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Biomacromolecules 2008, 9, 678–685
Structure of the O-Antigen of the Main Lipopolysaccharide Isolated from Sinorhizobium fredii SMH12 Francisco J. Fernández de Córdoba,† Miguel A. Rodríguez-Carvajal,† Pilar Tejero-Mateo,† Javier Corzo,‡ and Antonio M. Gil-Serrano*,† Department of Organic Chemistry, Faculty of Chemistry, University of Seville, 41071 Sevilla, Spain, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of La Laguna, E-38206 La Laguna, Spain Received September 11, 2007; Revised Manuscript Received November 30, 2007
The lipopolysaccharide of Sinorhizobium fredii SMH12, a wide-range host bacterium isolated from nodulated soybean plants growing in Vietnam, has been studied. Isolation of lipopolysaccharide by the phenol-water method leads to a mixture of two polysaccharides; polyacrylamide gel electrophoresis indicates that both are possibly lipopolysaccharides. The structures of the O-antigen of the main lipopolysaccharide and its deacetylated form are determined by sugar and methylation analysis, partial hydrolysis, lithium degradation, ESI-MS/MS, and NMR studies. Here we show that the fast-growing S. fredii SMH12 produces a lipopolysaccharide whose O-antigen has a repeating unit consisting of the trisaccharide f4)-R-D-GalpA-(1f3)-2-O-Ac-R-L-Rhap-(1f3)-2-O-Ac-R-DManp-(1f. The position O-6 of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in an approximate ratio 1:1:2. The tandem mass spectrometry studies rule out both an alternating and a random distribution of methyl groups and suggest the existence of zones in the polysaccharide rich in methyl groups interspersed with zones without methyl groups.
1. Introduction Sinorhizobium fredii SMH12 is a fast-growing Gram-negative soil bacterium that is able to enter into a nitrogen-fixing symbiosis with plants of the legume family,1 such as Glycine max (soy bean). The symbiotic bacterium-plant interaction results in the inclusion of highly differentiated bacterial cells (called bacteroids) in host-plant nodule cells, where atmospheric nitrogen is reduced to ammonia. Infection and nodule development is a species-specific process that is largely controlled by signal molecules from both the plant and the bacterium.2 In this cascade of biological signals, legume roots secrete a set of different flavonoids, a subset of which are able to induce the expression of rhizobial nodulation genes that are involved in the synthesis and secretion of lipo-chitin oligosaccharides (LCOs or Nod factors), which trigger nodule organogenesis in specific regions of the legume root. In addition to Nod factors, several rhizobial surface polysaccharides are necessary for successful nodulation, as they act as signal molecules and/or in preventing plant defense response.3–5 Exopolysaccharides (EPS), lipopolysaccharides (LPS), capsular polysaccharides (KPS or K-antigens), and cyclic β-glucans are rhizobial polysaccharides more commonly investigated for their roles in the establishment of an effective symbiosis.2,3,5,6 The cell wall of rhizobia, as in other Gram-negative bacteria, consists of an inner membrane (the cytoplasmic membrane) and an outer membrane, separated by the periplasmic space. The outer membrane is formed of proteins, LPSs, and phospholipids. Cyclic glucans are located in the periplasmic space. The extracellular matrix of the bacteria (usually termed “capsule”) contains acidic EPSs that are released into the cell’s milieu. In * Author to whom correspondence should be addressed. E-mail: agil@ us.es. Telephone: +34 954-557-150. Fax: +34 954-624-960. † University of Seville. ‡ University of La Laguna. In memoriam Dr. Corzo.
the genus Sinorhizobium, the outer membrane and the surrounding capsule are constituted in part by a complex array of LPS and KPS. LPSs are complex glycolipid molecules having three different regions distinguishable in their structure: lipid A, core, and O-chain. In rhizobia, the lipid A region is constituted by aminosugar residues that are O- and N-fatty acylated. The core region is located between the lipid A and the O-chain. This central region of the LPS is an oligosaccharide composed of different sugars and derivatives, including Kdo (3-deoxy-Dmanno-2-octulosonic acid). The O-chain polysaccharide is a polymerized repeating oligosaccharide whose composition is highly variable in different rhizobia. This O-chain is responsible for the antigenic properties of the LPS (the so-called O-antigen). Here we show that the fast-growing S. fredii SMH12, a widerange host bacterium isolated from nodulated soybean plants growing in Vietnam, produces an LPS whose O-antigen has a repeating unit consisting of the trisaccharide f4)-R-D-GalpA(1f3)-2-O-Ac-R-L-Rhap-(1f3)-2-O-Ac-R-D-Manp-(1f. The position O-6 of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in a ratio 1:1:2.
2. Materials and Methods A. Bacterial Culture Conditions and Isolation of Polysaccharides. S. fredii SMH12 was routinely grown at 28 °C in TY medium, as described by Beringer.7 For the isolation of bacterial polysaccharides, 10 L of TY liquid medium were inoculated with 100 mL of early stationary phase cultures of S. fredii SMH12 and incubated on an orbital shaker at 160 rpm for three days at 28 °C. After incubation, the cells were harvested by slow-speed centrifugation. Bacterial pellets were washed three times with 0.9% (w/v) NaCl, freeze-dried, and stored in sealed bottles at room temperature. The lipopolysaccharide was extracted from the freeze-dried bacterial cells (5 g) with 1:1 hot phenol-water mixture (100 mL),8 and the two phases were separated. The aqueous phase was dialyzed against water, freeze-dried, and
10.1021/bm701011d CCC: $40.75 2008 American Chemical Society Published on Web 01/22/2008
O-antigen of S. fredii SMH12 redissolved in 10 mM MgSO4 and 50 mM Tris-HCl solution (100 mL, pH 7.0); DNase (1 mg) and RNase (1 mg) were added, and the solution was stirred overnight at 5 °C. Proteinase K (2 mg) was added, and the solution was shaken for 24 h at 37 °C, dialyzed, and then freeze-dried. The lipopolysaccharide was chromatographed on Sephacryl S-500 (60 cm × 2.6 cm), using 0.05 M EDTA/triethylamine, pH 7.0 as eluent, and carbohydrates were detected using a refractive-index detector and the orcinol-sulfuric acid method on thin-layer chromatography plates. Fractions containing carbohydrates were dialyzed and freeze-dried. A 0.1% (v/w) solution of LPS fraction in 1% (v/v) AcOH was heated at 100 °C for 2.5 h, and the lipid A precipitated was separated by centrifugation. The supernatant was extracted with dichloromethane, and the aqueous phase containing the O-antigen and core oligosaccharides was fractionated by gel permeation chromatography on BioGel P-2 (1.6 cm × 60 cm) (from Bio-Rad) using water as eluent. B. SDS-Polyacrylamide Gel Electrophoresis (PAGE). Two different protocols have been used to analyze the bacterial lipopolysaccharide (LPS) and K-antigen polysaccharide (KPS) profiles. The first method allows the analysis of lipopolysaccharide. Bacterial cultures of strain SMH12 were grown on solid TY medium for four days at 28 °C. Then, bacterial cells were washed three times with 0.9% NaCl and pelleted by centrifugation. The bacterial pellet was resuspended and lysed by heating at 100 °C in 125 µL of 60 mM Tris/HCl/ 2% SDS (w/v)/1 mM EDTA, pH 6.8 for 5 min, and then diluted to 1 mL with the same buffer without SDS. The crude bacterial extract was treated with RNase, DNase, and proteinase K as described by Köplin et al.9 Sample solutions containing isolated LPS extract (1 mg) were dissolved in 100 µL of 60 mM Tris/HCl, 1 mM EDTA, pH 6.8, and diluted to 200 µL in the same buffer containing 2% SDS (w/v), 6% glycerol (v/v), 1% of 2-mercaptoethanol (v/v), and 1% (v/v) of a saturated solution of bromophenol blue in water. Electrophoresis of crude bacterial extracts or purified bacterial polysaccharide was performed on a 16.5% (w/v) polyacrylamide gel with the tricine buffer system described by Lesse et al.10 For visualization of lipopolysaccharides, gels were silver-stained as described by Kittelberger and Hilbink.11 The second method, PAGE without any detergent, was also carried out to analyze the possible KPS. In the absence of SDS, only the KPS migrates on the gel, whereas the LPS is stacked in the upper part of the gel and does not run any further. In this way, samples were analyzed by vertical electrophoresis (Bio-Rad), as described for extracellular polysaccharides from Bradyrhizobium.12 A continuous system was employed, using slab gels 0.75 mm thick. The acrylamide concentration was 18% (w/v), the acrylamide/N,N′-methylenebisacrylamide ratio being 30:0.8. The electrophoresis buffer was 50 mM Tris, 13 mM EDTA, and 15 mM boric acid (pH 8.5). Samples were dissolved in the same buffer and diluted (1:2) in 1 M sucrose in deionized water. Gels were fixed using Alcian Blue in acetic acid13 and stained by the silver method.11 C. Monosaccharide Analysis. Monosaccharides were identified on GLC-MS separation of their trimethylsilylated methyl glycosides obtained as described.14 The absolute configuration of monosaccharides was assigned following GLC-MS analysis of their trimethylsilylated (S)- and (R,S)-2butyl glycosides for galacturonic acid, rhamnose, and mannose. The trimethylsilylated 2-butyl glycosides were prepared as described.15 The absolute configurations of 6-O-methyl-mannose were determined following GLC-MS analysis of its permethylated (S)- and (R,S)-2-butyl glycosides: the polysaccharide was methanolyzed with HCl/MeOH 0.625 M for 16 h at 80 °C, and the released monosaccharides were methylated as described.16 Finally, the methylated monosaccharides were treated with HCl/(S)-2-butanol or HCl/(R,S)-2-butanol for 16 h at 80 °C and analyzed by GLC-MS. Derivatives from standard monosaccharides were prepared for comparison. Gas–liquid chromatography–mass spectrometry (GLC-MS) was performed on a Micromass AutoSpec-Q instrument fitted with an OV-1 column (25 m × 0.25 mm). The temperature program for separating
Biomacromolecules, Vol. 9, No. 2, 2008 679 the trimethylsilylated methyl glycosides and 2-butyl glycosides was isothermal at 150 °C for 2 min, followed by a 10 °C/min gradient up to 250 °C. The ionization potential was 70 eV, and spectra were recorded in low-resolution mode. D. Partial Degradations of the Polysaccharide. The polysaccharide was partially hydrolyzed with 0.5 M trifluoroacetic acid at 100 °C for 2 h. The resulting oligosaccharides were purified by size exclusion chromatography on a Biogel P-6 column (65 cm× 2 cm) using water as eluent. Oligosaccharides were analyzed by electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation (CIDMS/MS). Experiments were performed on an Applied API 2000 LCMS/MS system (Foster City, CA). Samples were dissolved in methanol: water (1:1, v/v) containing 0.1% formic acid and were continuously infused into the microspray ionspray source at a flow rate of 5 µL/min using the integral syringe drive. The instrument was operated in the positive mode with declustering potential of 120 V. The capillary was held at a voltage of 5500 V. The collision energy setting for tandem mass spectrometry experiments ranged from 25 to 80 V. Nitrogen was used as curtain and collision gases. Spectra were acquired using the Analyst QS software (Applied Biosystems). Reduced oligosaccharides were obtained by stirring the polysaccharide in ethylenediamine with lithium metal as described.17 The resulting residue was dissolved in 5 mL water, titrated to pH 4.5 with glacial acetic acid, and passed through a 10 mL column of Dowex AG-50W X12 (H+) ion-exchange resin, eluting with water. The reduced oligosaccharides were chromatographed on Biogel P-2, collecting the low-molecular-weight fraction. This fraction was analyzed by liquid secondary ionization mass spectrometry (LSIMS), performed on a Micromass AutoSpec-Q instrument in positive ion mode with a cesium ion gun at an acceleration voltage of 8 kV. Thioglycerol containing NaI as cationizing agent was used as matrix. The final concentration of samples was 10 mg/mL in water. E. Methylation Analysis. The vacuum-desiccated sample of polysaccharide was methylated by the method of Ciucanu and Costello,18 using either CH3I or C2H3I (for the location of endogenous methyl groups). The permethylated polysaccharide was carboxyl-reduced by treatment with 2 mg of NaB2H4 dissolved in 500 µL of ethanol:water (3:1, v/v) at room temperature overnight.19 Finally, the sample was hydrolyzed, reduced with NaB2H4, and acetylated as described.20 Gas–liquid chromatography–mass spectrometry was performed on a Micromass AutoSpec-Q instrument fitted with an OV-1 column (25 m × 0.25 mm). The temperature program for separating the partially methylated alditol acetates was isothermal at 120 °C for 1 min, followed by an 8 °C/min gradient up to 250 °C. The ionization potential was 70 eV, and spectra were recorded in low-resolution mode. F. Determination of Molecular Size. The molecular size of the O-antigen was determined by size exclusion chromatography on an HPLC Sugar Analyzer I system (Waters) fitted with a µBondagel E-linear column (300 mm × 3.9 mm) and monitored with a differential refractometer (Waters). The elution solvent was water at an isocratic flow rate of 0.5 mL/min. The column was calibrated with 503000, 252000, 110000, 70000, and 39100 Dalton dextrans from Sigma. G. NMR Spectroscopy. Samples were deuterium-exchanged several times by freeze-drying from 2H2O and then examined in solution (5 mg/750 µL) in 99.98% 2H2O. Spectra were recorded at 303 K on a Bruker AV500 spectrometer operating at 500.13 MHz (1H) and 125.75 MHz (13C). Chemical shifts are given in ppm, using the H2HO signal (4.75 ppm) (1H) and external dimethylsulfoxide (39.5 ppm) (13C) as references. The TOCSY was acquired using a data matrix of 256 × 2K points to digitize a spectral width of 4084 Hz; 16 scans per increment and an isotropic mixing time of 90 ms were used. The 2D homonuclear DQF-COSY was performed using the Bruker standard pulse sequence. A data matrix of 256 × 2K points was used to digitize a spectral width of 3280 Hz; 16 scans were used per increment. The 2D heteronuclear one-bond proton-carbon correlation experiment was registered in the 1H-detection mode via single-quantum coherence (HSQC). A data matrix of 256 × 1K points was used to digitize a
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Figure 2. 1H NMR (500 MHz) of (a) high-molecular-weight fraction (HMW) containing the O-antigen of S. fredii SMH12 (PS) and another minor polysaccharide; (b) deacetylated O-antigen (PSdAc).
Figure 1. Polyacrylamide gel electrophoresis analysis of surface polysaccharides of S. fredii SMH12. (a) Electrophoresis with SDS and silver-stained to visualize lipopolysaccharides. (b) Electrophoresis without SDS and Alcian Blue/silver-stained to visualize capsular polysaccharides. Lane 1: cell-wall extracts of S. fredii strain SMH12. Lane 2: high-molecular-weight fraction obtained by size exclusion chromatography of the phenol:water extract. Lane 3: low-molecularweight fraction obtained by size exclusion chromatography of the phenol-water extract.
spectral width of 3280 and 22522 Hz in F2 and F1; 64 scans were used per increment. 13C decoupling was achieved by the GARP scheme. Squared-cosine-bell functions were applied in both dimensions, and zero-filling was used to expand the data to 512 × 1K. This experiment was slightly modified by the implementation of an editing block in the sequence.21 The HMBC experiment was performed using the Bruker standard sequence with 256 increments of 1K real points to digitize a spectral width of 4084 × 30030 Hz; 64 scans were acquired per increment with a delay of 70 ms for evolution of long-range couplings. The coupled 2D heteronuclear one-bond proton-carbon correlation experiment was registered in the 1H-detection mode via single-quantum coherence (coupled HSQC). A data matrix of 256 × 2K points was used to digitize a spectral width of 2694 and 22522 Hz in F2 and F1; 48 scans were used per increment. Squared-cosine-bell functions were applied in both dimensions, and zero-filling was used to expand the data to 1K × 2K. The pure absorption NOESY experiment was performed with a mixing time of 150 ms. A data matrix of 256 × 2K points was used to digitize a spectral width of 4084 Hz; 32 scans were used per increment.
3. Results and Discussion S. fredii SMH12 bacteria were grown in TY medium. The lipopolysaccharide was extracted by the hot phenol-water method8 and then purified by ion exchange, dialysis, enzyme treatments, and size exclusion chromatography. The presence of LPS in the resulting extract was confirmed by SDS/PAGE analysis (Figure 1A), showing a characteristic LPS profile similar to that obtained from S. fredii SMH12 cells. Moreover, PAGE analyses carried out without SDS do not show KPS either in this extract or in S. fredii SMH12 cells (Figure 1B). Hydrolysis under mild acidic conditions released O-antigen, core, and Lipid A. The latter was removed by extraction with CH2Cl2, and the aqueous layer was submitted to size exclusion chromatography on Biogel P-2 (from Bio-Rad), giving a highmolecular-weight (more than 2000 Da) fraction (HMW) corresponding to the O-antigen. Estimation of apparent mean
Figure 3. 1H (500 MHz), 13C (125.75 MHz) multiplicity-edited HSQC spectrum of deacetylated O-antigen of S. fredii SMH12. Signals are assigned as follows: G: galacturonic acid; R: rhamnose; M: mannose; X: 6-O-methyl-mannose. Signals corresponding to methylene groups are shown as dotted lines.
molecular weight by size exclusion chromatography (HPLC) gave a value of 7 × 105 Da. Sugar analysis indicated that HMW contains rhamnose, mannose, galacturonic acid, and an unknown sugar (subsequently identified as 6-O-methyl-mannose) in a ratio 2.5:1.0: 0.9:1.3, respectively. The identification of 6-O-methyl-mannose was done on the basis of its chromatographic behavior in CGMS (a methyl group instead of a trimethylsilyl group reduces the retention time of the corresponding trimethylsilylated methyl glycoside by about one minute in the monosaccharide analysis), methylation analysis, and NMR. Methylation analysis of this fraction, having included a carboxyl reduction step prior to hydrolysis, gave the partially methylated and acetylated alditols: 1,2,5-tri-O-acetyl-1-deutero-3,4-di-O-methyl-rhamnitol (from a f2)-Rhap residue), 1,3,5-tri-O-acetyl-1-deutero-2,4-di-O-methyl-rhamnitol (from a f3)-Rhap residue), 1,3,5-tri-O-acetyl-1deutero-2,4,6-tri-O-methyl-mannitol (from a f3)-Manp or f3)6-O-MeManp residue), and 1,4,5,6-tetra-O-acetyl-1,6,6-trideutero2,3-di-O-methyl-galactitol (from f4)-GalpA or f5)-GalfA units). Lithium Degradation. The occurrence of galacturonic acid makes the partial degradation of HMW with lithium in ethylenediamine17 appropriate. Reduced oligosaccharides were obtained by stirring the polysaccharide in ethylenediamine with
O-antigen of S. fredii SMH12
Figure 4. Partial 1H (500 MHz) TOCSY and NOESY spectra of deacetylated O-antigen of S. fredii SMH12 (PSdAc). Signals are assigned as follows: G: galacturonic acid; R: rhamnose; M: mannose; X: 6-O-methyl-mannose.
lithium metal and further purification by chromatography. The monosaccharide analysis indicates that this fraction contains rhamnose, mannitol, and an alditol arising from the reduction of a 6-O-methylated sugar. Methylation analysis using C2H3I to locate the endogenous methyl groups gave the partially methylated and acetylated alditols: 1,5-di-O-acetyl-1-deutero-2,3,4-tri-O-trideuteromethylrhamnitol (from terminal nonreducing Rhap residues), 3-Oacetyl-1,2,4,5,6-penta-O-trideuteromethyl-mannitol (from reduced f3)-Manp residues), and 3-O-acetyl-6-O-methyl-1,2,4, 5-tetra-O-trideuteromethyl-mannitol (from reduced f3)-6-Omethyl-mannopyranose units). Both mannitol derivatives coeluted in the same chromatographic peak and differed only in electron-impact mass spectra. Analysis by liquid secondary ionization mass spectrometry (LSIMS) showed two peaks at m/z 351 and 365, corresponding to pseudomolecular ions ([M + Na]+) of 3-O-(rhamnopyranosyl)mannitol and 6-O-methyl-3-O-(rhamnopyranosyl)mannitol (for the sake of clarity, the numbering of the original sugar has been kept). As those residues linked to galacturonic acid units become alditols after lithium treatment, the above disaccharides indicate the sequences Rhap-(1f3)-Manp-GalA and Rhap-(1f3)-6-OMeManp-GalA. NMR. Fraction HMW was studied by NMR. In general, the 500 MHz 1H monodimensional spectrum (Figure 2) shows poorly resolved peaks. Regarding the anomeric region between 4.8 and 5.4 ppm, several signals can be assigned to anomeric protons, although a few of them present lower intensities. In this region, signals at 5.27 and 5.28 ppm do not correspond to anomeric protons, as the HSQC spectrum correlates them with carbon signals at about 68 ppm (not shown). A significant downfield shift of these methine signals can be explained by the presence of acetyl groups, which appear as intense singlet signals at 2.2 ppm. In the region between 3.2 and 4.5 ppm, where signals from most of the sugar protons are located, an intense singlet signal at 3.39 ppm was assigned to the methyl group of the 6-O-methyl-mannose. Finally, signals corresponding to methyl groups of rhamnose can be found at ca. 1.3 ppm.
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The presence of acetyl groups, together with methyl groups of rhamnose and 6-O-methyl-mannose, reduces the solubility of the polysaccharide in water, making the NMR study difficult. To increase this solubility, part of the polysaccharide was deacetylated by treatment with 12.5% NH4OH22 and further purified by size exclusion chromatography. The deacetylated polysaccharide obtained (PSdAc) was submitted to structural study by mass spectrometry and NMR. Structural Study of the Deacetylated Polysaccharide (PSdAc). Monosaccharide analysis of PSdAc indicates that it is composed of galacturonic acid, rhamnose, mannose, and 6-Omethyl-mannose in the ratio 1:1:0.5:0.5. Methylation analysis of PSdAc using C2H3I and including a carboxyl reduction step prior to hydrolysis, gave the partially methylated and acetylated alditols 1,4,5,6-tetra-O-acetyl-1,6,6-trideutero-2,3-di-O-trideuteromethyl-galactitol (coming from 4-linked galactopyranosuronic acid or 5-linked galactofuranosuronic acid; numbers follow the original sugar numbering), 1,3,5-tri-O-acetyl-1deutero-2,3-di-O-trideuteromethyl-rhamnitol (coming from 3-linked rhamnopyranose), 1,3,5-tri-O-acetyl-1-deutero-2,4, 6-tri-O-trideuteromethyl-mannitol (coming from 3-linked mannopyranose), and 1,3,5-tri-O-acetyl-1-deutero-2,4-di-O-trideuteromethyl-6-O-methyl-mannitol (coming from 3-linked mannopyranose methylated at O-6). When comparing these results with those obtained from fraction HMW, it can be seen that the polysaccharide PSdAc does not contain 2-linked rhamnopyranose units as well as that the ratio of this sugar has decreased. The explanation is that fraction HMW contains two polysaccharides: a major one (PS), composed of 3-linked rhamnose, 3-linked mannose, 3-linked 6-O-methyl-mannose, and 4- or 5-linked galacturonic acid, and whose deacetylation leads to polysaccharide PSdAc; the second one is a minor polysaccharide containing 2-linked rhamnose, which has been separated from the major one by the purification treatments after deacetylation. This paper describes the structure of the major polysaccharide PS and its deacetylated derivative, PSdAc. The structure of the minor polysaccharide is under study. The absolute configurations of monosaccharides in PSdAc were studied by the formation of (S)- and (R,S)-2-butyl glycosides, followed by analysis of their trimethylsilyl derivatives by GLC-MS. Results indicate that both mannose and galacturonic acid have a D absolute configuration, whereas rhamnose has an L configuration. The 6-O-methyl-mannose configuration was identified as D from analogous treatment of the permethylated derivative. NMR of PSdAc. The structure of the deacetylated O-antigen (PSdAc) was studied by NMR. Assignments of 1H and 13C NMR spectra were made on the basis of DQF-COSY, TOCSY, NOESY, multiplicity-edited HSQC, coupled HSQC, and HMBC experiments. The HSQC spectrum (Figure 3) shows three signals, δH 5.16/ δC 95.6 ppm, 5.01/96.2 ppm, and 4.92/100.8 ppm, corresponding to three anomeric protons/carbons. Besides other sugar proton signals, which are located between 4.6 and 3.4 ppm, an intense singlet signal appears at 3.41 ppm, which was assigned to an O-methyl group. The 1H NMR spectrum (Figure 2) also shows an intense doublet at δH 1.28 ppm, corresponding to the methyl group of rhamnose. Other deoxysugar signals can also be found in this region, although of lower intensity, coming from traces of the minor polysaccharide present in the HMW fraction. DQF-COSY and TOCSY (Figure 4) experiments enabled us to identify several sugar residues: crosspeaks in DQF-COSY correlate the signal at 5.16 ppm (H-1) with H-2 at 3.90 ppm, as well as H-2 with H-3 at 4.07 ppm. These last two signals show
682 Biomacromolecules, Vol. 9, No. 2, 2008 Table 1.
1
H- and
13
Fernández de Córdoba et al.
C NMR Chemical Shifts of Deacetylated O-Antigen (PSdAc) Isolated from S. fredii SMH12 position
residue f4)-R-D-GalpA f3)-R-L-Rhap f3)-R-D-Manp f3)-R-D-6MeManp a
Exchangeable assignment.
1
H 13 C 1 H 13 C 1 H 13 C 1 H 13 C b
1
2
3
4
5
6
Me
5.16 95.6 5.01 96.2 4.93a 100.8 4.91a 100.8
3.90 68.0 4.20 67.6 4.10 66.7 4.10 66.7
4.07 68.8 3.94 75.5 3.89 75.3 3.89 75.3
4.39 79.3 3.53 70.3 3.73 64.8 3.73 64.8
4.59 71.0 4.01 68.9 4.11 68.5 4.11 66.8b
174.0 1.28 17.2 3.81 60.7 3.69 71.5
3.41 58.8
Educated guess.
a large coupling constant 3J2,3 (∼10 Hz). The TOCSY spectrum shows a fourth signal at 4.39 ppm belonging to the same spin system with very small coupling constants. The spin system with a coupling pattern 3J2,3 large/3J3,4 small/3J4,5 small is characteristic of a galacto configuration in the pyranose form. Finally, H-5 (δH 4.59/δC 71.0 ppm) could be assigned by comparison with the expected chemical shifts in uronic acids23,24 and from intense NOESY crosspeaks with H-3 and H-4. HSQC enables the assignment of 13C signals. The downfield shift of C-4 (79.3 ppm) indicates the point of substitution. The very small coupling constant 3J1,2 (
