Full text of DOI:10.1007/s11745-001-0751-3

A Novel Sphingophosphonolipid Head Group 1-Hydroxy-2-
aminoethyl Phosphonate in Bdellovibrio stolpii
Yoko Watanabea, Masaharu Nakajimab, Tsutomu Hoshinoc, Koka Jayasimhulud,
Elwood E. Brooksd, and Edna S. Kaneshiroe,*
aDepartment of Medical Technology, School of Health Sciences, Faculty of Medicine, Niigata University, Niigata 951-8518,
Japan, bDepartment of Analytical Chemistry, Niigata College of Pharmacy, Niigata 950-2081, Japan, cDepartment
of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi-2 Niigata 950-2181, Japan,
and Departments of dChemistry and eBiological Sciences, University of Cincinnati, Cincinnati, Ohio 45221
ABSTRACT: Members of the bacterial genus Bdellovibrio in- ositolsphingolipids (8–20). Sphingolipids, their precursors and
clude strains that are free-living, whereas others are known to in-
vade and parasitize larger Gram-negative bacteria. The bacterium
can synthesize several sphingophospholipid compounds includ-
ing those with phosphoryl bonds as well as phosphonyl bonds. In
the present study, the dominant sphingophosphonolipid compo-
nent was isolated by column chromatography, and the long-chain
bases, fatty acids, and polar head groups were identified by thin-
layer and gas–liquid chromatographic procedures. The definitive
structural identity of the sphingolipid was established by nuclear
magnetic resonance and mass spectrometry of hydrolysis prod-
ucts and the intact compound. The compound was identified as
metabolic products (e.g., sphingosine, sphingosine-1-phos-
phate, and ceramide) play important roles in transmembrane
signaling and appear to be important in mammalian cell pro-
liferation, differentiation, aging, and apoptosis (21–24). There
is evidence that sphingolipid metabolism within host cell
membranes augments the ability for invasion by pathogens
(25,26). It has been suggested that sphingolipids may play a
role in the attack and invasion processes of Bdellovibrio (5).
Sphingolipids are not widely distributed among the
prokaryotes (27,28), and have been mainly studied among or-
N-2′-hydroxypentadecanoyl-2-amino-3,4-dihydroxyheptadecan- ganisms in the genus Sphingomonas (27). To date, there is a
1-phosphono-(1-hydroxy-2-aminoethane).
single report of sphingophosphonolipids in Bdellovibrio.
Steiner et al. (5) compared the phospholipids of two strains
of B. stolpii (B. bacteriovorus) to determine whether phos-
pholipids in the predator cell membrane could play a role in
Paper no. L8697 in Lipids 36, 513–519 (May 2001).
Members of the genus Bdellovibrio are small, comma- or the attack phase. Two strains, UKi2 (a facultative parasite)
crescent-shaped bacteria, commonly found in soil. Although and UKi1 (a free-living strain derived from the same obligate
many species are free-living, most are characterized by their parasite parent), were both grown axenically under the same
predatory behavior. They can attack other larger bacteria, and culture conditions. Three sphingophospholipids were de-
proliferate as parasites in the intraperiplasmic space of vari- tected in UKi2 but not in UKi1. The most abundant of the
ous Gram-negative species (1–5). Obligate and facultative three was designated as compound 9, and the other two, com-
parasitic strains have been reported. Much of the lipids in par- pounds 8 and 11. These components were identified as sphin-
asitically grown organisms are scavenged from the host bac- golipids by their resistance to alkaline hydrolysis, and by sev-
terium, but the parasite synthesizes its own distinct array of eral chromatographic properties. Based on reactions to differ-
lipids (2,3) and like eukaryotic cilia and flagella (6), the bac- ential hydrolysis, it was tentatively concluded that compound
terial flagellar domain differs in lipid composition compared 11 was a sphingolipid with a phosphoryl bond, and this minor
to the rest of the cell surface membrane (7). The free-living lipid component was not further characterized. The two major
and facultative parasitic strains can be grown in the labora- sphingolipids compounds 8 and 9 were tentatively identified
tory under axenic culture conditions, thus biochemical analy- as sphingophosphonolipids. C17 dihydrosphingosine and phy-
sis of these organisms eliminates the potential of contamina- tosphingosine were identified as the long-chain bases (LCB)
tion by host lipids (5).
of compounds 8 and 9, respectively. Both contained a similar
Sphingolipids have been reported in all groups of eukary- mixture of α-hydroxy fatty acids, dominated by a branched
otic organisms thus far analyzed, and they occur in the mem- C15 hydroxy fatty acid.
branes as phosphosphingolipids, glycosphingolipids, and in-
*To whom correspondence should be addressed.
E-mail: Edna.Kaneshiro@uc.edu
Abbreviations: 2-AEP, 2-aminoethylphosphonic acid; BSTFA, N,O-bis-
(trimethylsilyl)trifluoroacetamide; EI, electron impact; FAB, fast atom bom-
bardment; FAME, fatty acid methyl ester; GLC, gas–liquid chromatography;
HPTLC, high-performance thin-layer chromatography; LCB, long-chain
base; LSI, liquid secondary ion; MS, mass spectrometry; NMR, nuclear mag-
netic resonance; PHG, polar head group; TLC, thin-layer chromatography;
TMS, trimethylsilyl.
Phosphonolipids appear rare as naturally occurring com-
pounds. Most reports of this group of lipids have been from
studies of the ethanolamine glycerophosphonolipids and
sphingophosphonolipids in the ciliated protozoa Tetrahymena
and Paramecium (29–32). The discovery of sphingophospho-
nolipids in a bacterium was a novel and significant finding
(5), hence it is important that definitive structural characteri-
zations of these Bdellovibrio lipids be elucidated. In the
Copyright © 2001 by AOCS Press
513
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514
Y. WATANABE ET AL.
present study, we obtained the definitive structural identity of were extracted into CHCl3 and concentrated under N2. The
the major B. stolpii sphingolipid component, compound 9 (5) LCB were purified on an Iatrobeads column, eluting with
employing mass spectrometry (MS) and nuclear magnetic CHCl3/CH3OH/H2O/25% NH4OH (70:17.5:1.5:0.25, by vol).
resonance (NMR) spectroscopy.
The LCB fractions were identified by TLC, pooled, and dried
under N2. The LCB was acetylated in CH3OH/acetic anhydride
(4:1, vol/vol) and analyzed by GLC–MS. Alternatively, it was
converted to its O-trimethylsilyl (TMS) derivative with N,O-
MATERIALS AND METHODS
Organism. Bdellovibrio stolpii (ATCC 27052) was the same bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Tokyo Kasei
strain previously analyzed for sphingolipids (5). It was grown Co., Tokyo, Japan) and analyzed. The acetylated TMS LCB de-
axenically in 2-L flasks containing 660 mL of 1% (wt/vol) rivative was subjected to GLC–MS analysis and compared
Bactopeptone and 0.3% (wt/vol) yeast extract (Difco, Becton with derivatized authentic dihydrosphingosine, sphingosine,
Dickinson, MD). The cultures were grown at 30°C without and phytosphingosine (Sigma, St. Louis, MO).
shaking until stationary phase was achieved. Cultures were
centrifuged at 10,000 × g for 5 min, and the cell pellet was acetic acid, dried, redissolved in approximately 2 mL of H2O;
washed with physiological saline and recentrifuged. and the polar head group (PHG) was purified by cation ex-
The remaining water phase was neutralized with 1 M
Lipid extraction and purification of the major sphingolipid change chromatography (Amberlite IR-120B). Ethanolamine-
component. The cell pellet was immediately extracted with and phosphorus-positive fractions were pooled, lyophilized,
chloroform/methanol (2:1, vol/vol) and stored at −35°C. The and an aliquot was derivatized with BSTFA.
lipids from 18 L of culture were pooled, water (0.25 vol) was
The ceramide moiety was obtained by treating the intact
added to the extract, and the lower organic layer was recov- sphingophosphonolipid with HF (33). Fast-atom bombard-
ered and evaporated using a rotary evaporator. The residue ment (FAB)-MS was obtained by the JEOL Company (Tokyo,
was dissolved in the minimum amount of CHCl3/MeOH (2:1, Japan).
vol/vol). The sample was placed in an ice bath, and acetone
TLC. Phospholipids were separated by Silica Gel 60 high-
(3 vol) added. After standing overnight at 4°C, it was cen- performance TLC (HPTLC; Merck, Darmstadt, Germany)
trifuged, and the acetone-insoluble pellet containing the phos- using CHCl3/CH3OH/H2O (60:35:8, by vol). After the
pholipids was dissolved in CHCl3.
solvent reached 5 cm from the top of the plate, it was dried,
Adsorption column chromatography of the phospholipid then re-developed with CHCl3/CH3OH/acetic acid (90:2:8,
fraction was performed to isolate the major sphingophos- by vol). Development in the second dimension was with
phonolipid. The phospholipid fraction (approximately 1 g) n-propanol/n-propylamine/H2O (80:15:5, by vol). The sol-
in CHCl3 was loaded onto a 1.5 × 30.0-cm glass column vent system for LCB analysis was CHCl3/CH3OH/H2O/25%
containing Iatrobeads (6RS-8060, Iatron Laboratories, Tokyo, NH4OH (70:17.5:1.5:0.25, by vol).
Japan) and eluted with (i) 70 mL CHCl3; (ii) 70 mL of
The PHG was analyzed by cellulose TLC (Avicel, Asahi-
CHCl3/CH3OH, 95:5; (iii) 70 mL CHCl3/CH3OH, 4:1; (iv) Kasei, Tokyo, Japan) using authentic 2-aminoethylphospho-
140 mL CHCl3/CH3OH, 3:2; (v) 70 mL CHCl3/CH3OH, 2:3; nic acid (2-AEP, Sigma) as standard. After one-dimensional
(vi) 70 mL CHCl3/CH3OH, 1:4; (vii) 70 mL CHCl3/CH3OH, development with either 88% phenol/H2O (4:1, vol/vol),
1:9; and (viii) 70 mL CH3OH. After thin-layer chromatogra- isobutyric acid/H2O/25% NH4OH (66:33:1, by vol), or bu-
phy (TLC) analysis of all the fractions, those containing the tanol/acetic acid/H2O (5:3:1, by vol), plates were stained for
major sphingophosphonolipid were pooled and dried.
ethanolamine (ninhydrin) and phosphorus (34).
The sample was resuspended in n-propanol/n-propyl-
GLC–MS, FAB-MS, and liquid secondary ion MS (LSIMS)
amine/H2O (80:5:3, by vol), applied to a second Iatrobeads col- analysis. The hydrolysis products were analyzed by GLC–MS
umn, and the sphingophosphonolipids were eluted using the as FAME, the PHG TMS, and the LCB N-acetylated TMS (11)
same solvent system. After analyzing each fraction by TLC, the using a Hewlett-Packard 5890 Series II GLC equipped with
fractions containing only the major sphingphosphonolipid were an HP-1 capillary column and interfaced with a 5971 mass se-
pooled and dried to approximately 0.5 mL. The purified sphin- lective detector. The initial 2-min column temperature of
gophosphonolipid was recrystallized from 5 mL of ethyl ac- 100°C was increased at 10°C/min for 15 min and held at
etate/methanol (3:2, vol/vol). Approximately 200 mg of the pu- 250°C for 10 min. The helium carrier gas flow rate was 1.0
rified sphingophosphonolipid were obtained from 54 L of bac- mL/min. The MS electron-impact (EI) source and transfer line
terial cultures, representing approximately 2 kg wet wt of cells. temperatures were 280 and 190°C, respectively.
Analysis of fatty acid, polar head group (PHG), and LCB.
The underivatized ceramide was analyzed by FAB-MS on a
The purified sphingophosphonolipid was hydrolyzed with JEOL MS700 mass spectrometer. 3-Nitrobenzyl alcohol was
CH3OH/H2O/HCl (11:2.6:1, by vol) at 80°C overnight the matrix. Spectra were recorded in a positive ion mode at an
(11–13), and the fatty acid methyl esters (FAME) were ex- accelerating voltage of 6.0 kV. The intact sphingophosphono-
tracted into n-hexane for analysis by gas–liquid chromatogra- lipid was analyzed by LSIMS on a Kratos MS-890 mass spec-
phy (GLC) and MS. The remaining material was dried under trometer (Kratos, Manchester, United Kingdom) in the positive
N2 and dissolved in approximately 2 mL H2O. The pH was ion mode. The sample on a glycerol matrix was bombarded
then adjusted to 10.0–11.0 (with 1 M NaOH), and the LCB with cesium ions, transferring a proton to the intact molecule.
Lipids, Vol. 36, no. 5 (2001)

BDELLOVIBRIO PHOSPHONOLIPID
515
31P, 13C, 15N, and 1H NMR analysis of the PHG. The PHG from m/z 276 ion), 174 (C1 and C2 with their derivatives), 132
dissolved in D2O was analyzed by NMR spectroscopy (Bruker (C1 with its TMS group and C2 with an NH2 group), and 73
DMX600 spectrometer) by one- and two-dimensional (1-D (TMS). The fragmentation pattern and the mass spectrum are
and 2-D) analyses. Chemical shifts were estimated from the typical of the derivatized phytosphingosine (11).
external TMS for proton and carbon, from 85% H3PO4 for 31P,
and from 90% formamide (112.4 ppm) for 15N.
Analysis of hydrolysis products: PHG. The TMS deriva-
tive of the polar head group was analyzed by cellulose TLC
and by GLC–MS. A single TLC component was detected in
the sample after staining the plate with ninhydrin. By using
three different solvent systems, the polar head group from the
RESULTS
The phospholipid composition of B. stolpii. The major phos- B. stolpii sphingophosphonolipid migrated at a slower rate
pholipid components of B. stolpii UKi2, identified by 2-D than authentic 2-AEP or phosphorylethanolamine, indicating
HPTLC, were phosphatidylethanolamine, phosphatidylglyc- that the former was more polar (Table 1). It eluted earlier than
erol, diphosphatidylglycerol, and three sphingophospholipids. 2-AEP on an amino acid analyzer (data not shown), further
The total phospholipid profile in organisms analyzed in the demonstrating that they were distinct compounds. The GLC
present study was similar to that reported in the earlier study retention time of the TMS derivative of 2-AEP was 8.8 min.
of the UKi2 strain. After alkaline hydrolysis, three phospho- In contrast, the TMS derivative of B. stolpii sphingophospho-
rus-containing sphingolipids were left intact. The sphin- nolipid head group eluted at 8.2 min.
golipid, with similar chromatographic properties as the com-
ponent designated as compound 9 by Steiner et al. (5), was did not show the molecular ion at m/z 429, but the ion repre-
also identified as the dominant sphingolipid in this study.
senting (M+· − 15, loss of TMS methyl group) was observed
Analysis by GLC–MS of the TMS-derivatized head group
Analysis of hydrolysis products: N-linked fatty acids. The at m/z 414. The mass of the derivative indicated the presence
alkali-stable, acid-labile amide-linked fatty acids in the puri- of four TMS groups, which is only possible if there is a hy-
fied sphingophosphonolipid preparation were analyzed as droxyl group on C-2. This fragmentation pattern clearly
their methyl ester derivatives by GLC and quantified by inte- demonstrated that, unlike 2-AEP, a hydroxyl group was pres-
gration of peak areas obtained by using a total ion current de- ent in the head group of the B. stolpii sphingophosphonolipid.
tector. Results were reported as retention time in min (wt%): Other important ions observed included the ions at m/z 328
8.741 (3.4); 9.755 (3.8); 10.938 (89.6); 11.305 (3.3). By EI- [327 + H] (loss of C-2 and TMS amine group), and 204 (oxy-
MS, the molecular ion was observed at m/z 272. The frag- gen and N-TMS aminoethanol, which further indicates that
mentation pattern resulting from cleavage between the C-1 there must be a hydroxyl group at C-1, and that C-1 is directly
and C-2 positions produced the dominant m/z 213 ion (Figs. linked to the phosphorus).
1,2). This indicated the presence of a hydroxyl group neigh-
The 1H, 13C, 15N, and 31P NMR spectra of the PHG (dis-
boring the carboxyl group. The structure of the major fatty solved in D2O, pH 4.6) were obtained. Three protons were
acid in this preparation was shown by MS to be the straight- found at 3.27 (multiplet of eight peaks, Ha), 3.08 (multiplet
chained C15 α-hydroxy fatty acid. The other FAME compo- of eight peaks, Hb), and 3.87 ppm (multiplet of six peaks be-
nents were at too low a concentration for MS analysis. This cause Jbc and Jcf are equivalent in value) in the 1H NMR spec-
sphingophosphonolipid, which had previously been isolated trum (Fig. 3A). However, the multiple splits of each proton
by TLC and analyzed for its fatty acid composition, had 68% were collapsed to a multiplet of four peaks (Ha, Jab = 13.2 Hz
iso-branched α-hydroxy C15 fatty acid and 13% of an un- and Jac = 3.3 Hz), multiplet of four peaks (Hb, Jab = 13.2 Hz
branched α-hydroxy-C15 fatty acid (5). The apparent dispar- and Jbc = 10.0 Hz), and the multiplet of four peaks (Hc, Jac
=
ity can be explained by the large-scale column chromato- 3.4 Hz and Jbc = 10.0 Hz), respectively, by 31P decoupling ex-
graphic procedures used in the present study. Since some frac- periments (data not shown). The protons Ha and Hb are non-
tions containing other sphingolipids were discarded, the equivalent; both protons are bonded to Cd and exhibit a large
isolated population was enriched with the molecular species H-H coupling constant of 13.2 Hz. The Hc proton at 3.87 ppm
with the straight-chain C15 α-hydroxy fatty acid.
Analysis of hydrolysis products: LCB. The N-acetylated
TMS-derivatized LCB fraction was analyzed by GLC and
found to contain a single major component (not shown). The
GLC-EI-MS analysis of the N-acetylated TMS derivative of
TABLE 1
Thin-Layer Chromatographic Migrations of the Hydrolyzed Polar
Head Group of the Major Bdellovibrio stolpii Sphingophosphonolipid
the LCB (C17 phytosphingosine) did not indicate the molecu-
Rf
Bb
+
·
lar ion, but an ion at m/z 546 corresponding to [M − 15] typ-
ical of TMS derivatives (loss of a TMS methyl group). Other
ions detected were m/z 456 (loss of trimethylsilanol), 387
(loss of C1 and C2 with the attached N-acetyl and the TMS),
286 [285 + H] (C4 through C17 including the silylated hy-
droxyl group at C4), 276 (M+· − 285), 218 (loss of the amino
Head group
Aa
Cc
Authentic phosphorylethanolamine
Authentic 2-aminoethylphosphonate
0.42 0.50 0.19
0.32 0.53 0.27
Head group of B. stolpii sphingophosphonolipid 0.19 0.47 0.06
a88% phenol/water (4:1, vol/vol).
bIsobutyric acid/water/25% NH4OH (66:33:1, by vol).
acyl group at C2 from the m/z 276 ion), 187 (loss of O-TMS cButanol/acetic acid/water (5:3:1, by vol).
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517
FIG. 2. Fragmentation patterns of samples shown in Figure 1. (A) The amide-linked fatty acid methyl esters indicate it is an α-hydroxy 15-carbon
straight-chain acid. (B) The N-acetylated LCB TMS derivative was identified as C-17 phytospingosine. (C) The polar head group TMS derivative in-
dicated it was ethanolamine with a direct P–C bond and a hydroxyl group at C-1. (D) FAB mass spectrum of the underivatized ceramide moiety
verified the structural assignments of the fatty acid and LCB. (E) LSI mass spectrum of the underivatized intact sphingophosphonolipid verified the
structural assignments of the fatty acid (C15 α-hydroxy n-fatty acid), LCB (C17 phytosphingosine), and the polar head group (1-hydroxy-2-aminoethyl
phosphonate). For abbreviations see Figure 1.
resonated at a lower field, compared to those of Ha and Hb. atom in the PHG, as shown in Figure 3E. The chemical shifts
This suggested that Hc is bonded to carbon Ce, which is also and coupling constants of 1H, 13C, 15N, and 31P are summa-
bonded to the hydroxy group. All other protons in the head rized in Figure 3. The NMR data, together with those obtained
group were not observed due to the isotopic exchange with from the other analyses presented in this paper, unequivocally
deuterium atoms of the D2O solvent.
demonstrated that the PHG of the major B. stolpii phospho-
The proton-decoupled 13C NMR spectrum showed four nosphingolipid is 1-hydroxy-2-aminoethylphosphonic acid.
signals, with two peaks centered at 65.7 and 41.8 ppm. A
double-decoupled experiment (in which both the 1H and 31
Analysis by FAB-MS and LSIMS. The FAB-MS analysis of
underivatized ceramide released by HF digestion showed the
P
were decoupled from 13C) showed that the Ce-Pf coupling (M + H)+ ion at 544 Da. The fragmentation pattern confirmed
constants were JcePf = 155.1 Hz and Jc Pf = 8.8 Hz (Figs. the assignments of the LCB and the fatty acid moieties. The
3C,3D). The large coupling constant of d155.1 Hz indicated ion at m/z 566 is the result of the addition of a sodium ion to
that Ce is bonded to Pf. The chemical shifts of 65.7 and 41.8 the molecule. The ion at 526 Da is due to the loss of H2O from
ppm are also suggestive of the presence of Ce-OH and Cd-N the (M + H)+ ion. The ion at 304 Da is due to the protonated
centers, respectively. The 31P NMR spectrum showed a sin- LCB phytosphingosine (Figs. 1,2).
gle peak with a chemical shift of 16.2 ppm (Fig. 3B) corre-
The LSIMS analysis of the intact underivatized molecule
sponding to the chemical shift of the alkylphosphonic acid demonstrated that the ion at m/z 667 (M + H)+ was intense.
reference standard. 15N showed a single peak at 26.0 ppm.
The fragment ion at m/z 142 (141 + H)+ corresponds to the
1
The detailed analyses by 2D-NMR including H-13C, polar head group, and the ion at m/z 526 (667 − 141) corre-
1H-15N, and 1H-31P heteronuclear multiple bond correlation sponds to ceramide [loss of the polar head group from (M +
(pulse-field gradient mode) made clear the position of each H)+]. This analysis demonstrated that it is a phosphonolipid,
Lipids, Vol. 36, no. 5 (2001)

518
Y. WATANABE ET AL.
3,4-dihydroxyheptadecan-1-phosphono-(1-hydroxy-2-
aminoethane). It cannot be ruled out that other molecular
species were present in the spot or band on TLC plates. This
was the structure for the major component that was isolated
by the purification procedures used in the present study.
DISCUSSION
Sphingophosphonolipids have been isolated from various or-
ganisms (5,6,8,9,11,12,17,20,29–31), and occur as minor
lipid components in mammals (32). In most cases, the PHG
is 2-AEP, but 2-N-monomethyl-AEP has also been found
(11,12). Steiner et al. (5) found that the isolated head group
from the major sphingophosphonolipid of Bdellovibrio did
not migrate as far as 2-AEP in the two paper chromatographic
systems they used. They also noted that N-methyl derivatives
of authentic standards of 2-AEP had Rf values greater than un-
derivatized 2-AEP, thus eliminating N-methyl derivatives as
possible structures for the head group of the sphingophospho-
nolipid in this bacterium. The only previously reported iden-
tification of naturally occurring 1-hydroxy-2-AEP was a hy-
drolysis product of complex polysaccharides of the soil
amoeba Acanthamoeba castellanii (35). Thus, this is the first
report of a lipid with a phosphonoethanolamine head group
containing a hydroxyl group. The definitive structure of the
intact B. stolpii phosphonosphingolipid was here shown to be
N-2′-hydroxypentadecanoyl-2-amino-3,4-dihydroxyhepta-
decan-1-phosphono-(1-hydroxy-2-aminoethane).
The lipids of many bacterial lipids can have high propor-
tions of fatty acids with odd-numbered carbons (e.g., C15,
C17); branched (iso, anteiso), or with an α- or 2-hydroxy group
(36–39). There is evidence for high α-oxidation activity that
could account for the high proportions of α-hydroxy fatty
acids (36) in bacteria, including Cornebacterium (Arthrobac-
ter) simplex grown on 1-octadecene and other hydrocarbons
as the only sources of carbon (39). In C. simplex, radiolabeled
palmitate was mainly incorporated into α-hydroxypalmitate.
Furthermore, the fatty acid composition of lipopolysaccharide
from intraperiplasmically grown B. stolpii (bacteriovorus) was
found to be highly enriched in β-hydroxy fatty acids (7). The
discovery of the hydroxylated ethanolamine head group in this
B. stolpii sphingophosphonolipid suggests that oxidative reac-
tions in aerobic bacteria may play greater roles in several
metabolic pathways than earlier appreciated.
FIG. 3. Nuclear magnetic resonance (NMR) spectra of the polar head
group. (A) Proton NMR spectrum showing peaks at 0.87 ppm for Hc,
3.37 ppm for Ha, and 3.08 ppm for Hb. (B) 31P NMR spectrum. The sin-
gle peak present was at 16.2 ppm, the chemical shift for a direct P–C
bond. (C) The decoupled 13C spectrum showing the single peak of
Ce (bonded to Pf) at 65.65 ppm and the single peak of Cd (bonded to
Ng) at 41.8 ppm. (D) The coupled 13C spectrum showing two peaks
at 65.05 ppm and 66.3 ppm from Ce (bonded to Pf), and two peaks be-
tween 41.8 ppm and 41.9 ppm from Cd (bonded to Ng). (E) Summary of
analyses by two-dimensional (2-D) NMR showing coupling between
atoms in the polar head group. HMBC, heteronuclear multiple bond
correlation.
ACKNOWLEDGMENT
Supported in part by NIH grant RO1 AI29316 (ESK).
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and More Complex Sphingolipids, J. Bioeng. Biomembr. 23, [Received November 29, 2000, and in revised form March 15, 2001;
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Lipids, Vol. 36, no. 5 (2001)