Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada, K1A 0R6
Received on July 13, 2004; revised on October 1, 2004; accepted on November 5, 2004
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Abstract |
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Key words: core oligosaccharide / lipopolysaccharide / mass spectrometry / nuclear magnetic resonance / Pasteurella multocida
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Introduction |
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Vaccination of animals with killed bacteria is practiced and is sometimes effective in controlling clinical disease, but it is not uncommon for vaccinated flocks to suffer outbreaks. Empirically derived, live, avirulent strains have been used as vaccines in both poultry and cattle, but the basis for attenuation is not known, so it is therefore not surprising that reversion to virulence occurs. P. multocida isolates may be grouped serologically based on their capsular antigens into five serogroups A, B, DF using a passive hemagglutination test with erythrocytes sensitized with capsular antigen. Structural information is available for the capsules of serogroups A (hyaluronic acid) (Rosner et al., 1992), D (heparin), and F (chondroitin) (De Angelis et al., 2002), each being an example of the well-studied glycosaminoglycans. Somatic (lipopolysaccharide, LPS) typing can also be used for the identification of strains, and there have been two main systems reported. The Namioka system is based on a tube agglutination test and is able to recognisz 11 serotypes (Namioka, 1978
), whereas the Heddleston system uses a gel diffusion precipitation test, can recognize 16 serotypes, and is currently the preferred method (Heddleston et al, 1972
).
In 1981 a standard system for the identification of P. multocida serotypes was recommended that utilized both the Carter capsular typing identified by the letters A, B, D, E and F, and the Heddleston somatic typing system identified by numbers (Carter and Chengappa, 1981). P. multocida expresses LPS molecules that do not have the polymeric O-antigen, so-called rough LPS (Rimler, 1990
). Little is known about the structure and composition of P. multocida LPS. There is presently only published data on the LPS isolated from two serotype A strains of P. multocida and these contain a triheptose unit linked to a 2-keto-3-deoxyoctulosonic acid (Kdo) residue (Erler et al., 1981
, 1986
, 1988
). The genome sequence of P. multocida strain Pm70 was released in 2001 and some homologies to LPS biosynthetic genes were identified (May et al., 2001
). This study was therefore undertaken to determine the structure of the core oligosaccharide region of the genome strain LPS.
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Results |
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O-deacylated LPS (LPS-OH) was prepared and fractionated by gel filtration chromatography and analyzed by capillary electrophoresis (CE) mass spectrometry (MS) (Table I). A simple mass spectrum was observed with one major triply charged ion of m/z 1173.8 corresponding to a molecule of 3525 amu consistent with a composition of HexNAc2, Hep4, Hex6, phosphoethanolamine (PEtn), Kdo, P, Lipid A-OH with low amounts of ions consistent with the loss or gain of one PEtn residue from the major species indicated by m/z 1132.93 and 1215.23. CE-tandem MS (MS/MS) analysis (data not shown) on the triply charged ion m/z 1173.8 gave a singly charged peak of m/z 951 and a doubly charged ion of m/z 1236.5, confirming the size of the O-deacylated lipid A as 952 amu and the core OS as 2475 amu. The O-deacylated lipid A basal species (952 amu) consists of a disaccharide of N-acylated (3-OH C 14:0) glucosamine residues, each residue substituted with a phosphate molecule. Interestingly, small amounts of glycoforms that would correspond to the losses of a hexose and phosphate molecules with the concomitant gain of a Kdo molecule were indicated by m/z 1125.73 and 1166.73, suggesting the presence of two distinct arrangements of the Kdo region of the molecule with either a Kdo-P or a Kdo-Kdo arrangement (Table I). A similar mixture of one and two Kdo residue-containing glycoforms were observed in the CE-MS spectrum of KOH treated LPS (Table I). Electrospray ionizationMS and CE-MS analyses of the fractionated core OS sample revealed a mass of 2492 Da, consistent with a composition of HexNAc2, Hep4, Hex6, PEtn, Kdo (Table I; Figure 1).
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CE-MS/MS analyses in positive ion mode was performed on the core OS to obtain information as to the location of some of the functional groups in the OS molecule. MS/MS analysis on the doubly charged ion at m/z 1246.52+ revealed several product ions (Figure 2). Dominant were the singly charged ions corresponding to a HexNAc residue at 203.5+ and two HexNAc residues at 407.5+. Other product ions were also identified and correspond to the compositions indicated in Figure 2. Following precursor ion scanning for an ion with m/z 316 (which corresponds to a Hep-PEtn group) the PEtn moiety of the core OS was localized to a heptose residue (Hep) of the inner core by virtue of the identification of hydrated doubly charged ions of m/z 817.52+ (Hex3, Hep4, Kdo, PEtn), 898.52+ (Hex4, Hep4, Kdo, PEtn), 979.52+ (Hex5, Hep4, Kdo, PEtn), 1059.52+ (Hex6, Hep4, Kdo, PEtn), and 1263.52+ (HexNAc2, Hex6, Hep4, Kdo, PEtn) (Figure 3). Precursor ion scanning for an ion with m/z 407 (which corresponds to a HexNAc-HexNAc group) revealed a singly charged ion of 893+ that corresponds to a composition of Hex3, HexNAc2 (data not shown). These two precursor ion experiments therefore suggested an inner core composition of Hex3, Hep4, Kdo, and PEtn with an outer core extension of 3Hex and 2HexNAc residues.
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Methylation analysis was performed on the core OS to determine the linkage pattern of the molecule revealing the presence of terminal Glc, 6-substituted Glc, 4-substituted Glc, 4-substituted Gal, 3-substituted Gal, terminal LD-Hep and 4,6-disubsituted LD-Hep, in the approximate molar ratio of 2:1:1:1:1:1:1 with lesser amounts of terminal Gal, 2-substituted LD-Hep, 3,4-disubsituted LD-Hep and 3,4, 6-tri-substituted LD-Hep also observed. Additionally, by comparison to the permethylated alditol acetates from Actinobacillus pleuropneumoniae serotype 2, the retention time for a 4,6 disubstituted LD-Hep residue resolves from a 4,6 disubstituted DD-Hep residue, confirming the assignment and consistent with the absence of DD-Hep in the LPS of P. multocida.
To elucidate the exact locations and linkage patterns of the OS, nuclear magnetic resonance (NMR) studies were performed on the OS fraction that gave the most resolved and homogeneous spectrum and the fully deacylated (KOH-treated) material (Figure 4). The assignment of 1H resonances of the OS and KOH treated LPS was achieved by correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) (Figure 5a) experiments with reference to the structurally related core OS from Mannheimia haemolytica and A. pleuropneumoniae (Brisson et al., 2002; St. Michael et al., 2004
) (Table II).
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In the 1H-NMR spectrum of the Pm70 OS and KOH-treated LPS, spin systems arising from heptose residues (Hep I [E], Hep II [F] Hep III [G], and Hep IV [J]) were readily identified from their anomeric 1H resonances at 5.10OS (OS sample)/5.19KOH (KOH-treated sample) (Hep I, E), 5.87OS/5.74KOH (Hep II, F), 5.23OS&KOH (Hep III, G), and 5.12OS&KOH (Hep IV, J) ppm coupled with the appearance of their spin systems, which pointed to manno-pyranosyl ring systems. Heterogeneity was observed for the anomeric protons of Hep I in the OS sample due to the presence of various rearrangement products of the neighboring Kdo molecule following acid hydrolysis (assignments from the major Hep I signal in the OS sample are detailed). The -configurations were evident for the heptosyl residues from the occurrence of intraresidue nuclear Overhauser effects (NOEs) between the H1 and H2 resonances only. Two of the remaining residues in the
-anomeric region at 5.22OS&KOH ppm (Glc II, H) and 5.09OS/5.53KOH ppm (GalNAc II/GalN II, P) were determined to be gluco- and galacto-pyranose sugars, respectively, based on the appearance of their spin systems. The galacto-configured residue at 5.09OS/5.53KOH ppm was determined to be an amino sugar by virtue of its 1H resonance of its H-2 proton at 4.24OS/3.65KOH ppm correlating to a 13C chemical shift of 50.4OS/51.5KOH ppm in 13C-1H heteronuclear single quantum coherence (HSQC) experiments. The 13C chemical shift was consistent with a nitrogen substituted carbon atom. The remaining residue in the
-anomeric region at 4.95OS/4.99KOH ppm (Gal II, N) was determined to be a galacto-pyranose sugars, based on the appearance of its characteristic spin system to the H-4 resonance in a TOCSY experiment. The remainder of the anomeric resonances in the low-field region (4.456.00 ppm) of the spectrum were all attributable to ß-linked residues by virtue of the chemical shifts of their anomeric 1H resonances and in the case of resolved residues their high J1,2 (
8 Hz) coupling constants. Resonances at 4.65OS&KOH ppm (Glc I, I), 4.70 OS&KOH ppm (Glc III, K), and 4.69OS&KOH ppm (Glc IV, L) were assigned to the gluco-configuration from the appearance of their spin systems. The remaining resonances in the low-field region at 4.53 OS&KOH ppm (Gal I, M) and 4.73OS/5.01KOH ppm (GalNAc I/GalN I, O) were assigned to galacto-pyranosyl residues from the appearance of characteristic spin systems to the H-4 resonances in a TOCSY experiment. The galacto-configured residue at 4.73OS/5.01KOH ppm was determined to be an amino sugar by virtue of its 1H resonance of its H-2 proton at 4.12/3.53 ppm correlating to a 13C chemical shift of 51.9OS/53.1KOH ppm in 13C-1H HSQC experiments. The 13C chemical shift was consistent with a nitrogen substituted carbon atom. Signals for the CH2 protons of the PEtn moiety were observed in the OS sample at 3.27 and 4.16 ppm that correlated to characteristic 13C chemical shifts of 40.1 and 62.2 ppm, respectively, in a 13C-1H HSQC experiment. Additionally, characteristic signals for acetyl groups of the amino sugars were observed in the OS sample at 2.06 and 2.04 ppm that correlated to 13C chemical shifts of 22.4 and 22.1 ppm, respectively, in a 13C-1H HSQC experiment.
The sequence of the glycosyl residues in the OS was determined from the interresidue 1H-1H NOE measurements between anomeric and aglyconic protons on adjacent glycosyl residues and confirmed and extended the methylation analysis data. The linkage pattern for the Pm70 deacylated OS was determined in this way (Figure 5b; Table II). Thus the occurrence of intense transglycosidic NOE connectivities between the proton pairs Hep III (G) H-1 and Hep II (F) H-2, Hep II (F) H-1 and Hep I (E) H-3, and Hep I (E) H-1 and Kdo (C) H-5 established the sequence and points of attachment of these three LD-heptose residues as the frequently observed triheptosyl moiety. This linkage pattern is commonly encountered in the inner core OS from Haemophilus influenzae and M. haemolytica (Brisson et al, 2002; Cox et al., 2001a
). Furthermore, interresidue NOEs between the anomeric protons Hep III (G) H-1 and Hep II (F) H-1 provided confirmation of the 1,2-linkage (Romanowska et al., 1988
). Examination of NOE connectivities from H-1 of Glc I (I) illustrated that this glucose residue was connected to Hep I (E) at the 4-position by virtue of interresidue NOEs to Hep I (E) H-4 and Hep I (E) H-6. The appearance of an interresidue NOE to H-6 is a common occurrence for 4-substituted heptose residues (Backman et al., 1988
).
The occurrence of a long-range NOE connectivity between H-1 of Glc I (I) and H-1 of Glc II (H) suggested that the -configured glucose residue (Glc II [H]) was substituting Hep I at the 6-position, as has been observed previously for the OS from both M. haemolytica and A. pleuropneumoniae (Brisson et al., 2002
; St. Michael et al., 2004
). Examination of NOE connectivities from H-1 of Glc II (H) confirmed that this glucose residue was connected to Hep I (E) at the 6-position by virtue of interresidue NOEs to Hep I (E) H-6. Similarly to Glc I (I) a long-range NOE connectivity was observed between the anomeric 1H resonances of Glc I (I) and Glc II (H). Examination of NOE connectivities from H-1 of Hep IV (J) revealed that this heptose residue was connected to Glc I (I) at the 6-position by virtue of interresidue NOEs to Glc I (I) H-6a and H-6b. This inner core glycose structure has been observed previously for both M. haemolytica and A. pleuropneumoniae; however, in both cases the HepIV residue (J) was of the D-glycero-D-manno configuration, whereas here the HepIV residue (J) is of the L-glycero-D-manno configuration because this was the only configuration of heptose residue identified in sugar analysis.
The linkage pattern of the outer core residues was deduced from NOE connectivities and 13C-1H-heteronuclear multiple bond correlation (HMBC) evidence, in conjunction with the methylation analysis data. The two gluco-configured residues (K and L) with their anomeric proton resonances at 4.70OS&KOH/4.69OS&KOH ppm showed inter-NOE connectivities to the H-4 and H-6 proton resonances of the Hep IV residue (J) at 4.15OS&KOH and 4.31OS&KOH ppm, consistent with the 4,6-disubstituted LD-heptose residue observed in methylation analysis and similar to the arrangement of the DD-HepIV previously observed in A. pleuropneumoniae serotype 2 (St. Michael et al., 2004). By virtue of a 1H-13C HMBC experiment (Figure 6) it could be determined that Glc III (K) was linked to HepIV (J) at the 4-position and Glc IV (L) was linked to HepIV (J) at the 6-position. An inter-NOE connectivity from the anomeric proton resonance of Gal I (M) at 4.53OS&KOH ppm to the H-4 proton resonance of Glc IV (L) suggested that Gal I (M) substituted Glc IV (L) at the 4-position. An inter-NOE connectivity from the anomeric proton resonance of Gal II (N) to the H-4 proton resonance of Gal I (M) suggested that Gal II (N) substituted Gal I (M) at the 4-position, consistent with methylation analysis data that had resolved two 4-linked hexose residues, that is, a 4-linked glucose and a 4-linked galactose residue. An inter-NOE connectivity from the anomeric proton resonance of GalNAc I/GalN I (O) to the H-3 proton resonance of Gal II (N) suggested that GalNAc I/GalN I (O) substituted Gal II (N) at the 3-position, confirmed by methylation analysis that had identified a 3-linked hexose residue. Finally, an inter-NOE connectivity from the anomeric proton resonance of GalNAc II/GalN II (P) to the H-3 proton resonance of GalNAc I/GalN I (O) suggested that GalNAc II/GalN II (P) substituted GalNAc I/GalN I (O) at the 3-position, consistent with MS/MS identification of a HexNAc-HexNAc disaccharide. Confirmation of the 3-position of Hep II (F) as the location of PEtn substitution was obtained from 31P-1H-HSQC and 31P-1H-HSQC-TOCSY experiments on the OS sample. The HSQC experiment identified a cross-peak from the phosphorus signal to the proton resonance at 4.41 ppm, which had been assigned to the 3-position of the Hep II residue (F), and this was confirmed and extended in the HSQC-TOCSY experiment that revealed the H-2 and H-1 proton resonances of Hep II (F) at 4.31 and 5.87 ppm, respectively (data not shown).
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The linkages of the Hep I-Kdo-Lipid A region (E-C-B-A) were confirmed from analyses on the KOH-treated LPS as L--D-Hep I-(1-5)-
-Kdo4P-(2-6)-ß-GlcN4P-(1-6)-
-GlcN1P (Table II). A two-Kdo-containing fraction was isolated by chromatography and identified by MS, but insufficient amounts were available for NMR analysis.
The availability of the genome sequence for the Pm70 strain (May et al., 2001), along with a thorough knowledge of the OS structure, coupled with information about the glycosyltransferases that put similar OS structures together in the structurally and taxonomically related species enabled several candidate glycosyltransferases for the biosynthesis of the Pm70 OS to be identified (Figure 7). Several putative heptosyltransferases were identified in the Pm70 genome sequence and included a Hep III Tase PM1294, a Hep II Tase PM1844, two Hep I Tases PM1302 and PM1843, and a Hep IV Tase PM1144. The best homolog of each of these genes in the databases are detailed in Table III, and the high degree of homology suggests that the candidate PM gene would have the indicated function.
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Previously our group and collaborators showed that PM1294 was the Hep III Tase in another P. multocida strain VP161 (Harper et al., 2004). The gene that adds the
-glucose (H) to the 6-position of Hep I (E) is not known but there is a good homolog PM1306 to the ß-glucosyltransferase that substitutes Hep I (E) at the 4-position. The Kdo transferase was readily identified as PM1305 due to an extensive number of excellent homologs; however, only one Kdo transferase homolog has been identified in the P. multocida genome. Interestingly, several genes involved in the biosynthesis of the Kdo (C), Hep I (E), Glc I (I) region are clustered in this region of the chromosome. Putative glycosyltransferases for the outer core region also appear to be clustered in a region of the chromosome ranging from PM1138 to PM1144. This clustering of glycosyltransferase genes is not always observed in this genus, and indeed the glycosyltransferase for H. influenzae OS are liberally scattered throughout the genome. Another locus of putative glycosyltransferases can be found in another region of the P. multocida genome from PM0506 to PM0512. This locus lines up very well with the so-called lsg locus from both H. influenzae and Haemophilus ducreyi. The role of this locus in the biosynthesis of H. influenzae OS has been the matter of some discussion in the literature without any clear-cut role being assigned (Cox et al., 2001b
; Phillips et al., 1996
, 2000
). However, in the midst of this Pm70 locus is one sialyltransferase homolog (PM0508) of the three sialyltransferase homologs in the Pm70 genome (also PM0188 and PM1174). Structural studies are ongoing in our laboratory to see if Pm70 LPS can be sialylated under the appropriate growth conditions. Candidate genes for the formation of the activated nucleotide sugar donors are also detailed in Table III.
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Discussion |
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Based on the Pm70 OS structure, homologs to glycosyltransferases with similar specificities were identified in the Pm70 genome. Considering the novel finding of two LPS populations depending on the presence of one or two Kdo residues, it was interesting to note that only one homolog to Kdo transferases was identified in the P. multocida genome. This is not surprising because the pleiotropic nature of Kdo transferases is well documented, that is, the same Kdo transferase can transfer a Kdo residue to lipid A and a second Kdo residue to the initial Kdo residue. The novel feature of P. multocida LPS, though, is that populations with both one and two Kdo residues have been identified that has not been observed previously to our knowledge. However, it was observed that there were two good homologs in the Pm70 genome for Hep I (E) to Kdo (C) (-1,5 heptosyltransferase) transferases. One transferase had the highest homology to HI 0261 (OpsX) of H. influenzae wherein the LPS structure contains one Kdo residue; the second transferase had the highest homology to WaaC from Escherichia coli and Klebsiella pneumoniae that have two Kdo residues in their LPS structures. The identification of two Hep I transferases is therefore consistent with the presence of two structural arrangements in the Kdo region of the Pm70 LPS, and it will be intriguing to understand the regulation of the Kdo transferase from this organism.
This study has structurally characterized the core OS region of the genome strain of P. multocida and identified putative glycosyltransferases for the complete biosynthesis of the core OS.
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Materials and methods |
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Isolation and purification of LPS
Following washing of the lyophilized cell mass (70 g) with organic solvents yielding
50 g, the LPS was isolated from 10 g of the washed, lyophilized cell mass by the hot water/phenol method (Westphal and Jann, 1965
). The aqueous phase was dialyzed against water and lyophilized. The dried sample was dissolved in water to give a 12% solution (w/v) and treated with DNase (0.01 mg/ml) and RNase (0.01 mg/ml) for 3 h at 37°C, then treated with proteinase K (0.01 mg/ml) for 3 h. The dialyzed, dried sample was dissolved in water to make a 1% solution and ultracentrifuged at 45,000 following a low-speed spin at 8000 to remove any insoluble material (124 mg). The LPS pellet from the 45,000 spin was redissolved in water and lyophilized (180 mg). Purified LPS (75 mg) was treated with anhydrous hydrazine with stirring at 37°C for 1 h to prepare LPS-OH. The reaction was cooled in an ice bath, gradually cold acetone (70°C, 5 vols) was added to destroy excess hydrazine, and the precipitated LPS-OH was isolated by centrifugation (60 mg). The sample was then purified down a Bio-Gel P-2 column as described.
The core OS was isolated by treating the purified LPS (100 mg) with 1% acetic acid (10 mg ml1, 100°C, 1.5 h) with subsequent removal of the insoluble lipid A by centrifugation (5000 g). The lyophilized OS (50 mg) was further purified down a Bio-Gel P-2 column with individual fractions lyophilized. Fully deacylated LPS was isolated by treatment of LPS-OH (20 mg) with 4 N KOH at 125°C for 16 h and following neutralization were fractionated by anion-exchange liquid chromatography as described previously (Vinogradov and Bock, 1999).
Analytical methods
Sugars were determined as their alditol acetate derivatives by GLC-MS (Sawardeker et al., 1965). Samples were hydrolyzed for 4 h using 4 M trifluoroacetic acid at 100°C, then reduced (NaBD4) overnight in H2O and acetylated with acetic anhydride at 100°C for 2 h using residual sodium acetate as catalyst. The GLC-MS was equipped with a 30 M DB-17 capillary column (180°C to 260°C at 3.5°C/min), and MS was performed in the electron impact mode on a Varian Saturn II mass spectrometer. Methylation analysis was carried out by the NaOH/dimethyl sulfoxide/methyl iodide procedure (Ciucanu and Kerek, 1994
) and analyzed by GLC-MS as described. Absolute configurations were determined by GLC analysis of butyl glycoside derivatives (St. Michael et al., 2004
).
MS
Electrospray ionizationMS was performed in the negative ion mode on a VG Quattro Mass Spectrometer (Micromass, Manchester, U.K.) by direct infusion of samples in 25% aqueous acetonitrile containing 0.5% acetic acid. CE-MS was performed on a Prince CE system (Prince Technologies, Netherlands) coupled to an API 3000 mass spectrometer (Applied Biosystem/Sciex, Concord, Canada) via a microspray interface. A sheath solution (isopropanol-methanol, 2:1) was delivered at a flow rate of 1 µl/min to a low dead volume tee (250 µm ID, Chromatographic Specialties, Belleville, Ontario). All aqueous solutions were filtered through a 0.45-µm filter (Millipore, Bedford, MA) before use. An electrospray stainless steel needle (27-gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column. The separations were obtained on about 90-cm length bare fused-silica capillary using 10 mM ammonium acetate/ammonium hydroxide in deionized water, pH 9.0, containing 5% methanol. A voltage of 20 kV was typically applied at the injection site. The outlet of the capillary was tapered to 15 µm ID using a laser puller (Sutter Instruments, Novato, CA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z unit in full-mass scan mode. The MS/MS data were acquired with dwell times of 1.0 ms per step of 1 m/z unit. Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell were mass analyzed by scanning the third quadrupole.
NMR
NMR experiments were acquired on Varian Inova 400, 500, and 600 MHz spectrometers using a 5 mm or 3 mm triple resonance (1H, 13C, 31P) probe "(Varian, Palo Alto, CA)". The lyophilized sugar sample was dissolved in 600 ml (5 mm) or 140 ml (3 mm) of 99% D2O. The experiments were performed at 25°C with suppression of the deuterated H2O signal at 4.78 ppm. The methyl resonance of acetone was used as an internal reference at 2.225 ppm for 1H spectra and 31.07 ppm for 13C spectra. Standard homo- and heteronuclear correlated 2D pulse sequences from Varian, COSY, TOCSY, NOE spectroscopy, 13C-1H HSQC, 13C-1H HSQC-TOCSY, and 13C-1H HMBC were used for general assignments. The 2D 1H-31P HSQC experiment was acquired on a Varian Inova 400 spectrometer for 6 h. The coupling constant was optimised at 12 Hz by performing an array of 1D HSQC experiments. The sweep width in the F2 (1H) dimension was 6.0 ppm and in the F1 (31P) dimension was 16.2 ppm. Water presaturation during the relaxation delay was 1.5 s, acquisition time in t2 was 0.21 s, and 32 increments with 180 (HMQC) scans per increment were obtained. The 2D 1H-31P HSQC-TOCSY experiment was acquired on a Varian Inova 400 spectrometer for 8 h using the same parameters as the HSQC experiment with a TOCSY mixing time of 80 ms.
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Acknowledgements |
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Abbreviations |
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References |
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