Two-dimensional ¹H-¹H COSY analysis of the purified novel lipid at 500 MHz. The COSY experiment establishes the connectivities among the key glycerol, phosphoethanolamine, GlcNAc sugar, and acyl ¹H resonances (also see Table 3). COSY, correlation spectroscopy NMR; GlcNAc, N-acetylglucosamine.

ESI-MS/MS of [M-H]⁻ at m/z 837.53, a major molecular species of the ethanolamine-phosphate containing glycosyldiacylglycerol from C. tetani ATCC 10779. The proposed structure of a typical phosphoethanolamine GlcNAc-diacylglycerol (Fig. 1A, spot 6) is shown in the inset, along with the numbering scheme used for the NMR analysis.

Partial HMQC and HMBC spectra of the purified novel lipid. A: Single bond ¹H-¹³C correlations of the glycerol, phosphoethanolamine, and GlcNAc moiety are shown. The single carbon-decoupled cross peak at 3.30/49.9 ppm is due to residual methanol solvent. B: This expansion shows multibond correlations from the glycerol H3 protons to the GlcNAc C-1 carbon, and from the GlcNAc H-1 to the glycerol C-3, thus confirming the proposed linkage of the glycerol to the C-1 carbon of the sugar (Fig. 2, inset). The two cross peaks at 49.9 ppm (from carbon coupling) are due to residual methanol solvent.

Analysis by 2D-TLC of [¹⁴C]labeled lipids from C. tetani. The lipids were subjected to hydrolysis with HCl fumes between the first and second chromatographies, as described in Materials and Methods. A: Polar lipids of C. tetani ATCC 10779. Based on their positions and staining, the major lipids were tentatively identified as (1) GlcNAcDAG, (2) unknown, (3) cardiolipin, (4) PtdGro, (5) PtdEtn, (6) the novel phosphorus-containing glycosyl-diacylglycerol, and (7) phosphatidic acid. B: Polar lipids of C. tetani ATCC 454. Compound (1) aldehyde from GlcNAcDAG plasmalogen, (2) GlcNAcDAG, (3) lyso-GlcNAcDAG from GlcNAcDAG plasmalogen, (4 and 5) aldehydes from PtdGro and cardiolipin plasmalogens, (6) unknown, (7) unknown, (8) unknown, (9) cardiolipin, (10) PtdGro, (11) lyso-cardiolipin from cardiolipin plasmalogen, (12) lyso-PtdGro from PtdGro plasmalogen, (13) unknown, (14 and 15) aldehydes from PtdEtn plasmalogen, (16) PtdEtn, (17) lyso-PtdEtn from PtdEtn plasmalogen, (18) aldehyde from phosphorus-containing GlcNAcDAG plasmalogen, (19) lyso-PtdEtn, (20) phosphorus-containing GlcNAcDAG, (21) lyso form of 20 resulting from hydrolysis of the plasmalogen form, (22) and phosphatidic acid. The long arrows indicate the chromatographic first and second dimensions. The materials in the upper left corners are presumed to be neutral lipids that were not further analyzed.

³¹P NMR spectrum, selective inverse ³¹P-decoupled-¹H-detected difference spectroscopy, and partial ¹H NMR spectrum of the purified novel lipid. A: Two ³¹P NMR signals in 75:25 ratio are detected. The large ³¹P NMR signal arises from the purified novel lipid, while the small signal arises from lyso-phosphatidylethanolamine, which elutes with the purified lipid. The chemical shifts (1.511 and 1.242 ppm) are consistent with the presence of one monophosphodiester in each compound. B: The selective inverse decoupled difference ¹H NMR spectrum obtained with on and off resonance ³¹P decoupling of the 1.511 ppm phosphorus signal showed that the 6-CH₂ group of the putative N-acetylglucosamine is linked via a phosphodiester to the α-methylene unit of the phosphoethanolamine group. C: The partial one-dimensional 500 MHz 1H NMR spectrum of the donor lipid shows six GlcNAc and various glycerol and phosphoethanolamine proton signals (see Table 3). The resonances at 4.6 ppm and 3.3 ppm are due to residual water and methanol solvent signals. The small resolved multiplets at 3.99 and 3.86 ppm arise from impurities, presumably from co-eluting lysophosphatidylethanolamine, the presence of which is well detected in the ³¹P nmr spectrum of Panel A. Note that an impurity peak also overlaps H-5 near 3.66 ppm. The proposed structure of the novel lipid is shown in the inset to Fig. 2 along with the numbering scheme.