-D-Fucp4NAc1

4→3)-β-D-ManpNAcA-(1→4)-β-D-ManpNAcA-(1→3)-β-D-GlcpNAc-(1→

� 2013 Elsevier Ltd. All rights reserved.

The genus Cellulophaga belongs to the family Flavobacteriaceaeof the phylum Bacteroidetes. It was created by Johansen et al.1 toaccommodate the heterotrophic aerobic Gram-negative yellow/or-ange pigmented gliding and agarolytic bacteria of marine origin.Currently this genus comprises seven species: Cellulophaga algicola,Cellulophaga baltica, Cellulophaga fucicola, Cellulophaga lytica, Cellul-ophaga pacifica,2 Cellulophaga tyrosinoxydans3 and Cellulophagageojensis.4 Earlier we published the structures of the O-polysaccha-ride chain (O-specific polysaccharide, OPS) of lipopolysaccharidesfrom C. baltica5 and C. fucicola.6 Acidic character of the studied Cel-lulophaga O-polysaccharides is defined by the presence of uronicacid (GlcA in C. baltica) or nonulosonic acid (Pse in C. fucicola). Inthis paper we present the structure of Cellulophaga pacificaO-polysaccharide, which contains two types of rarely occurringcomponents, 4-acetamido-D-fucose and 2-acetamido-2-deoxy-D-mannuronic acid.

The O-polysaccharide (OPS) of C. pacifica was obtained by mildacid degradation of the lipopolysaccharide (LPS), isolated from

dried bacterial cells by hot phenol/water extraction. Sugar analysisby GLC of the alditol acetates derived after full acid hydrolysis ofthe OPS revealed GlcN only. Moreover, methanolysis of the OPS fol-lowed by GLC–MS analysis of acetylated methyl glycosides showedthe presence of 4-acetamido-4,6-dideoxy-hexose (identified asFuc4NAc, vide infra) and 2-acetamido-2-deoxy-hexuronic acid(identified as ManNAcA, vide infra). Methylation analysis of theOPS resulted in the identification of 4,6-dideoxy-2,3-di-O-methyl-4-(N-methyl)acetamidohexose (derived from Fuc4NAc),2-deoxy-4,6-di-O-methyl-2-(N-methyl)acetamidohexose (derivedfrom 3-substituted GlcN) and 2-acetamido-2-deoxy-hexuronicacid (derived from 3,4-disubstituted ManNAcA). Subsequent GLCanalysis of the components derivatized with (S)-2-octanol fol-lowed by acetylation showed that GlcN has the D configuration.Absolute configurations of ManNAcA and Fuc4N were establishedby measurement of the specific optical rotation value of the OPSand oligosaccharide obtained by Smith degradation (see below).

The 13C NMR spectrum of the OPS (Fig. 1A) indicated a tetrasac-charide repeating unit with four anomeric signals at d 98.1, 100.1and 100.6 (double intensity). It also showed one signal of CH3–Cgroup for 6-deoxy sugar at d 16.8, four carbons bearing nitrogen

Figure 1. 13C NMR spectra of O-polysaccharide (A) and Smith-degraded polysaccharide (B). Arabic numerals refer to the carbons in the sugar residues denoted as described inTable 1.

70 A. V. Perepelov et al. / Carbohydrate Research 372 (2013) 69–72

at d 50.5–56.6, 12 other ring carbons in the region d 67.3–80.5, car-bonyl groups in the region of d 175.8–176.9 and four N-acetyl sig-nals (Me) at d 23.3–24.0. The absence from signals of non-anomericsugar carbons at a lower field than d 81 in the 13C NMR spectrumdemonstrated the pyranoid form of all sugar residues.

Six signals were observed in a low-field region of the 1H NMRspectrum of the OPS at d 4.55–5.41, which included only four sig-nals for anomeric protons at d 4.59, 4.78, 4.90 and 5.41; the othertwo signals at d 4.55 and 4.56 belonged to non-anomeric protons(see below). The high-field region of the spectrum contained foursignals for N-acetyl groups at d 2.01–2.07 and one doublet for aCH3 group at d 1.04.

The 1H and 13C NMR spectra of the OPS were assigned using 2Dhomonuclear COSY, TOCSY, ROESY, heteronuclear 1H, 13C HSQC,1H, 13C HMQC-TOCSY and 1H, 13C HMBC experiments (see Table 1).The spin system of GlcNAc was identified by H-1/H-2 up to H-6correlations found in the TOCSY spectrum. The COSY and TOCSY

Table 11H and 13C NMR data of the OPS and Smith degraded PS of C. pacifica (d, ppm)

Sugar residue 1 2 3

OPSa-D-Fucp4NAc-(1? 1H 5.41 3.65 3.92A 13C 100.1 69.9 69.9?3,4)-b-D-ManpNAcAI-(1? 1H 4.78 4.59 4.36B 13C 100.6 50.5 76.6?4)-b-D-ManpNAcAII-(1? 1H 4.90 4.56 3.90C 13C 100.6 53.7 71.5?3)-b-D-GlcpNAc-(1? 1H 4.59 3.77 3.69D 13C 98.1 56.6 80.5

Smith-degraded PS?3)-b-D-ManpNAcA-(1? 1H 4.65 4.54 4.01B 13C 100.2 51.3 78.2?4)-b-D-ManpNAcA-(1? 1H 4.87 4.55 3.86C 13C 100.5 54.1 71.7?3)-b-D-GlcpNAc-(1? 1H 4.66 3.70 3.68D 13C 99.3 56.5 80.3

spectra showed cross peaks of H-1/H-2 up to H-4 for the sugar resi-due with the galacto configuration (Fuc4NAc). The COSY and ROESYspectra allowed the assignment of its remaining signals using H-6/H-5 and H-6/H-4,H-5 correlations at d 1.04/4.07 and d 1.04/4.20,4.07, respectively. The b-GlcNAc residue was confirmed by cor-relation between proton at the nitrogen-linked carbon with the cor-responding carbon at d 3.77/56.6 (H-2/C-2). Relatively small 3JH-1,H-2

coupling constant (ca. �3 Hz) determined from the 1H NMR spec-trum and H-4/C-4 cross-peak at d 4.20/55.2 proved the a-Fucp4NAcresidue.7

Two last residues were assigned to 2-acetamido-2-deoxy-man-nuronic acids (ManNAcAI and ManNAcAII) basing on the chemicalshifts and coupling constant values. Relatively small 3JH-1,H-2 cou-pling constants (ca. �2 Hz) determined from the 1H NMR spec-trum, H-2/C-2 cross-peaks at d 4.59/50.5 and 4.56/53.7 (found inthe HSQC experiment) and H-5/C-6 cross-peaks at d 3.97/174.7and 3.97/174.1 (found in the HMBC experiment) are characteristic

4 5 6 (6a,6b) CO (NAc) CH3 (NAc)

4.20 4.07 1.04 2.0655.2 67.3 16.8 176.9 23.33.89 3.97 2.0572.7 77.0 174.6 175.8 23.43.84 3.97 2.0578.7 75.6 174.1 176.8 23.43.68 3.50 3.70; 3.85 2.0173.5 75.8 61.5 175.7 24.0

3.64 3.72 2.0668.7 78.2 176.8 176.8 23.53.78 3.78 2.0578.7 77.5 176.4 175.8 23.93.69 3.51 3.71; 3.85 2.0173.8 75.7 61.5 176.8 23.5

Figure 2. Part of a 1H, 13C HMBC spectrum of the OPS from C. pacifica. The corresponding parts of the 1H and 13C NMR spectra are displayed along the horizontal and verticalaxes, respectively. Arabic numerals before and after oblique stroke refer to protons and carbons, respectively, in the sugar residues denoted by letters as shown in Table 1.

A. V. Perepelov et al. / Carbohydrate Research 372 (2013) 69–72 71

for 2-amino-2-deoxy-mannuronic acids. The NMR chemical shiftsfor H-5 and C-5 compared with published data8 showed thatManNAcAI and ManNAcAII are b-linked.

Relatively low-field positions of the signals for C-3 and C-4 ofManNAcAI, C-4 of ManNAcAII and C-3 of GlcNAc at d 76.6, 72.7,78.7 and 80.5, respectively, in the 13C NMR spectrum of the OPS, ascompared with their positions in the spectra of the correspondingnon-substituted monosaccharides8,9 demonstrated the modes of su-gar glycosylation. A 2D ROESY experiment revealed strong interres-idue cross-peaks between the anomeric protons and protons at thelinkage carbons at d 5.41/3.89; 4.78/3.84; 4.90/3.69 and 4.59/4.36,which were assigned to Fucp4NAc H-1, ManpNAcAI H-4; ManpNAcAI

H-1, ManpNAcAII H-4; ManpNAcAII H-1, GlcNAc H-3; GlcNAc H-1,ManpNAcAI H-3 correlations, respectively. The monosaccharide se-quence thus determined was confirmed by a heteronuclear 1H, 13CHMBC experiment, which showed correlations between the ano-meric protons and linkage carbons (Fig. 2) and vice versa.

In order to confirm this structure and determine the absoluteconfiguration of both ManNAcA residues, the OPS was subjectedto Smith degradation. Studies of the resultant Smith-degradedpolysaccharide by 1D and 2D NMR spectroscopy as describedabove for the OPS, including the full assignment of 1H and 13CNMR signals (Table 1), enabled elucidation of the followingstructure:

?3)-b-ManpNAcAI-(1?4)-b-ManpNAcAII-(1?3)-b-D-GlcpNAc-(1?

The Smith-degraded polysaccharide had specific optical rota-tion values [a]D �47� (water). Capsular antigen of Neisseriameningitides serogroup K of the structure ?3)-b-D-ManpNAcA-(1?4)-b-D-ManpNAcA-(1? and b-D-GlcNAc-O-Me had [a]D �46�and �43�, respectively.8,10 According to these data and Klyne’srule the calculated optical rotation [a]D of the Smith-degradedpolysaccharide should be �47�, that is in good agreement withexperimental data and, thus, demonstrated that both ManNAcAresidues have the D configuration (in case of L-ManNAcA, [a]D ofSmith-degraded polysaccharide should be +15�).

As compared to the HSQC spectrum of the OPS, the H-4/C-4cross-peak of ManpNAcAI in the Smith degraded PS shifted fromupfield from d 3.89/72.7 to 3.64/68.7 (Fig. 1B). This displacementconfirmed that Fuc4NAc linked at position 4 of ManpNAcAI. The va-lue [a]D +42� was determined for isolated D-Fuc4NAc7 and the cal-culated optical rotation [a]D of the OPS should be �21� accordingto Klyne’s rule in case of D-Fucp4NAc, that is in good agreementwith experimental data (�24�). Therefore, the absolute configura-tion of Fuc4NAc is D and the OPS of C. pacifica has the followingstructure:

To our knowledge, this structure is unique among the knownbacterial polysaccharide structures. It is interesting to note thatall four residues presented in the OPS of C. pacifica are amino sug-ars and hitherto both rare sugars, 2-acetamido-2-deoxy-D-mannu-ronic acid and 4-acetamido-4,6-dideoxy-D-galactose, were foundonly once in the enterobacterial common antigen.11

1. Experimental

1.1. Bacterial strains and isolation of lipopolysaccharide

Cellulophaga pacifica strain KMM 3664T (=JCM 11735T=LMG21938T)2 from the Collection of Marine Microorganisms of thePacific Institute of Bioorganic Chemistry, Far East Branch of RussianAcademy of Sciences was isolated from a sea water sample thatwas collected during June 2000 in Amursky Bay, Gulf of Peterthe Great, Sea of Japan, Pacific Ocean, from a depth of 5 m. The

72 A. V. Perepelov et al. / Carbohydrate Research 372 (2013) 69–72

bacterium was cultivated for 48 h at ambient temperature on themedium consisting of (L�1) 5 g Bacto Peptone (Difco), 2 g BactoYeast Extract (Difco), 1 g glucose, 0.02 g KH2PO4 and 0.05 gMgSO4�7H2O in 50% (v/v) natural seawater and 50% (v/v) distilledwater. LPS was isolated in a yield of 7.5% from dried bacterial cellsby hot phenol/water extraction12 and purified by treatment withcold aq 50% CCl3CO2H, followed by dialysis of the supernatant.

1.2. Degradation of lipopolysaccharide

Delipidation of the lipopolysaccharide of C. pacifica (250 mg)was performed with aqueous 2% HOAc (100 �C, 2 h). The precipi-tate was removed by centrifugation (15,000 g, 30 min). The super-natant was fractionated by GPC on columns of TSK-50 and TSK-40sequentially to give the high-molecular weight O-polysaccharide(78 mg, OPS). The optical rotation of the OPS, ½a�20

D �24� (water),was measured on a Jasko DIP-360 polarimeter (Japan).

1.3. Chemical analyses

The polysaccharide was hydrolysed with 2 M CF3COOH (120 �C,2 h). The monosaccharides were reduced with 0.25 M NaBH4 in aq1 M ammonia (25 �C, 1 h), acetylated with a 1:1 (v/v) mixture ofpyridine and acetic anhydride (120 �C, 0.5 h) and were analysedby GLC. Methanolysis of the polysaccharide (1 mg) was carriedout using 1 M HCl–MeOH (100 �C, 3 h) followed by acetylationwith Ac2O in pyridine (120 �C, 30 min) with subsequent GLC–MSanalysis. The absolute configurations of the GlcN was determinedby GLC of acetylated (S)-(+)-2-octyl glycosides according to pub-lished methods.13,14 GLC was performed on a Hewlett–Packard5890 chromatograph equipped with an Ultra-1 column using atemperature gradient of 160–290 �C at 10 �C min�1.

Methylation of the OPS was performed with CH3I in dimethylsulfoxide in the presence of sodium methylsulfinylmethanide.15

Partially methylated monosaccharides were derived by methanol-ysis under the same conditions as in sugar analysis, acetylated andanalysed by GLC–MS on a Hewlett–Packard 5890 chromatograph(USA) equipped with a capillary column HP 5 MS and connectedwith a Hewlett–Packard 5973 mass-spectrometer (USA) using thesame chromatographic conditions as in GLC.

1.4. Smith degradation

Smith degradation of OPS (100 mg) was performed with 0.1 MNaIO4 for 74 h at room temperature in the dark. After the reduction

with NaBH4 and dialysis modified polysaccharide was hydrolysedwith aq 1% HOAc for 1.5 h at 100 �C. Smith degraded PS C. pacifica(20 mg) was isolated by GPC on TSK HW-40 gel. The optical rota-tion of the Smith-degraded polysaccharide, ½a�20

D �47� (water),was measured on a Jasko DIP-360 polarimeter (Japan).

1.5. NMR spectroscopy

The sample was deuterium exchanged by freeze drying fromD2O and then examined in a solution of 99.96% D2O, using internalTSP (dH 0.00) and acetone as reference (dH 2.225, dC 31.45). NMRspectra were recorded at 30 �C on a Bruker DRX-500 spectrometerequipped with a SGI Indy/Irix 5.3 workstation and XWINNMR soft-ware. Mixing times of 100 and 200 ms were used in ROESY andTOCSY experiments, respectively.

Acknowledgements

This work was supported by Grants 09-III-A-05-142 FEB RASand NSS-2383.2008.4.

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