Structural Analysis of the Capsular Polysaccharide from

Biomacromolecules 2005, 6, 1448-1456

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Structural Analysis of the Capsular Polysaccharide from Sinorhizobium fredii HWG35 Miguel A. Rodrı´guez-Carvajal,† Joao A. Rodrigues,‡ Marı´a E. Soria-Dı´az,† Pilar Tejero-Mateo,† Ana Buendı´a-Claverı´a,§ Rocı´o Gutie´ rrez,§ Jose E. Ruiz-Sainz,§ Jane Thomas-Oates,‡ and Antonio M. Gil-Serrano*,† Departamento de Quı´mica Orga´ nica, Facultad de Quı´mica, and Departamento de Microbiologı´a, Facultad de Biologı´a, Universidad de Sevilla, 41071 Sevilla, Spain, and Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom Received November 19, 2004; Revised Manuscript Received January 26, 2005

We have determined the structure of a capsular polysaccharide from Sinorhizobium fredii HWG35. This polysaccharide was isolated following the standard protocols applied for lipopolysaccharide isolation. On the basis of monosaccharide analysis, methylation analysis, mass spectrometric analysis, one-dimensional 1H and 13C NMR, and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the following disaccharide repeating unit: f6)-2,4-di-O-methyl-R-D-Galp-(1f4)-β-D-GlcpA-(1f. Strain HWG35 produces a capsular polysaccharide that does not show the structural motif (sugar-Kdx) observed in those S. fredii strains that, while effective with Asiatic soybean cultivars, are unable to form nitrogen-fixing nodules with American soybean cultivars. Instead, the structure of the capsular polysaccharide of S. fredii HWG35 is in line with those produced by strains HH303 (rhamnose and galacturonic acid) and B33 (4-O-methylglucose-3-O-methylglucuronic acid), two S. fredii strains that form nitrogen-fixing nodules with both groups of soybean cultivars. Hence, in these three strains that effectively nodulate American soybean cultivars, the repeating unit of the capsular polysaccharide is composed of two hexoses, one neutral (methylgalactose, rhamnose, or methylglucose) and the other acidic (glucuronic, galacturonic, or methylglucuronic acid). Introduction Sinorhizobium fredii is a Gram-negative soil bacterium that is able to form root-nodule symbioses with numerous members of the plant family Leguminosae,1 such as Glycine max (soybean). This 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, a utilizable nitrogen source for the plant. The formation of legume nodules is the result of a complex plant-microbe interaction which is induced, and controlled, by reciprocal signal exchange between the partners.2 In this cascade of biological signals, legume roots secrete a set of different flavonoids, a subset of which are able to induce the transcription of bacterial genes essential for nodulation. Many of these genes (nod) are responsible for the synthesis and secretion of lipo-chitin oligosaccharides, collectively known as “LCOs” or “Nod factors”, that trigger nodule organogenesis in specific regions of the legume root.3 Bacterial LCOs are necessary, but not sufficient, for the formation of nitrogen-fixing nodules. In addition to nod genes and those directly involved in nitrogen fixation (fix and nif genes), other bacterial determinants are * Author for correspondence. Tel. no. +34 954557150, fax no. +34 954624960, e-mail [email protected]. † Departamento de Quı´mica Orga ´ nica, Universidad de Sevilla. ‡ University of York. § Departamento de Microbiologı´a, Universidad de Sevilla.

required for the formation of stable nitrogen-fixing nodules. Bacterial polysaccharides, such as β-glucans, acidic exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS), are clearly among those determinants that are essential for the establishment of an effective symbiosis.2,4-6 The cell wall of Gram-negative bacteria is formed of the inner membrane (the cytoplasmic membrane) and the outer membrane, with a periplasmic space between them. The cytoplasmic membrane is formed of phospholipids and lipoproteins. The outer membrane consists of proteins, LPS, and phospholipids. β-glucans are mainly located in the periplasmic space. The extracellular matrix of the bacteria (usually termed capsule) contains acidic EPS that is released into the cell’s milieu. In bacterial strains belonging to the Sinorhizobium genus, the outer membrane and the surrounding capsule are composed in part of a complex array of LPS and KPS. Although these polysaccharides appear to play multiple roles in nodule formation, there is the general agreement that one of these is to prevent the triggering of plant-defense responses that could abort rhizobial invasion of the root.4-7 Reports describing the structures of K-antigenic polysaccharides from several S. fredii and S. meliloti strains have shown that there are clear differences among them.5,8,9 In many of them, however, a conserved structural motif becomes evident since the repeating unit is formed of variable

10.1021/bm049264u CCC: $30.25 © 2005 American Chemical Society Published on Web 04/07/2005

Structure of the KPS from S. fredii HWG35

glycosyl residues linked to Kdo or a Kdo-related residue (Kdx). This conserved structural motif (hexose-Kdx) appears in three (AK631, NRG185, and NRG247) out of four S. meliloti strains studied so far. The exception is S. meliloti 1021, which produces a homopolymeric KPS composed of Kdo.10 The hexose-Kdx subunit is also present in the KPS of the broad host-range Sinorhizobium sp. NGR234 and in S. fredii strains USDA201, USDA205, USDA257, and USDA208.5,8,9 These S. fredii strains form nitrogen-fixing symbioses with soybean Asiatic cultivars but fail to nodulate American soybean cultivars.11 In contrast, the S. fredii wildtype strains HH103, HH303, and B33, which form Fix+ nodules with Asiatic and American soybean cultivars, produce KPS repeating units that do not show the sugarKdx motif.8,12,13 Here we show that the S. fredii strain HWG35 produces a capsular polysaccharide with a repeating unit consisting of the disaccharide f6)-2,4-di-O-methyl-R-D-Galp-(1f4)β-D-GlcpA-(1f. This strain apparently does not produce a Kdx-containing KPS and is also able to form nitrogen-fixing nodules on both Asiatic and American soybean cultivars. Experimental Section General Methods. Gas-liquid chromatography-mass spectrometry (GLC-MS) was performed with a Micromass AutoSpec-Q instrument fitted with an OV-1 column (25 m × 0.25 mm). The temperature program for separating the trimethylsilylated methyl glycosides was isothermal at 150 °C for 2 min followed by a 10 °C/min gradient up to 250 °C, while that for 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 protocol for analyzing the permethylated 2-octyl glycosides and trimethylsilylated butyl glycoside derivatives was isothermal at 130 °C for 3 min followed by a gradient of 3 °C/min up to 150 °C and then 10 °C/min up to 250 °C. Mass spectra were recorded using low-resolution electron ionization with an ionization potential of 70 eV. 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 permethylated (S)- and (R,S)-2-octylglycosides and trimethylsilylated (S)- and (R,S)-2-butylglycosides. The polysaccharide was methanolyzed with 0.625 M HCl/MeOH for 16 h at 80 °C, and the released monosaccharides were methylated as described.13 The permethylated monosaccharides were treated with 0.625 M HCl/(S)-2-octanol and 0.625 M HCl/(R,S)-2octanol (generated with acetyl chloride). The trimethylsilylated-2-butylglycosides were prepared as described.15 Bacterial Culture Conditions and Isolation of Polysaccharides. S. fredii HWG35 was routinely grown at 28 °C in TY medium as described by Beringer.16 For the isolation of bacterial polysaccharides, 10 L of TY liquid medium were inoculated with 100 mL of early-stationary-phase cultures of HWG35 and incubated on an orbital shaker at 160 rev/ min for 3 days at 28 °C. After incubation, the cells were

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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 polysaccharide was extracted from the freeze-dried bacterial cells (5 g) with 1:1 hot phenol-water mixture (100 mL),17 and the two phases were separated. The aqueous phase was dialyzed against water, freeze-dried, and 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 polysaccharide was chromatographed on Sephacryl S-500 (60 × 2.6 cm) using 0.05 M ethylenediaminetetraacetic acid (EDTA)-triethylamine (pH 7.0) as the eluent, and carbohydrates were detected using a refractive index detector and the orcinolsulfuric acid method on thin-layer chromatography plates. Fractions containing carbohydrates were dialyzed and freezedried. Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE). Two different protocols have been used to analyze the bacterial LPS and K-antigenic polysaccharide (KPS) profiles. In one of them the LPS is visualized. In the other, the KPS, but not the LPS, is the polysaccharide that fully runs into the gel. In the first method, bacterial cultures of strain HWG35 were grown on solid TY medium for 4 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% (w/v) SDS/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 Ko¨plin et al.18 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.19 For visualization of LPS, gels (Figure 1A) were silver stained as described by Kittelberger and Hilbink.20 The second method, PAGE without any detergent, was carried out to facilitate the visualization of KPS. In the absence of SDS, only the KPS migrates on the gel (the LPS is stacked in the upper part of the gel and does not run any further). In this way, samples of K-antigenic polysaccharide were analyzed using vertical electrophoresis (Bio-Rad), as described for extracellular polysaccharides from Bradyrhizobium.21 A continuous system was employed, using 0.75-mmthick slab gels. 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 acid22 and stained by the silver method.20 Mass Spectrometric Analysis. Procedures of varying harshness were carried out over different time periods to partially release oligosaccharides from the polysaccharide: autolysis in water at 60 °C and incubation in 1% acetic acid at 100 °C or 0.5 M trifluoroacetic acid (TFA) at 100 °C for

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Figure 1. Silver-stained (A) and Alcian Blue/silver-stained (B) PAGE of whole-cell extracts of S. fredii HWG35 and fractions isolated by GPC of the polysaccharide. Panel A: Silver-stained SDS-PAGE of cell wall extracts of S. fredii strain HWG35 (lane 4); F1, highmolecular-weight fraction (lane 1); F2, medium-molecular-weight fraction (lane 2); and F3, low-molecular-weight fraction (lane 3). Panel B: Alcian Blue/silver-stained PAGE of cell wall extracts of S. fredii strain HWG35; F1, high-molecular-weight fraction (lane 1); F2, medium-molecular-weight fraction (lane 2); and F3, low-molecularweight fraction (lane 3).

1.5 h. All these experiments were performed by addition of 100 µL of the desired solvent to 100 µg of isolated polysaccharide. Following partial hydrolysis, electrosprayquadrupole-orthogonal-time-of-flight (ES-Q-o-TOF) MS and collision-induced dissociation (CID-MS/MS) experiments were performed using an Applied Biosystems Q-Star Pulsar i (Warrington, U.K.). Samples were dissolved in methanol/ water (50:50, v/v) containing 0.1% formic acid and were continuously infused into the microspray ion source at a flow rate of 1 µL‚min-1 using the integral syringe drive. The instrument was operated in the positive mode with declustering and focusing potentials of 65 and 265 V, respectively. The capillary was held at a voltage of 5500 V. The collision energy setting for tandem mass spectrometry experiments was varied between 25 and 80 V. Nitrogen was used as curtain gas and as collision gas. Spectra were acquired using the Analyst QS software (Applied Biosystems). Calibration of the instrument was performed using a mixture of maltooligosaccharides (Sigma, Poole, Dorset, U.K.), following recording the electrospray mass spectrum and subsequent CID product ion spectra. Methylation Analysis. The vacuum-desiccated sample of polysaccharide was methylated by the method of Ciucanu and Kerek,23 although using C2H3I instead of CH3I to enable location of endogenous methyl groups. The permethylated polysaccharide was purified by gel-exclusion chromatography on Sephadex LH-20, using MeOH/CH2Cl2 (1:1, v/v) as the eluent. It was then carboxyl-reduced by treatment with 2 mg of NaB2H4 dissolved in 500 µL of ethanol/water (75: 25, v/v) at room temperature overnight.24 Finally, the sample was hydrolyzed, reduced with NaB2H4, and acetylated. NMR Spectroscopy. The samples were deuteriumexchanged several times by freeze-drying from 2H2O and then examined in solution (10 mg/600 µL) in 99.98% 2H2O. Spectra were recorded at 303 K on a Bruker AMX500 spectrometer operating at 500.13 MHz (1H) and 125.75 MHz

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(13C). Chemical shifts are given in ppm, using the H2HO signal (4.75 ppm; 1H) and external dimethyl sulfoxide (39.5 ppm; 13C) as references. Selective excitation one-dimensional experiments were carried out by application of the DANTE-Z pulse train (n ) 300, τ ) 100 µs, θ ) 0.3°).25 This train was also concatenated to a total correlation spectroscopy (TOCSY) sequence (isotropic mixing times of 37.6, 70.6, 117.6, 150.5, and 188.2 ms)26 to yield the one-dimensional TOCSY subspectra. The number of accumulated scans was 128. The two-dimensional TOCSY was acquired using a data matrix of 512 × 1K points to digitize a spectral width of 3030 Hz; 32 scans per increment and a isotropic mixing time of 148.2 ms were used. The two-dimensional homonuclear double-quantum-filtered correlation spectroscopy (DQFCOSY) was performed using the Bruker standard pulse sequence. A data matrix of 512 × 1K points was used to digitize a spectral width of 2392 Hz; 48 scans were used per increment. The two-dimensional heteronuclear one-bond proton-carbon correlation experiment was registered in the 1 H-detection mode via single-quantum coherence (HSQC). A data matrix of 256 × 1K points was used to digitize spectral widths of 3012 and 16 350 Hz in F2 and F1; 64 scans were used per increment with a delay between scans of 1 s and a delay corresponding to a J value of 150 Hz. 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.27 The heteronuclear multiple-bond correlation (HMBC) experiment was performed using the Bruker standard sequence with 256 increments of 1K real points to digitize a spectral width of 3012 × 25 154 Hz. A total of 128 scans were acquired per increment with a delay of 80 ms for evolution of long-range couplings. The pure absorption two-dimensional nuclear Overhauser effect spectroscopy (NOESY) experiment was performed with a mixing time of 200 ms. A data matrix of 512 × 1K points was used to digitize a spectral width of 3012 Hz; 32 scans were used per increment with a delay between scans of 1 s. Results Isolation and Purification of the Polysaccharides Produced by S. fredii HWG35. The polysaccharides (mainly LPS and KPS) were extracted from bacterial pellets of S. fredii strain HWG35 using the hot phenol water method. Contaminating nucleotides and proteins were removed by enzyme treatment, and the polysaccharides present in the sample were then fractionated using gel-permeation chromatography (GPC). Three fractions containing polysaccharides, assessed using both a refractive index detector and orcinol staining, were isolated on the basis of their molecular masses. These three fractions were then investigated for the presence of LPS and/ or KPS using two different PAGE protocols. SDS/PAGE with the tricine buffer system followed by silver staining was carried out to visualize the LPS component of the samples (Figure 1A). In this method, K-antigenic polysac-

Structure of the KPS from S. fredii HWG35

charides escape from the gel during the washing steps before silver staining. The second method allows the visualization of KPS because PAGE is carried out in the absence of any detergent (the absence of SDS means that LPS molecules become stacked in the upper part of the gel) and, in addition, K-antigens are fixed with Alcian blue, so that they cannot escape from the gel before silver staining (Figure 1B). SDS/PAGE of fractions 1 and 2 (Figure 1A, lanes 1 and 2) shows that these fractions contain different molecular species (most probably LPS) that are clearly distinguishable by their electrophoretic mobility: (a) slow-migrating material, that probably corresponds to complete LPS molecules (smooth LPS) containing lipid-A, the core oligosaccharide and varying numbers of repeating subunits of the O-antigen portion, and (b) fast-migrating material, that probably corresponds to shorter LPS molecules (rough LPS) in which only one subunit of O-antigen, or zero, is present. Fraction 1 (Figure 1A, lane 1) seems to contain less LPS than fraction 2 (lane 2), and their electrophoretic patterns resemble that observed for crude bacterial cell-wall extracts (Figure 1, lane 4), although the slow-migrating material in fractions 1 and 2 is not clearly resolved in a ladder of discrete bands. Fraction 3 (Figure 1A, lane 3) did not show any silver-stained material, indicating that the amount of LPS in the sample (if any) is negligible. PAGE experiments without SDS and fixation with Alcian blue before silver staining showed that samples of fraction 1 run as a dark smear that covers the whole lane (Figure 1B). This silver-stained material that is present all along the lanes corresponds to a polysaccharide that is lost from the gel if the fixing steps with Alcian blue are omitted. Fraction 1 appears to contain more Alcian blue fixable material, most probably KPS, than fractions 2 and 3. Fraction 3 appears to contain only putative KPS. However, when it was desalted, by GPC on BioGel P-2, the amount of carbohydrate recovered was too small to allow studies aimed at structural determination. Thus, fraction 1 (which is the fraction richest in KPS, although containing some contaminating LPS) was chosen for the structural determination of the bacterial K-antigenic polysaccharide. Monosaccharide Composition Analysis. GLC-MS analysis of the trimethylsilylated methyl glycosides obtained after methanolysis of fraction 1 revealed that it contains, as major components, glucuronic acid and two peaks that could not be identified as arising from any common monosaccharide (results not shown). The corresponding mass spectra of these peaks contain a relatively intense fragment ion at m/z 146, which is characteristic of methylated trimethylsilylated methyl glycosides. In addition, the retention times are shorter than those of nonmethylated and monomethylated monosaccharide derivatives. Tentatively, we propose that these two peaks belong to a dimethylated monosaccharide. In addition we identified as minor components Rha, Glc, mono-and dimethylated hexose, and Kdo, probably arising from the contaminating LPS. Mass Spectrometric Analysis. The KPS was then subjected to mass spectrometric analysis following depolymerization to generate manageably sized oligosaccharide fragments. Autolysis and mild acid hydrolysis were performed

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but failed to release small oligosaccharides detectable by electrospray mass spectrometry (ES-MS). Partial hydrolysis was then accomplished using 0.5 M TFA at 100 °C for 1.5 h. ES-MS analysis yielded [M + Na]+ ions for a di- and tetrasaccharide (Figure 2). To determine the structure of the released oligosaccharides, they were induced to fragments following mass spectrometric ionization (ES), on collision with molecules of a neutral collision gas in the process of CID. This process results in the formation of structurally diagnostic fragments that are analyzed using tandem mass spectrometry. The product ion spectrum obtained is shown in Figure 3. The most intense fragments in the resulting tandem mass spectra result from the cleavage of glycosidic bonds. The ion at m/z 773 for the tetrasaccharide fragments to yield Y ions, which indicate an alternating sequence of hexuronic acid (HexA) and a residue with mass 190 Th [consistent with a hexose residue (Hex) plus 28 Da]. A 28 Th mass difference could suggest either an ethyl ether or two methyl ethers. Because the Y3 ion (m/z 597) loses 14 and 32 Th, consistent with β-cleavage and β-elimination, respectively, of a methyl group and not 28 and 46 Th, we assign a dimethylated instead of an ethylated hexose. Methylation Analysis. The position of the methyl groups as well as the position of the glycosidic linkages in the polysaccharide were determined by methylation analysis. The polysaccharide was trideuteriomethylated, to identify the position of the endogenous methyl groups, and carboxylreduced using NaB2H4, to identify the position of the carboxyl group. Finally, it was hydrolyzed, reduced with NaB2H4, and acetylated. GLC-MS analysis of the resulting partially trideuteriomethylated alditol acetates showed the presence of a 1,5,6-tri-O-acetyl-1-deuterio-3-O-trideuteriomethyl-2,4-di-O-methylhexitol (Figure 4), derived from a 6-linked 2,4-di-O-methylhexopyranose and a 1,4,5,6tetra-O-acetyl-1,6,6-trideuterio-2,3-di-O-trideuteriomethylhexitol. The latter could arise from a 4-linked glucopyranosyluronic acid or a 5-linked glucofuranosyluronic acid. NMR Analysis. The 1H NMR spectrum (Figure 5, top) of this fraction shows two intense signals (at δH 3.57 and 3.54 ppm) that are assigned to two methoxyl groups. The 1 H NMR spectrum also has signals for two anomeric protons at δH 5.73 ppm (corresponding to an R-anomer) and δH 4.56 ppm (corresponding to a β-anomer), in a 1:1 ratio. These data and the monosaccharide composition analysis and methylation analysis data suggest that the sample consists of a polysaccharide built up of a disaccharidic repeating unit composed of glucuronic acid and a dimethylated hexose. The 13C NMR spectrum (data not shown) has two signals at δC 60.6 and 57.0 ppm, assigned to the methyl groups, eight signals in the region between δC 68 and δC 78 ppm, two signals at δC 101.9 and 94.9 ppm, assigned to anomeric carbons, and a signal at 174.2 ppm which was assigned to a carbonyl group. The NMR analysis allowed us to determine the configuration of the di-O-methylhexose and to confirm the linkage position in the polysaccharide. Thus, the chemical shifts for the 1H and 13C resonances of the polysaccharide chain were assigned (Table 1) from the 1H and one-dimensional TOCSY,

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Figure 2. ES-Q-o-TOF MS of the partially hydrolyzed capsular polysaccharide from S. fredii HWG35.

Figure 3. Positive ion mode CID product ion spectrum of the sodiated molecule [M + Na]+ of the tetrasaccharide, obtained on partial hydrolysis of the capsular polysaccharide. The spectrum was obtained on collision of the ion at m/z 773.

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C, DQF-COSY (Figure 5), NOESY, HSQC, and HMBC spectra (Figure 6). The spectrum obtained in the DQF-COSY experiment allowed us to correlate the anomeric signals with

their spin system. In the case of the residue with R anomericity (residue A) we identified all the protons, from H-1 to both H-6s. This fact means that residue A, with R

Structure of the KPS from S. fredii HWG35

Figure 4. EI-MS and fragmentation scheme for partially methylated alditol acetates obtained on methylation analysis of the polysaccharide. The spectrum corresponds to 1,5,6-tri-O-acetyl-1-deuterio-3-Otrideuteriomethyl-2,4-di-O-methylhexitol, derived from a 6-linked 2,4di-O-methylhexopyranose.

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O-methyl hexose residue. The 3J ) 10.2 and 2.6 for H-2 indicate the trans-diaxial relative disposition of H-2 and H-3.28 The values of 3J (