The total syntheses of D-erythro-sphingosine, N-palmitoylsphingosine (ceramide), and glucosylceramide (cerebroside) via an azidosphingosine analog

Article abstract of DOI:10.1016/S0009-3084(01)00152-9

The total synthesis of D-erythro-sphingosine (9) was performed by a chirospecific method starting from D-galactose via an azidosphingosine intermediate to give highly homogeneous ( > 99.9% C18:1) sphingosine base (9) which contained no observable olefin isomerization by product and was demonstrated to be optically pure by a novel method utilizing Mosher's acid. Ceramide (10) was prepared from this sphingosine (9) with highly homogeneous (99.8% C16:0) palmitic acid by two methods. The cerebroside glucosylceramide (23) was the next sphingolipid in this series to be synthesized in a highly homogeneous form. These three sphingolipids are currently being used for biophysical studies of the structures of their hydrated bio-molecular assemblies.

Full text of DOI:10.1016/S0009-3084(01)00152-9

Chemistry and Physics of Lipids
111 (2001) 111–138
www.elsevier.com/locate/chemphyslip
The total syntheses of
D-erythro-sphingosine,
N-palmitoylsphingosine (ceramide), and glucosylceramide
(cerebroside) via an azidosphingosine analog
Richard I. Duclos Jr *
Department of Physiology and Biophysics, Boston Uni6ersity School of Medicine, 715 Albany Street, Boston,
MA 02118-2526, USA
Received 31 August 2000; received in revised form 10 April 2001; accepted 10 April 2001
Abstract
The total synthesis of
D
-erythro-sphingosine (9) was performed by a chirospecific method starting from D-galactose
via an azidosphingosine intermediate to give highly homogeneous (\99.9% C18:1) sphingosine base (9) which
contained no observable olefin isomerization by product and was demonstrated to be optically pure by a novel
method utilizing Mosher’s acid. Ceramide (10) was prepared from this sphingosine (9) with highly homogeneous
(99.8% C16:0) palmitic acid by two methods. The cerebroside glucosylceramide (23) was the next sphingolipid in this
series to be synthesized in a highly homogeneous form. These three sphingolipids are currently being used for
biophysical studies of the structures of their hydrated bio-molecular assemblies. © 2001 Elsevier Science Ireland Ltd.
All rights reserved.
Keywords: Sphingosine; Ceramide; Cerebroside; Glucosylceramide; Azidosphingosine; Mosher’s acid
1. Introduction
Sphingosine, ceramide, and glucosylceramide
are found in various cellular locations and are
Abbre6iations: Aq, aqueous; Ar, aryl; bT, bath temperature;
interconverted by their metabolic pathways (Bell
et al., 1993a,b; Kolter and Sandhoff, 1999), which
also notably include sphingomyelin (Hannun,
1994; Perry and Hannun, 1998). Sphingosine is
produced as a product of sphingolipid catabolism
(Buehrer and Bell, 1993; Kok et al., 1997; Rother
et al., 1992). Sphingosine has been the subject of
considerable research interest since the initial re-
port of its inhibition of protein kinase C activity
de, diastereomeric excess; DMF, dimethylformamide; DMSO,
dimethylsulfoxide; DSC, differential scanning calorimetry; ee,
enantiomeric excess; ESI, electrospray ionization; FA, fatty
acyl; FAB, fast atom bombardment; hex, hexanes; MALDI,
matrix-assisted laser desorption ionization; MS, mass spec-
trometry; MTPA, a-methoxy-a-(trifluoromethyl)phenylacetyl;
TEA, triethylamine; THF, tetrahydrofuran.
* Corresponding author. Tel.: +1-617-6384255; fax: +1-
617-6384041.
E-mail address: duclos@med-biophd.bu.edu (R.I. Duclos,
Jr).
0009-3084/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(01)00152-9

112
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
(Hannun et al., 1986) and subsequent reports of
other biological activities which have been re-
viewed (Hannun and Linardic, 1993; Igarashi,
1997). Sphingosine and ceramide are important
minor components of membranes (Vance and
Vance, 1996). Ceramide is also a principal inter-
cellular component of the epidermis (Elias and
Menon, 1991; Fuchs, 2000). Ceramide is biosyn-
thesized at the cytosolic surface of the endoplas-
mic reticulum (Rosenwald and Pagano, 1993; van
Echten and Sandhoff, 1993) and is a metabolic
intermediate having important roles in signal
transduction (Hannun, 1997; Kro¨nke, 1999;
Mathias et al., 1998; Merrill and Hannun,
2000a,b) including regulating apoptosis (Ariga et
al., 1998; Hannun and Obeid, 1995). Glucosylce-
ramide, anabolized at an early Golgi apparatus
compartment (Chujor et al., 1998), is an impor-
tant glycosphingolipid metabolic intermediate
(Kanfer, 1983a; Sandhoff and van Echten, 1993;
van Echten and Sandhoff, 1993; Young, 1993).
Glucosylceramide is the major cerebroside present
outside of the central nervous system, however,
glucosylceramide is only present in trace amounts,
except in individuals afflicted with Gaucher’s dis-
ease having insufficient glucocerebrosidase activ-
ity (Beutler and Grabowski, 1995; Kolter and
Sandhoff, 1999; Kanfer, 1983b). There is always
some heterogeneity of these endogeneous sphin-
golipids in living organisms due to chain length
and unsaturation in the sphingosine base, and
also due to chain length, unsaturation, and a-hy-
droxylation of the fatty acyl-containing ceramide
and glucosylceramide (cerebroside).
target molecules. There are many reports of sph-
ingosine syntheses, which have been recently re-
viewed (Byun and Bittman, 1993; Devant, 1992;
Gigg and Gigg, 1990; Hoffman and Tao, 1998;
Jung and Schmidt, 1999; Koskinen and Koskinen,
1998; Merrill and Hannun, 2000a; Nugent and
Hudlicky, 1998). With few exceptions, the syn-
thetic routes have been chirospecific, utilizing nat-
urally occurring optically active starting materials
including L-serine or carbohydrates, either as the
sources of what ultimately become the chiral cen-
ters of sphingosine 9 or to induce diastereoselec-
tivity. Few sphingosine syntheses satisfy the
criteria of optical purity (ꢀ99% de, ꢀ99% ee)
with complete control of the unsaturation and
feasibility on large scales. All syntheses which
utilized L-serine as a starting material were elimi-
nated from consideration, since the Garner alde-
hyde was reportedly only prepared between
95–98% ee (Dondoni and Perrone, 1999; Garner
and Park, 1987, 1991; Meffre et al., 1994). Opti-
cally pure sphingosine 9 has been obtained by
chirospecific syntheses from various chiral starting
materials 1. There have been a variety of method-
ologies reported for complete control of the trans
double bond 2. The superior sphingosine 9 syn-
theses obtained both chiral centers by efficient
carbohydrate degradation that neither jeopardized
the optical purity of sensitive chiral centers with
heterogeneous reactions containing strong base,
nor jeopardized the trans double bond with acidic
conditions. Proceeding through azidosphingosine
analogs has also allowed flexibility in synthetic
methodologies, which may be of particular impor-
tance in some glycosphingolipid syntheses.
Intermediate compounds have often been used
to establish optical purity (de, ee) in syntheses of
sphingosine 9, or optical purity has been esti-
mated by the optical rotation method for sphin-
gosine 9 itself or of the triacetate derivative. It has
always been relatively easy to chromatographi-
cally demonstrate that the erythro-sphingosine
enantiomeric pair was free of the threo-sphin-
gosine enantiomers. (Pagano and Martin, 1988;
Sambasivarao and McCluer, 1963; Sarmientos et
al., 1985). However, it has been difficult to sepa-
In conjunction with continuing studies of mem-
brane structure and function (Saxena et al., 1999,
2000; Shah et al., 1995a,b), research quantities of
optically pure and highly homogeneous
D-ery-
thro-sphingosine ((2S,3R,4E)-2-amino-4-oc-
tadecene-1,3-diol, (E)-sphing-4-enine, C18H37NO2,
Sph) 9, ceramide ((2S,3R,4E)-2-(hexadecanoy-
lamido)-4-octadecene-1,3-diol, N-palmitoylsphin-
gosine,
((2S,3R,4E)-b-
decanoylamido)-4-octadecene-1,3-diol,
broside, GlcCer) 23 were required. There has al-
ready been considerable body of earlier
synthetic work directed at each of these three
Cer)
10,
-glucopyranosyl-(1%l1)-2-(hexa-
cere-
and
glucosylceramide
D
a
rate
D
-erythro-sphingosine from the enantiomeric
-threo-
L
-erythro-sphingosine, and to separate
D

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
113
sphingosine from the enantiomeric
L
-threo-sphin-
NMR spectra of ceramides have already been
reported (Sarmientos et al., 1985; Tkaczuk and
Thornton, 1981). The optically pure and highly
homogeneous synthetic ceramide 10 prepared by
this method was recently studied by DSC and
X-ray diffraction, and the structural and ther-
motropic properties upon hydration were re-
ported in detail (Shah et al., 1995a). The 13C
NMR spectra of glucosylceramides (cerebrosides)
have also already been reported (Koerner et al.,
1979; Sarmientos et al., 1985; Taki et al., 1992).
The optically pure and highly homogeneous syn-
thetic glucosylceramide (cerebroside) 23 prepared
by this method was also recently studied by DSC
and X-ray diffraction (XRD) (Saxena et al.,
1999).
gosine. The assays of the optical purity of each of
the four diastereomeric sphingosines as a variety
of derivatives have been performed by a nuclear
magnetic resonance (NMR) method which uti-
lized a lanthanide-induced shift (LIS) study (Hoff-
man and Tao, 1998) and by chromatographic
methods on normal (Boutin and Rapoport, 1986;
Pagano and Martin, 1988) and chiral (Kawamura
et al., 1996) stationary phases. It is extremely
important to be able to analyze for the optical
purity (de, ee) of sphingosine base due to the
potential for variations in the biological activities
of the four diastereomers of sphingosine. With the
notable exception of protein kinase C interaction
(Igarashi et al., 1990; Merrill et al., 1989; Sachs et
al., 1996; Smith et al., 2000) the four
diastereomers of sphingosine (Ariga et al., 1998;
Buehrer and Bell, 1992, 1993; Olivera et al., 1994;
Pushkareva et al., 1995) ceramide (Ariga et al.,
1998; Karasavvas et al., 1996; Moesby et al.,
1995; Pagano and Martin, 1988; Paul et al., 1996;
Van Overmeire et al., 2000), and other related
sphingolipids (Igarashi et al., 1990; Karasavvas et
al., 1996; Kok et al., 1997; Moesby et al., 1995;
Motoki et al., 1995) generally demonstrate
stereoselective biological activities.
2. Experimental procedures
2.1. General
Toluene and THF were distilled from ben-
zophenone ketyl. Reaction solvents including
CH2Cl2, hexanes (hex), and CCl4 were dried by
distillation from P2O5. Pyridine was distilled from
CaH2. Residueless t-BuOH was obtained after
simple distillation. Reactions were carried out un-
der argon with magnetic stirring at ambient tem-
perature, unless reported as bath temperature
(bT). Organic phases were dried over Na2SO4,
followed by paper filtration and solvent removal
by rotary-evaporation under reduced pressure. All
solvent ratios were by volume. Silica gel 60 (35–
70 mm, irregular particle) from E. Merck (Darm-
stadt, Germany) activated at 120°C for 12 h was
used for all chromatographic separations unless
otherwise noted. Acid-sensitive lipids required the
use of silica gel, which had been activated accord-
ing to a slight modification of a earlier reported
method (Hirsch and Ahrens, 1958) to eliminate
the cis/trans isomerization of these certain ex-
tremely acid-sensitive sphingolipids during chro-
matography. The silica gel (800 ml) was slurried
to a total volume of 1500 ml with MeOH, and the
cloudy MeOH was decanted. The mixture was
again slurried to 1400 ml with more MeOH and
collected on paper with a Buchner funnel. Slow
There have been a number of earlier syntheses
of the desired N-palmitoylsphingosine (ceramide)
10 (Dong, 1990; Kanemitsu and Sweeley, 1986;
Koike et al., 1986; Ohashi et al., 1988a, 1989;
Shah et al., 1995a; Shibuya et al., 1992) target
compound. Also, an HPLC method for determin-
ing the optical purity of ceramides was recently
reported (Bakke et al., 1998). The ceramide 10
was recently used as an intermediate in the chem-
ical total synthesis of ganglioside GM3 (Duclos,
2000). There have also been a few earlier synthe-
ses of the N-palmitoyl analog of the glucosylce-
ramide (cerebroside) 23 (Schmidt and Kla¨ger,
1985; Schmidt and Zimmermann, 1986b, 1988;
Shibuya et al., 1992; Zimmermann et al., 1988)
target compound.
1
The H and 13C NMR spectra of sphingosine 9
(Kisic et al., 1995; Sarmientos et al., 1985) and the
biophysical studies of sphingosine 9 by differential
scanning calorimetry (DSC) and X-ray diffraction
(Ko, 1997) have recently been detailed. The 13C

114
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
suction was maintained while the silica gel was
washed (2×250 ml) with MeOH. When the silica
gel appeared dry, it was slurried (2×450 ml) with
Et2O in the Buchner funnel followed by slow
suction filtration. The air dried powder was dried
in a vacuum desiccator at 100 mm for several
hours, then activated at 10 mm for several days.
Acid-sensitive lipids were often protected during
silica gel chromatography by the use of TEA or
aqueous NH4OH in the elution mixture. All reac-
tion products were purified to homogeneity as
judged by TLC on silica gel plates, which were
visualized by the reported charring method (Bit-
man and Wood, 1982). HPLC columns used were
Lichrosorb Si-60 of 5 or 10 mm particles. Product
solutions were microfiltered through 0.5 mm
Teflon membranes (Alltech). NMR utilized Me4Si
(TMS) as an internal reference, except for
DMSO-d6 where the solvent peaks at l 2.50 for
1H and l 39.50 for 13C were used. Chemical shifts
were often assigned with the assistance of COSY
and HMQC spectroscopy. Where possible, spe-
cific rotation data was obtained under conditions
of solvent, concentration, and temperature corre-
sponding to previous literature reports. Fast atom
bombardment (FAB), matrix assisted laser des-
orption ionization (MALDI), and electrospray
ionization (ESI) mass spectrometry (MS) tech-
niques were utilized. The major observable molec-
ular ion and selected fragments and clusters have
been reported. The notations for fragment ions
have been previously reported (Costello and Vath,
1990; Domon and Costello, 1988). The primed (%)
fragment notation indicates the loss of H2O (MW
18). The fragmentations of these sphingolipids
have been discussed in several literature reports
(Ann and Adams, 1993; Ariga et al., 1980; Cos-
tello and Vath, 1990; Domon and Costello, 1988;
Domon et al., 1990; Hara and Taketomi, 1986;
Hemling et al., 1984; Kushi et al., 1989; Murphy,
1993; Perreault and Costello, 1996; Shoyama et
al., 1978; Suzuki et al., 1989, 1991).
mol, 600 mol%), and fused ZnCl2 (38.8 g, 0.285,
100 mol%) were vigorously mechanically shaken
for 14 h. Three parallel batches were run, com-
bined, extracted with H2O (3×300 ml), and back-
washed with 600 ml of hexanes. The aqueous
solution was stirred mechanically during the addi-
tion of 1.01 l of 15% aq Na2CO3 during which the
ZnCO3 precipitate formed. The mixture was suc-
tion filtered, the precipitate washed with H2O
(2×100 ml), and the filtrates were combined and
washed once with 600 ml of hexanes. The aqueous
solution was frozen and lyophilized. The white
solid residue was boiled twice with 2 l portions of
40:60 EtOH/EtOAc. The combined extracts were
filtered through a short column and concentrated
to give 106 g of crude product. Three hot filtra-
tions/recrystallizations from EtOH/EtOAc gave a/
b-2 (39.91 g, 0.149 mol, 13%) as a white solid
which was an 85:15 a/b mixture by NMR in
DMSO-d6. The epimeric mixture was high melting
with a strong rotation, and was free of any other
impurities by NMR. This material was compara-
ble to earlier reported (Byun and Bittman, 1993;
Dong, 1990; Gros and Deulofeu, 1962, 1964; Kiso
et al., 1986a; Paca´k and Cerny´, 1963; Schmidt and
Zimmermann, 1986a, 1988; Zimmermann and
Schmidt, 1988) preparations and was used with-
out further purification: a/b-2 m.p. 171–173°C;
[a]2D5 +132° (c 1.0 MeOH). a-2 1H NMR
(DMSO-d6) l 7.30–7.50 (m, 5H, Ar), 5.53 (s, 1H,
CH6 Ph); Gal, 5.05 (dd, 1H, J1,1-OH=4.2, J1,2=3.4
Hz, 1), 6.26 (d, 1H, J1,1-OH=4.2 Hz, 1-OH), 3.62
(ddd, 1H, J1,2=3.4, J2,2-OH=6.7, J2,3=9.4 Hz,
2), 4.41 (d, 1H, J2,2-OH=6.7 Hz, 2-OH), 3.74–
3.77 (m, 1H, 3), 4.59 (d, 1H, J3,3-OH=6.2 Hz,
3-OH), 4.11 (d, 1H, J3,4=2.6 Hz, 4), 3.77 (s, 1H,
5), 3.96 (d, 1H, J6a,6b=12.1 Hz, 6a), 4.05 (d, 1H,
J
6a,6b=12.1 Hz, 6b); 13C NMR (DMSO-d6) l
138.73, 128.45, 127.78 (×2), 126.21 (×2) [Ar×
6]; 99.66 (CHPh); Gal, 93.07 (1), 68.42 (2), 67.65
6
1
(3), 76.67 (4), 61.96 (5), 68.88 (6). b-2 H NMR
(DMSO-d6) l 7.30–7.50 (m, 5H, Ar), 5.55 (s, 1H,
CH6 Ph); Gal, 4.34 (dd, 1H, J1,1-OH=6.9, J1,2=7.5
2.2. 4,6-Benzylidene-h/i-
D
-galactose (h/i-2)
Hz, 1), 6.56 (d, 1H, J1,1-OH=6.9 Hz, 1-OH),
3.28–3.34 (m, 1H, 2), 4.77 (d, 1H, J2,2-OH=5.2
Hz, 2-OH), 3.40–3.46 (m, 1H, 3), 4.76 (d, 1H,
A mixture of -galactose (1) (50.0 g, 0.278 mol,
D
100 mol%, assayed by Sigma to contain B0.01%
glucose), redistilled benzaldehyde (177 g, 1.67
J
3,3-OH=6.4 Hz, 3-OH), 4.00–4.10 (m, 3H, 4, 6a,
6b), 3.45 (br s, 1H, 5); 13C NMR (DMSO-d6) l

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
115
138.67, 128.46, 127.76 (×2), 126.21 (×2) [Ar×
6]; 99.63 (CHPh); Gal, 97.22 (1), 71.55 (2), 72.05
(3), 76.10 (4), 65.74 (5), 68.80 (6).
100 mol%) as a solid yellow foam. A mechanically
stirred mixture of tetradecyltriphenylphospho-
nium bromide (4) (91.84 g, 0.170 mol, 115 mol%)
in 1200 ml of anhydrous degassed PhMe under
argon was cooled below −25°C (internal temper-
ature) with a −29°C (bT, o-xylene/N2) bath and
then PhLi, prepared from bromobenzene (103.62
g, 0.660 mol, 4.46 mol%) and lithium (9.16 g, 1.32
mol, 892 mol%) in 280 ml of anhydrous Et2O, was
added over 15 min. The reaction mixture was
stirred for an additional 45 min, then a solution
of 3 in 212 ml of anhydrous THF was added over
30 min. The reaction mixture was stirred for an
additional 20 min at −29°C (bT). The reaction
was quenched with 210 ml of MeOH followed by
350 ml of H2O and allowed to stir at ambient
temperature for 14 h. The organic phase was
concentrated by rotoevaporation at 45°C. Chro-
matography (use of MeOH activated silica gel
recommended) with 80:20:0.25, hex/EtOAc/TEA
gave nearly homogeneous 5 (25.11 g, 64.6 mmol,
44%) (Byun and Bittman, 1993; Dong, 1990; Jung
and Schmidt, 2000; Kiso et al., 1986a; Kumar and
Schmidt, 1998; Ohashi et al., 1989, 1988b;
Schmidt and Zimmermann, 1986a, 1988; Yadav et
al., 1993; Yamanoi et al., 1989; Zimmermann and
Schmidt, 1988) as a white solid: TLC (50:50:0.25,
hex/EtOAc/TEA) Rf=0.55; m.p. 55–56°C Lit.
(Kiso et al., 1986a) m.p. 53.5°C; Lit. (Zimmer-
mann and Schmidt, 1988) m.p. 54–55°C; Lit.
(Dong, 1990) m.p. 56.0–58.0°C); Lit.(Kumar and
Schmidt, 1998) m.p. 56–57°C); 1H NMR
(DMSO-d6) d 7.50 (br d, 2H, J=7.8 Hz, Ar),
6
2.3. Tetradecyltriphenylphosphonium bromide (4)
Tetradecylbromide was prepared from tetrade-
cyl alcohol by the method used for the hexadecyl
analog (Abdelmageed et al., 1989) and was
demonstrated to be 0.1% dodecylbromide, 99.9%
tetradecyl bromide, and 0.0% hexadecylbromide
by gas chromatography (5% DEGS-PS on 100/
120 supelcoport at 125°C). A mixture of tetrade-
cylbromide (97.89 g, 0.353 mol, 100 mol%) and
triphenylphosphine (92.60 g, 0.353 mol, 100
mol%) were heated at 170°C (bT) for 2 days then
dissolved in 350 ml of warm Me2CO and the
product crystallized by the addition of 950 ml of
warm anhydrous Et2O. A second crystallization
from Me2CO/Et2O gave 4 (140.48 g, 0.260 mol,
74%) as a white crystalline solid, which was free
of alkyl halide and triphenylphosphine: TLC
(Me2CO) Rf 0.06; m.p. 83–85°C (Lit. (Reist and
1
Christie, 1970) m.p. 94–96°C); H NMR (CDCl3)
l 7.70–7.90 (m, 15H, Ar), 3.74 (m, 2H, 1), 1.55–
1.68 (m, 4H, 2, 3), 1.25–1.42 (m, 20H, 4–13), 0.87
1
(t, 3H, J13,14=6.5 Hz, 14); H NMR (C6D6) l
8.07–8.11 (m, 6H, Ar), 7.35–7.50 (m, 9H, Ar),
4.60 (m, 2H, 1), 1.79–1.88 (m, 2H, 2), 1.54–1.65
(m, 2H, 3), 1.15–1.35 (m, 20H, 4–13), 0.92 (t, 3H,
J13,14=6.5 Hz, 14).
2.4. (2R,3R,4E)-1,3-Benzylidene-4-octadecene-
1,2,3-triol (5)
7.35–7.47 (m, 3H, Ar), 5.61 (s, 1H, CH6 Ph); Sph,
4.02 (dd, 1H, J1a,1b=11.8, J1a,2=1.4 Hz, 1a), 4.06
(dd, 1H, J1a,1b=11.8, J1b,2=1.4 Hz, 1b), 3.40 (m,
1H, 2), 4.38 (br d, 1H, J3,4=6.6 Hz, 3), 5.62 (dd,
1H, J3,4=6.6, J4,5=15.5 Hz, 4), 5.74 (dt, 1H,
4,6-Benzylidene-a/b- -galactose (a/b-2) (39.58
D
g, 0.148 mol, 100 mol%) in 1000 ml of 0.067 M
phosphate buffer at pH 7.8 was stirred mechani-
cally, and NaIO4 (72.72 g, 0.340 mol, 230 mol%)
was added slowly over 30 min. The pH was
maintained above 7.75 with 50 ml of 6N NaOH.
The mixture was concentrated by rotoevaporation
at 45°C (bT) and the solvent was removed com-
pletely by lyophilization for 14 h. The off-white
solid residue was dissolved in dry THF, filtered
through paper, the solvent removed, and the
residue dried under vacuum for 4 h to give crude
J4,5=15.5, J5,6=6.6 Hz, 5), 2.03 (dt, 2H, J5,6=
6.6, J6,7=7.4 Hz, 6), 1.37 (m, 2H, 7), 1.24–1.35
(m, 20H, 8–17), 0.88 (t, 3H, J17,18=6.9 Hz, 18);
13C NMR (DMSO-d6) d 138.78, 128.44, 127.76
(×2), 126.27 (×2) [Ar×6]; 100.13 (C6 HPh); Sph,
72.00 (1), 65.06 (2), 79.91 (3), 127.72 (4), 132.69
(5), 31.71 (6), 28.43–28.93 (7–15), 31.19 (16),
21.99 (17), 13.84 (18); [a]2D5 0° (c 0.50, CHCl3) Lit.
(Kiso et al., 1986a) [a]D −0.6° (c 0.506, CHCl3);
Lit. (Zimmermann and Schmidt, 1988) [a]D22
3.8° (c 0.5, CHCl3).
−
2,4-benzylidene- -threose (3) (31.43 g, 0.151 mol,
D

116
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
2.5. (2R,3R,4E)-1,3-Benzylidene-2-
methanesulfonyl-4-octadecene-1,2,3-triol (6)
bient temperature and the salt removed by
filtration through a short column (86 g) eluting
with 90:10 hex/EtOAc. The eluent was concen-
trated and the residue chromatographed (90:10
hex/EtOAc). Homogeneous azide 7 (1.56 g, 3.77
mmol, 21%) (Byun and Bittman, 1993; Dong,
1990; Jung and Schmidt, 2000; Ohashi et al.,
1989, 1988b; Schmidt and Zimmermann, 1986a,
1988; Yadav et al., 1993; Zimmermann and
Schmidt, 1988) was obtained by HPLC (22×300
mm, Si-60, 5 mm, 70:30 hexane/CH2Cl2): TLC
(50:50 CH2Cl2/hexane) Rf=0.37, (50:50 hex/
EtOAc) Rf=0.74; m.p. 43°C (Lit. (Dong, 1990;
Ohashi et al., 1989; Zimmermann and Schmidt,
To a mixture of alcohol 5 (7.28 g, 18.7 mmol,
100 mol%) and TEA (13.0 ml, 9.44 g, 93.3 mmol,
500 mol%) was added 80 ml of CH2Cl2 (freshly
doubly distilled from P2O5) and the mixture
stirred at −23°C (bT, CCl4/N2). A solution of
methanesulfonyl chloride (2.89 ml, 4.28 g, 37.5
mmol, 200 mol%) in 10 ml of CH2Cl2 was added
dropwise over 5 min. The reaction mixture was
allowed to warm to 0°C over 10 min, then al-
lowed to stir for an additional 30 min. The reac-
tion mixture was concentrated and the crude
residue chromatographed (70:30:0.5, hex/EtOAc/
TEA). The product was rechromatographed
(75:25, hex/EtOAc, 35°C) and fractions confirmed
1
1988) oil); H NMR (DMSO-d6) l 7.36–7.41 (m,
5H, Ar), 5.60 (s, 1H, CH
1a,1b=9, J1a,2=9 Hz, 1a), 4.28 (dd, 1H, J1a,1b
9, J1b,2=3.5 Hz, 1b), 3.64 (ddd, 1H, J1a,2=9,
1b,2=3.5, J2,3=8.8 Hz, 2), 4.13 (dd, 1H, J2,3
8.8, J3,4=7.6 Hz, 3), 5.57 (dd, 1H, J3,4=7.6,
4,5=15.5 Hz, 4), 5.92 (dt, 1H, J4,5=15.5, J5,6
6 Ph); Sph, 3.60 (dd, 1H,
J
=
1
to be homogeneous by H NMR were combined
to give mesylate 6 (7.53 g, 16.1 mmol, 86%) (Byun
and Bittman, 1993; Ohashi et al., 1989, 1988b;
Schmidt and Zimmermann, 1986a) as a white
crystalline solid: TLC (70:30 hex/EtOAc) Rf=
0.26, (50:50 hex/EtOAc) Rf=0.53; m.p. 105–
107°C (Lit. (Ohashi et al., 1989) m.p.
J
=
J
=
6.8 Hz, 5), 2.06 (dt, 1H, J5,6=6.8, J6,7=7.2 Hz,
6), 1.37 (m, 2H, 7), 1.24–1.35 (m, 20H, 8–17),
0.85 (t, 3H, J17,18=6.8 Hz, 18); 13C NMR
(DMSO-d6) l 137.61, 128.70, 127.94 (×2), 126.01
1
81.5–83.0°C); H NMR (DMSO-d6) l 7.40–7.47
(m, 5H Ar), 5.75 (s, 1H, CH
6
Ph), 3.21 (s, 3H, Ms);
(×2) [Ar×6]; 99.81 (C6 HPh); Sph, 67.97 (1),
Sph, 4.26 (d, 1H, J1a,1b=13.0 Hz, 1a), 4.31 (d,
1H, J1a,1b=13.0 Hz, 1b), 4.67 (br s, 1H, 2), 4.70
(d, 1H, J3,4=6.0 Hz, 3), 5.56 (dd, 1H, J3,4=6.0,
56.45 (2), 80.44 (3), 126.64 (4), 136.32 (5), 31.63
(6), 28.08–28.87 (7–15), 31.16 (16), 21.95 (17),
1
13.80 (18); H NMR (CDCl3) l 7.48 (br d, 2H,
J4,5=15.6 Hz, 4), 5.84 (dt, 1H, J4,5=15.6, J5,6
=
J=7.8 Hz, Ar), 7.32–7.38 (m, 3H, Ar), 5.49 (s,
6.7 Hz, 5), 2.06 (dt, 2H, J5,6=6.7, J6,7=7.0 Hz,
6), 1.37 (m, 2H, 7), 1.24–1.35 (m, 20H, 8–17),
0.88 (t, 3H, J17,18=6.5 Hz, 18); 13C NMR
(DMSO-d6) l 137.98, 128.69, 127.93 (×2), 125.99
1H, CH
6
Ph); Sph, 3.61 (dd, 1H, J1a,1b=11.1,
1a,2=10.8 Hz, 1a), 4.34 (dd, 1H, J1a,1b=11.1,
1b,2=5.2 Hz, 1b), 3.47 (ddd, 1H, J1a,2=10.8,
1b,2=5.2, J2,3=9.5 Hz, 2), 4.06 (dd, 1H, J2,3
J
J
J
=
(×2) [Ar×6]; 99.74 (C
6
HPh), 37.84 (Ms); Sph,
9.5, J3,4=7.4 Hz, 3), 5.59 (dd, 1H, J3,4=7.4,
4,5=15.4 Hz, 4), 5.98 (dt, 1H, J4,5=15.4, J5,6
6.7 Hz, 5), 2.11 (dt, 2H, J5,6=6.7, J6,7=7.2 Hz,
6), 1.42 (quint, 2H, J6,7=J7,8=7.2 Hz, 7), 1.24–
1.35 (m, 20H, 8–17), 0.88 (t, 3H, J17,18=6.9 Hz,
18); 13C NMR (CDCl3) l 137.33, 129.10, 128.30
68.92 (1), 74.97 (2), 77.22 (3), 125.49 (4), 134.11
(5), 31.51 (6), 28.24–28.91 (7–15), 31.17 (16),
21.96 (17), 13.81 (18).
J
=
2.6. (2S,3R,4E)-2-Azido-1,3-benzylidene-4-
octadecene-1,3-diol (trans-7)
(×2), 126.16 (×2) [Ar×6]; 101.12 (C6 HPh); Sph,
69.11 (1), 57.48 (2), 81.79 (3), 125.85 (4), 137.79
(5), 32.51 (6), 28.66–29.64 (7–15), 31.91 (16),
22.68 (17), 14.10 (18); IR (NaCl, cm−1) 2105
(N3); [a]2D4 −11° (c 3.0, CHCl3) (Lit.) (Zimmer-
mann and Schmidt, 1988) [a]2D0 −11.7° (c 3,
CHCl3); Lit. (Yadav et al., 1993) [a]D −33.4° (c
0.7, CHCl3).
A stirred mixture of mesylate 6 (8.28 g, 17.7
mmol, 100 mol%) and NaN3 (11.2 g, 172 mmol,
1000 mol%) in 300 ml of DMSO was heated at
95°C (bT) for 3 days until the starting material 6
was completely consumed by TLC (50:50 hex/
EtOAc). The reaction mixture was cooled to am-

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
117
2.7. (2S,3R,4Z)-2-Azido-1,3-benzylidene-4-
octadecene-1,3-diol (cis-7)
The solution was stirred for 14 h, then quenched
with TEA (0.738 ml, 0.536 g, 5.30 mmol, 109
mol%). The mixture was concentrated and filtered
through a short column (97.5:2.5 CH2Cl2/MeOH).
The crude product was re-chromatographed
(70:30 CH2Cl2/EtOAc) to give azidosphingosine 8
(1.52 g, 4.67 mol, 96%) (Dong, 1990; Ito et al.,
1988; Jung and Schmidt, 2000; Kiso et al.,
1986a,b; Kumar and Schmidt, 1998; Ohashi et al.,
1989; Schmidt and Zimmermann, 1986b, 1988;
Somfai and Olsson, 1993; Yadav et al., 1993;
Zimmermann and Schmidt, 1988) as a white solid
that was homogeneous: TLC (70:30 CH2Cl2/
EtOAc) Rf 0.30; m.p. 53–54°C (Lit. (Kiso et al.,
1986a,b) m.p. 50.5–51.5°C; Lit. (Zimmermann
and Schmidt, 1988) m.p. 49–50°C; Lit. (Dong,
1990) m.p. 49.0–50.0°C; Lit. (Kumar and
A small amount of the cis-azide cis-7 was iso-
lated in a nearly homogeneous form as a white
solid: TLC (50:50 hex/CH2Cl2) Rf=0.29; 1H
NMR (DMSO-d6) l 7.34–7.41 (m, 5H, Ar), 5.64
(s, 1H, CH
6
Ph); Sph, 3.65 (dd, 1H, J1a,1b=16.8,
1a,2=10 Hz, 1a), 4.30 (dd, 1H, J1a,1b=16.8,
1b,2=11.3 Hz, 1b), 3.66 (ddd, 1H, J1a,2=10,
1b,2=11.3, J2,3=7.4 Hz, 2), 4.52 (dd, 1H, J2,3
J
J
J
=
7.4, J3,4=9.3 Hz, 3), 5.48 (dd, 1H, J3,4=9.3,
4,5=10.6 Hz, 4), 5.74 (dt, 1H, J4,5=10.6, J5,6
7.6 Hz, 5), 2.18 (dddd, 1H, J5,6a=7.6, J6a,6b=14,
6a,7a=J6a,7b=7 Hz, 6a), 2.21 (dddd, 1H, J5,6b
J
=
J
=
7.6, J6a,6b=14, J6b,7a=J6b,7b=7 Hz, 6b), 1.37 (m,
2H, 7), 1.24–1.35 (m, 20H, 8–17), 0.85 (t, 3H,
1
J
17,18=6.8 Hz, 18); 13C NMR (DMSO-d6) l
Schmidt, 1998) m.p. 49°C); H NMR (DMSO-d6)
137.64, 128.66, 127.90 (×2), 125.96 (×2) [Ar×
6]; 99.74 (CHPh); Sph, 67.96 (1), 56.50 (2), 75.27
(3), 126.39 (4), 136.42 (5), 27.46 (6), 28.44–28.94
(7–15), 31.20 (16), 21.99 (17), 13.80 (18); 1H
NMR (CDCl3) l 7.47 (br d, 2H, J=7.8 Hz, Ar),
l 3.39 (ddd, 1H, J1a,1b=11, J1a,1-OH=5, J1a,2=8
Hz, 1a), 3.57 (ddd, 1H, J1a,1b=11, J1b,1-OH=5,
6
J
1b,2=4 Hz, 1b), 4.87 (t, 1H, J1a,1-OH=J1b,1-OH
5 Hz, 1-OH), 3.32 (ddd, 1H, J1a,2=8, J1b,2=4,
2,3=5.4 Hz, 2), 4.01 (ddd, 1H, J2,3=6.4, J3,3-
OH=4.8, J3,4=6.8 Hz, 3), 5.11 (d, 1H, J3,3-OH
=
J
7.32–7.38 (m, 3H, Ar), 5.52 (s, 1H, CH
6
Ph); Sph,
=
3.64 (dd, 1H, J1a,1b=11.1, J1a,2=10.8 Hz, 1a),
4.36 (dd, 1H, J1a,1b=11.1, J1b,2=5.2 Hz, 1b),
3.50 (ddd, 1H, J1a,2=10.8, J1b,2=5.2, J2,3=9.5
Hz, 2), 4.43 (dd, 1H, J2,3=9.5, J3,4=8.6 Hz, 3),
5.51 (dd, 1H, J3,4=8.6, J4,5=10.9 Hz, 4), 5.82
(dt, 1H, J4,5=10.9, J5,6=7.5 Hz, 5), 2.21 (dddd,
1H, J5,6a=7.5, J6a,6b=14, J6a,7a=J6a,7b=7 Hz,
4.8 Hz, 3-OH), 5.42 (dd, 1H, J3,4=6.8, J4,5=15.4
Hz, 4), 5.62 (dt, 1H, J4,5=15.4, J5,6=6.7 Hz, 5),
1.99 (dt, 2H, J5,6=6.7, J6,7=7 Hz, 6), 1.33 (m,
2H, 7), 1.20–1.35 (m, 20H, 8–17), 0.86 (t, 3H,
J
17,18=6.7 Hz, 18); 13C NMR (DMSO-d6) l 60.67
(1), 68.14 (2), 70.89 (3), 129.79 (4), 132.08 (5),
31.52 (6), 28.34–28.90 (7–15), 31.19 (16), 21.98
1
6a), 2.26 (dddd, 1H,
J5,6b=7.5,
J6a,6b=14,
(17), 13.83 (18); H NMR (CDCl3) l 3.78 (d, 2H,
J6b,7a=J6b,7b=7 Hz, 6b), 1.43 (m, 2H, 7), 1.24–
J1,2=5.1 Hz, 1), 3.49 (dt, 1H, J1,2=5.1, J2,3=5.5
1.40 (m, 20H, 8–17), 0.88 (t, 3H, J17,18=6.9 Hz,
Hz, 2), 4.24 (dd, 1H, J2,3=5.5, J3,4=7.2 Hz, 3),
5.53 (dd, 1H, J3,4=7.2, J4,5=15.4 Hz, 4), 5.82
(dt, 1H, J4,5=15.4, J5,6=6.6 Hz, 5), 2.07 (dt, 2H,
18); 13C NMR (CDCl3) l 137.31, 129.09, 128.29
(×2), 126.13 (×2) [Ar×6]; 101.09 (C6 HPh); Sph,
69.13 (1), 57.46 (2), 76.53 (3), 125.58 (4), 137.83
(5), 28.29 (6), 29.28-29.67 (7–15), 31.92 (16),
22.68 (17), 14.11 (18).
J5,6=6.6, J6,7=6.6 Hz, 6), 1.20–1.40 (m, 22H,
7–17), 0.88 (t, 3H, J17,18=6.3 Hz, 18); 13C NMR
(CDCl3) l 62.56 (1), 66.83 (2), 73.70 (3), 128.13
(4), 135.99 (5), 31.97 (6), 28.99–29.72 (7–15),
32.36 (16), 22.72 (17), 14.12 (18); [a]2D4 −33.0° (c
2.8. (2S,3R,4E)-2-Azido-4-octadecene-1,3-diol
(azidosphingosine) (8)
4.00, CHCl3) (Lit. (Kiso et al., 1986a) [a]D
−
33.3° (c 0.441, CHCl3); Lit. (Kiso et al., 1986b)
[a]D −33° (c 0.4, CHCl3); Lit. (Ito et al., 1988)
[a]D20-22 −34.1° (c 1.1, CHCl3); (Lit.) (Zimmer-
mann and Schmidt, 1988) [a]2D0 −32.9° (c 4,
CHCl3); Lit. (Kumar and Schmidt, 1998) [a]D20
−29.1° (c 1, CHCl3).
A solution of benzylidene azidosphingosine 7
(2.02 g, 4.88 mmol, 100 mol%) in 30 ml of 1:2
CH2Cl2/MeOH was stirred vigorously and a solu-
tion of p-TsOH-H2O (0.504 g, 2.65 mol, 54 mol%)
in 10 ml of MeOH was added rapidly dropwise.

118
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
2.10. (2S,3R,4E)-2-(Hexadecanoylamido)-4-
octadecene-1,3-diol (ceramide) (10)
2.9. (2S,3R,4E)-2-Amino-4-octadecene-1,3-diol
(D
-erythro-sphingosine) (9)
2.10.1. From sphingosine 9
To a stirred solution of azidosphingosine 8
A mixture of sphingosine 9 (1.07 g, 3.57 mmol,
100 mol%) in 40 ml of THF and NaOAc–3H2O
(19.4 g, 0.142 mol, 3980 mol%) in 19 ml of H2O
was stirred vigorously during the addition of
palmitoyl chloride (1.4 g, 5.1 mmol, 143 mol%,
prepared from palmitic acid, a sample of which
had been methylated and determined to be 0.04%
myristic acid, 99.81% palmitic acid, and 0.15%
stearic acid by gas chromatography using 5%
DEGS-PS on 100/120 supelcoport at 160°C) in 20
ml of THF over 5 min. The reaction mixture was
stirred vigorously for an additional hour. The
THF was separated, and the aqueous phase was
given two additional extractions with 99.5:0.5
CH2Cl2/TEA. The organic extracts were com-
bined, concentrated by rotoevaporation, and
(1.51 g, 4.64 mol, 100 mol%) in 25 ml of pyridine
was added 10 ml of H2O and H2S gas was bub-
bled through the solution for 2 days. The reaction
mixture was concentrated at 35°C (bT). Chro-
matography (2:1 CHCl3/MeOH) gave the crude
product which was re-chromatographed (60:30:5
CHCl3/MeOH/H2O) to give sphingosine 9 (1.32 g,
4.41 mmol, 95%) (Dong, 1990; Hirata et al., 1991;
Jung and Schmidt, 2000; Kiso et al., 1986a,b; Li
and Wu, 1996; Schmidt and Zimmermann, 1986a;
Shibuya et al., 1992; Yadav et al., 1993; Zimmer-
mann and Schmidt, 1988) as a white solid that
was homogeneous: TLC (60:30:5 CHCl3/MeOH/
H2O) Rf 0.29, (60:30:5 CHCl3/MeOH/aq NH4OH)
Rf 0.64; m.p. 75–80°C (Lit. (Kiso et al., 1986a,b)
m.p. 81.5–82.5°C; Lit. (Zimmermann and
Schmidt, 1988) m.p. 79–82°C; Lit. (Dong, 1990)
m.p. 75.0–77.0°C; Lit. (Hirata et al., 1991) m.p.
72–74°C; Lit. (Yadav et al., 1993) m.p. 78.4–
80.8°C; Lit. (Li and Wu, 1996) m.p. 73–75°C); 1H
filtered through
a short column (94.5:5:0.5
CH2Cl2/MeOH/TEA). The crude mixture of ce-
ramide 10 and palmitic acid was filtered through
an ion exchange column (Whatman DEAE DE-
52, acetate form, 90:10 CH2Cl2/MeOH). The
crude product was rechromatographed (95:5:0.1
CH2Cl2/MeOH/TEA, 35°C) and micropore
filtered to give homogeneous ceramide 10 (1.46 g,
2.71 mmol, 76%) (Dong, 1990; Kanemitsu and
Sweeley, 1986; Koike et al., 1986; Ohashi et al.,
1988a, 1989; Shah et al., 1995a; Shibuya et al.,
1992) as a white solid after lyophilization from
t-butanol solution: TLC (95:5:0.1 CH2Cl2/MeOH/
TEA) Rf 0.14; m.p. 100.5–101.5°C Lit. (Kane-
mitsu and Sweeley, 1986) m.p. 81–84°C; Lit.
(Koike et al., 1986) m.p. 93–94°C; Lit. (Ohashi et
al., 1988a, 1989) m.p. 95.0–96.0°C; Lit. (Dong,
1990 m.p. 100.0–102.0°C; Lit. (Shibuya et al.,
1992) m.p. 95–96°C; Lit. (Shah et al., 1995a) m.p.
100.0–102.0°C); 1H NMR (DMSO-d6, 45°C) l
Sph, 3.46 (br dd, 1H, J1a,1b=10.7, J1a,2=5 Hz,
1a), 3.49 (br dd, 1H, J1a,1b=10.7, J1b,2=5 Hz,
NMR (DMSO-d6, 32°C) l 3.28 (dd, 1H, J1a,1b
10.7, J1a,2=7.2 Hz, 1a), 3.45 (dd, 1H, J1a,1b
=
=
10.7, J1b,2=4.5 Hz, 1b), 2.68 (ddd, 1H, J1a,2=7.2,
1b,2=4.5, J2,3=5.2 Hz, 2), 3.87 (dd, 1H, J2,3
5.2, J3,4=6.5 Hz, 3), 5.44 (dd, 1H, J3,4=6.5,
4,5=15.4 Hz, 4), 5.57 (dt, 1H, J4,5=15.4, J5,6
J
=
J
=
6.6 Hz, 5), 1.99 (dt, 2H, J5,6=6.6, J6,7=7.0 Hz,
6), 1.32 (m, 2H, 7), 1.15–1.30 (m, 20H, 8–17),
0.86 (t, 3H, J17,18=6.8 Hz, 18); 13C NMR
(DMSO-d6, 32°C) l 62.03 (1), 57.19 (2), 72.11 (3),
130.54 (4), 131.22 (5), 31.64 (6), 28.50–28.95 (7–
1
15), 31.20 (16), 21.98 (17), 13.81 (18); H NMR
(CDCl3) l 3.65 (dd, 1H, J1a,1b=11, J1a,2=5.8 Hz,
1a), 3.70 (dd, 1H, J1a,1b=11, J1b,2=3 Hz, 1b),
2.92 (m, 1H, 2), 4.11 (m, 1H, 3), 5.47 (dd, 1H,
J
3,4=7, J4,5=15.4 Hz, 4), 5.76 (dt, 1H, J4,5
=
15.4, J5,6=6.7 Hz, 5), 2.05 (dt, 2H, J5,6=6.7,
1b), 3.64 (dddd, 1H, J1a,2=5, J1b,2=5, J2,2-NH
=
J
6,7=7.3 Hz, 6), 1.37 (m, 2H, 7), 1.20–1.40 (m,
8.7, J2,3=6 Hz, 2), 7.26 (d, 1H, J2,2-NH=8.7 Hz,
2-NH), 3.90 (br dd, 1H, J2,3=6, J3,4=6.8 Hz, 3),
5.38 (dd, 1H, J3,4=6.8, J4,5=15.4 Hz, 4), 5.53
(dt, 1H, J4,5=15.4, J5,6=6.6 Hz, 5), 1.94 (dt, 2H,
20H, 8–17), 0.88 (t, 3H, J17,18=6.9 Hz, 18); 13C
NMR (CDCl3) l 63.33 (1), 56.26 (2), 74.72 (3),
128.83 (4), 134.79 (5), 32.37 (6), 29.19–29.71 (7–
15), 31.94 (16), 22.70 (17), 14.13 (18).
J
5,6=6.6, J6,7=7.5 Hz, 6), 1.19–1.34 (m, 22H,

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
119
7–17), 0.86 (t, 3H, J17,18=6.9 Hz, 18); FA, 2.03
(ddd, 1H, J2a,2b=14.2, J2a,3a=J2a,3b=7.5 Hz, 2a),
2.06 (ddd, 1H, J2a,2b=14.2, J2b,3a=J2b,3b=7.5
Hz, 2b), 1.45 (m, 2H, 3), 1.19–1.34 (m, 24H,
4–15), 0.86 (t, 3H, J15,16=6.9 Hz, 16); 13C NMR
(DMSO-d6, 45°C) l Sph, 60.61 (1), 55.16 (2),
71.19 (3), 131.18 (4), 130.62 (5), 31.48 (6), 28.40–
28.90 (7–15), 31.05 (16), 21.82 (17), 13.63 (18);
FA, 171.87 (NCꢀO), 35.43 (2), 25.14 (3), 28.40–
mol%), concentrated, and chromatographed
(94.5:5:0.5 CH2Cl2/MeOH/TEA). The crude
product
was
rechromatographed
(95:5:0.1
CH2Cl2/MeOH/TEA, 35°C) and micropore
filtered to give homogeneous ceramide 10 (Dong,
1990; Kanemitsu and Sweeley, 1986; Koike et al.,
1986; Ohashi et al., 1988a, 1989; Shah et al.,
1995a; Shibuya et al., 1992) (87 mg, 0.16 mol,
80%) as a white solid after lyophilization from
t-butanol solution and was identical to ceramide
10 prepared from sphingosine 9.
1
28.90 (4–13), 31.05 (14), 21.82 (15), 13.63 (16); H
NMR (CDCl3) l Sph, 3.69 (dd, 1H, J1a,1b=11.3,
J1a,2=3.2 Hz, 1a), 3.94 (dd, 1H, J1a,1b=11.3,
J1b,2=3.7 Hz, 1b), 3.89 (dddd, 1H, J1a,2=3.2,
J1b,2=3.7, J2,2-NH=7.3, J2,3=3.4 Hz, 2), 6.22 (d,
2.11. (2S,3R,4E)-2-Amino-1,3-benzylidene-4-
octadecene-1,3-diol (11)
1H, J2,2-NH=7.3 Hz, 2-NH), 4.30 (dd, 1H, J2,3
3.4, J3,4=6.4 Hz, 3), 5.51 (dd, 1H, J3,4=6.4,
4,5=15.4 Hz, 4), 5.77 (dt, 1H, J4,5=15.4, J5,6
6.7 Hz, 5), 2.03 (dt, 2H, J5,6=6.7, J6,7=7.2 Hz,
6), 1.35 (m, 2H, 7), 1.20–1.34 (m, 20H, 8–17),
0.86 (t, 3H, J17,18=6.8 Hz, 18); FA, 2.21 (t, 2H,
=
To a stirred homogeneous faint yellow solution
of (2S,3R,4E)-azide 7 (80 mg, 0.19 mmol, 100
mol%) in 3 ml of i-propanol was added sodium
borohydride (26 mg, 0.69 mmol, 360 mol%) and
the reaction mixture was refluxed for 48 h. The
reaction mixture was cooled to ambient tempera-
ture, concentrated by rotoevaporation to dryness,
5 ml of H2O was then added and the mixture
extracted with 99.5:0.5 CH2Cl2/TEA (3×10 ml).
The organic extracts were combined, dried, and
concentrated to give a light yellow solid. Chro-
matography (74.5:25:0.5 CH2Cl2/EtOAc/TEA)
first eluted a trace amount of the cis-isomer: TLC
(74.5:25:0.5 CH2Cl2/EtOAc/TEA) Rf 0.17; 1H
NMR (CDCl3) l 7.25–7.55 (m, 5H, Ar), 5.55 (s,
J
=
J
2,3=7.5 Hz, 2), 1.62 (quint, 2H, J2,3=J3,4=7.5
Hz, 3), 1.20–1.34 (m, 24 H, 4–15), 0.86 (t, 3H,
15,16=6.9 Hz, 16); 13C NMR (CDCl3) l Sph,
J
62.56 (1), 54.73 (2), 74.63 (3), 128.98 (4), 134.23
(5), 32.33 (6), 29.22–29.73 (7–15), 31.98 (16),
22.72 (17), 14.10 (18); FA, 173.97 (NCꢀO), 36.92
(2), 25.82 (3), 29.22–29.73 (4–13), 31.98 (14),
22.72 (15), 14.10 (16); IR (KBr, cm−1) 3300 (br,
OH), 1642 (CꢀO); [a]2D6 −6° (c 1.0, 2:1 CHCl3/
MeOH), [a]2D6 −3° (c 0.66, CHCl3) (Lit. (Kane-
mitsu and Sweeley, 1986) [a]2D3 −1.9° (c 1.0,
CHCl3); Lit. (Koike et al., 1986) [a]2D5 −4.1° (c
0.66, CHCl3); Lit. (Ohashi et al., 1988a, 1989) [a]D28
−5.8° (c 0.98, 9:1 CHCl3/MeOH); Lit. (Shibuya
et al., 1992) [a]D23 −5.4° (c 0.9, 9:1 CHCl3/
MeOH)); ESIMS (pos) m/z 538 (M+H)+,
520 (M+H%)+, 264 (W¦)+; MALDIMS m/z 560
(M+Na)+, 538 (M+H)+, 520 (M+H%)+. Anal.
Calcd. for C34H67NO3: C, 75.92; H, 12.55; N,
2.60. Found: C, 76.27; H, 12.56; N, 2.94.
1H, CH6 Ph); Sph, 3.56 (dd, 1H, J1a,1b=11, J1a,2=
11 Hz, 1a), 4.30 (dd, 1H, J1a,1b=11, J1b,2=4.9
Hz, 1b), 2.89 (ddd, 1H, J1a,2=11, J1b,2=4.9,
J2,3=9 Hz, 2), 4.23 (dd, 1H, J2,3=9, J3,4=9 Hz,
3), 5.51 (dd, 1H, J3,4=9, J4,5=11 Hz, 4), 5.79 (dt,
1H, J4,5=11, J5,6=7.4 Hz, 5), 2.20 (m, 2H, 6),
1.20–1.50 (m, 22H, 7–17), 0.88 (t, 3H, J17,18=6.3
Hz, 18). Further elution gave amine 11 (74 mg,
0.19 mmol, 100%) (Byun and Bittman, 1993;
Ohashi et al., 1989, 1988b) as a white solid which
was nearly homogeneous: TLC (74.5:25:0.5
CH2Cl2/EtOAc/TEA) Rf 0.14: m.p. (argon) 48–
49°C; 1H NMR (CDCl3) l 7.25–7.55 (m, 5H, Ar),
2.10.2. From benzylidene ceramide 12
To a stirred solution of benzylidene ceramide 12
(125 mg, 0.200 mmol, 100 mol%) in 9 ml of 1:1
CH2Cl2/MeOH was added p-TsOH–H2O (7.6 mg,
0.040 mmol, 20 mol%) and the reaction mixture
stirred for 14 h. The reaction mixture was
quenched with TEA (74 ml, 54 mg, 0.54 mmol, 270
5.52 (s, 1 H, CH
11.0, J1a,2=10.5 Hz, 1a), 4.28 (dd, 1H, J1a,1b
11.0,
1a,2=10.5, J1b,2=4.9, J2,3=9.4 Hz, 2), 3.81 (dd,
1H, J2,3=9.4, J3,4=7.6 Hz, 3), 5.52 (dd, 1H,
6
Ph); Sph, 3.51 (dd, 1H, J1a,1b
=
=
J1b,2=4.9 Hz, 1b), 2.86 (ddd, 1H,
J

120
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
J
3,4=7.6, J4,5=15.4 Hz, 4), 5.89 (dt, 1H, J4,5
=
=
2H, 3), 1.20–1.40 (m, 24H, 4–15), 0.88 (t, 3H,
15,16=6.4 Hz, 16); 13C NMR (CDCl3) l 137.84,
129.02, 128.29 (×2), 126.36 (×2) [Ar×6];
101.40 (CHPh); Sph, 69.71 (1), 47.04 (2), 81.85
(3), 126.60 (4), 137.03 (5), 32.39 (6), 26.07–29.73
(7–15), 31.97 (16), 22.71 (17), 14.10 (18); FA,
172.91 (NCꢀO), 36.91 (2), 25.76 (3), 26.07–29.73
(4–13), 31.97 (14), 22.71 (15), 14.10 (16); IR
(KBr, cm−1) 3294 (br, NH), 1641 (C=O); [a]2D4
+10.0 (c 1.0, 2:1 CHCl3/MeOH). Anal. Calcd.
for C41H71NO3: C, 78.66; H, 11.43; N, 2.24.
Found: C, 78.93; H, 11.61; N, 2.17.
15.4, J5,6=6.7 Hz, 5), 2.09 (dt, 2H, J5,6=J6,7
J
6.7 Hz, 6), 1.20–1.50 (m, 22H, 7–17), 0.88 (t, 3H,
17,18=6.3 Hz, 18); 13C NMR (CDCl3) l 137.34,
128.97, 128.26 (×2), 126.20 (×2) [Ar×6];
101.25 (CHPh); Sph, 70.97 (1), 48.52 (2), 83.97
J
6
6
(3), 125.98 (4), 138.09 (5), 32.45 (6), 28.87–29.66
(7–15), 31.91 (16), 22.67 (17), 14.09 (18); [a]D25
+52° (c 1.0, 2:1 CHCl3/MeOH). Anal. Calcd. for
C25H41NO2: C, 77.47; H, 10.66; N, 3.61. Found:
C, 77.36; H, 10.69; N, 3.99.
2.12. (2S,3R,4E)-1,3-Benzylidene-2-
(hexadecanoylamido)-4-octadecene-1,3-diol
(benzylidene ceramide) (12)
2.13. General procedure for h-methoxy-h-
(trifluoromethyl)phenylacetamides (13)
To a stirred solution of amine 11 (84 mg, 0.22
mmol, 100 mol%) in 10 ml of anhydrous CH2Cl2
at −19°C (bT, CCl4/N2) was added anhydrous
diisopropylethylamine (0.77 ml, 0.44 mmol, 200
mol%) slowly dropwise. After 10 min, a solution
of palmitoyl chloride (76 mg, 0.28 mmol, 130
mol%, prepared from palmitic acid, a sample of
which had been methylated and determined to be
0.04% myristic acid, 99.81% palmitic acid, and
0.15% stearic acid by gas chromatography using
5% DEGS-PS on 100/120 supelcoport at 160°C)
in 5 ml of CH2Cl2 was then added over 5 min.
After 30 min, the reaction was warmed to ambient
temperature. The reaction was complete by ana-
lytical TLC after an additional 90 min. The reac-
To a stirred solution of sphingosine 9 (100
mol%) and TEA (400 mol%) in CH2Cl2 was
added a solution of MTPA chloride in CH2Cl2.
After 1 h, the homogeneous, clear, and colorless
reaction mixture was concentrated. The product
was then chromatographed (95:5:0.5 CHCl3/
MeOH/TEA), and yields were in the range of
90–100%. It should be noted that the (aR)-
MTPA chloride reagent yields the (aS)-amide 13
configuration, and likewise for the enantiomeric
(aS)-MTPA chloride Mosher’s reagent.
2.13.1. (hS,2S,3R)-2-[h-Methoxy-h-
(trifluoromethyl)phenylacetamido]-4-octadecene-
1,3-diol ((hS)-MTPA-D-erythro-Sph)
tion
mixture
was
concentrated
and
((hS,2S,3R)-13)
1
chromatographed (94.75:5:0.25 CH2Cl2/EtOAc/
TEA) to give 1,3-benzylidene ceramide 12 (133
mg, 0.21 mmol, 95%) (Ohashi et al., 1989, 1988b)
as a white solid which was homogeneous: TLC
(92:7.5:0.5 CH2Cl2/EtOAc/TEA) Rf 0.35; m.p.
(argon) 112°C (Lit. (Ohashi et al., 1989) m.p.
77.3–78.9°C); 1H NMR (CDCl3) l 7.49 (br d, 2H,
J=7.7 Hz, Ar), 7.32–7.40 (m, 3H, Ar), 5.52 (s,
TLC (95:5:0.5 CHCl3/MeOH/TEA) Rf 0.30; H
NMR (CDCl3) l 3.43 (s, 3H, OMe), 7.35–7.60
(m, 6H, Ar, 2-NH), 3.71 (dd, 1H, J1a,1b=11.4,
J
J
J
1a,2=3.2 Hz, 1a), 4.01 (dd, 1H, J1a,1b=11.4,
1b,2=3.5 Hz, 1b), 3.95 (m, 1H, 2), 4.40 (dd, 1H,
2,3=3.8, J3,4=6.5 Hz, 3), 5.54 (dd, 1H, J3,4
=
6.5, J4,5=15.4 Hz, 4), 5.82 (dt, 1H, J4,5=15.4,
5,6=6.6 Hz, 5), 2.05 (dt, 2H, J5,6=6.6, J6,7=7.3
J
1H, CH
J
J
6
Ph); Sph, 3.60 (dd, 1H, J1a,1b=10.8,
1a,2=10 Hz, 1a), 4.39 (dd, 1H, J1a,1b=10.8,
1b,2=4.1 Hz, 1b), 3.95–4.15 (m, 2H, 2, 3), 5.08
Hz, 6), 1.37 (m, 2H, 7), 1.26 (br s, 20H, 8–17),
0.88 (t, 3H, J17,18=6.9 Hz, 18).
(br s, 1H, 2-NH), 5.53 (dd, 1H, J3,4=6.9, J4,5
=
2.13.2. (hS,2R,3S)-2-[h-Methoxy-h-
15.4 Hz, 4), 5.82 (dt, 1H, J4,5=15.4, J5,6=6.6 Hz,
5), 2.04 (dt, 2H, J5,6=6.6, J6,7=6.8 Hz, 6), 1.20–
1.50 (m, 22H, 7–17), 0.88 (t, 3H, J17,18=6.4 Hz,
18); FA, 2.13 (t, 2H, J2,3=7.4 Hz, 2), 1.59 (m,
(trifluoromethyl)phenylacetamido]-4-octadecene-
1,3-diol ((hS)-MTPA-L-erythro-Sph)
((hS,2R,3S)-13)
TLC (95:5:0.5 CHCl3/MeOH/TEA) Rf 0.30; H
1

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
121
NMR (CDCl3) l 3.43 (s, 3H, OMe), 7.35–7.60
2.14. (hS,2S,3R)-1,3-Di-(trimethylsilyl)-2-[h-
methoxy-h-(trifluoromethyl)phenylacetamido]-4-
(m, 5H, Ar), 3.71 (dd, 1H, J1a,1b=11.4, J1a,2=3.2
Hz, 1a), 4.01 (dd, 1H, J1a,1b=11.4, J1b,2=3.5 Hz,
1b), 3.95 (m, 1H, 2), 7.51 (d, 1H, J2,2-NH=8 Hz,
2-NH), 4.32 (dd, 1H, J2,3=4.2, J3,4=6.5 Hz, 3),
5.51 (dd, 1H, J3,4=6.5, J4,5=15.4 Hz, 4), 5.78
(dt, 1H, J4,5=15.4, J5,6=6.7 Hz, 5), 2.03 (dt, 2H,
octadecene-1,3-diol ((hS)-MTPA-D-erythro-Sph-
TMS2) ((hS,2S,3R)-14)
To a stirred solution of diol (aS,2S,3R)-13, 400
mol% of TEA, and 200 mol% of imidazole in
CH2Cl2 at 0°C was added 400 mol% of
trimethylsilylchloride. The cloudy reaction was
stirred for 1 h, then warmed to room temperature.
The mixture became homogeneous and was
stirred for an additional 30 min. The solvent was
removed and the mixture chromatographed (80:20
hex/EtOAc) to elute the product (aS,2S,3R)-14
(Rf 0.66) followed by (aS,2S,3R)-1-(trimethylsi-
lyl)-2-[a-methoxy-a-(trifluoromethyl)phenylaceta-
mido]-4-octadecene-1,3-diol (Rf 0.48), (aS,2S,3R)-
3-(trimethylsilyl)-2-[a-methoxy-a-(trifluoromethyl)
phenylacetamido]-4-octadecene-1,3-diol (Rf 0.37),
and starting material (aS,2S,3R)-13 (Rf 0.04) by
TLC (80:20 hex/EtOAc).
J
5,6=6.7, J6,7=7.2 Hz, 6), 1.38 (m, 2H, 7),
1.26 (br s, 20H, 8–17), 0.88 (t, 3H, J17,18=6.9 Hz,
18).
2.13.3. (hR,2S,3R)-2-[h-Methoxy-h-
(trifluoromethyl)phenylacetamido]-4-octadecene-
1,3-diol ((hR)-MTPA-D-erythro-Sph)
((hR,2S,3R)-13)
(aR)-MTPA- -erythro-Sph (aR,2S,3R)-13 was
D
identical to its enantiomer (aS)-MTPA-
Sph (aS,2R,3S)-13.
L-erythro-
2.13.4. (hS,2S,3S)-2-[h-Methoxy-h-
(trifluoromethyl)phenylacetamido]-4-octadecene-
1,3-diol ((hS)-MTPA-L-threo-Sph)
((hS,2S,3S)-13)
2.14.1. (hS,2R,3S)-1,3-Di-(trimethylsilyl)-2-[h-
methoxy-h-(trifluoromethyl)phenylacetamido]-4-
1
TLC (95:5:0.5 CHCl3/MeOH/TEA) Rf 0.30; H
NMR (CDCl3) l 3.42 (s, 3H, OMe), 7.35–7.60
(m, 6H, Ar, 2-NH), 3.82 (dd, 1H, J1a,1b=11.2,
octadecene-1,3-diol ((hS)-MTPA-L-erythro-Sph-
TMS2) ((hS,2R,3S)-14)
J
J
J
1a,2=4.6 Hz, 1a), 3.85 (dd, 1H, J1a,1b=11.2,
1b,2=4.3 Hz, 1b), 3.99 (m, 1H, 2), 4.56 (dd, 1H,
The diol (aS,2R,3S)-13 was derivatized by the
method previously detailed and chromatographed
(80:20 hex/EtOAc) to elute the product
(aS,2R,3S)-14 (Rf 0.65) followed by (aS,2R,3S)-1-
(trimethylsilyl)-2-[a-methoxy-a-(trifluoromethyl)-
phenylacetamido]-4-octadecene-1,3-diol (Rf 0.43),
(aS,2R,3S)-3-(trimethylsilyl)-2-[a-methoxy-a-(tri-
fluoromethyl)phenylacetamido]-4-octadecene-1,3-
diol (Rf 0.37), and starting material (aS,2R,3S)-13
(Rf 0.04) by TLC (80:20 hex/EtOAc).
2,3=2.6, J3,4=6.3 Hz, 3), 5.50 (dd, 1H, J3,4
=
6.3, J4,5=15.5 Hz, 4), 5.78 (dt, 1H, J4,5=15.5,
5,6=6.8 Hz, 5), 2.04 (dt, 2H, J5,6=6.8, J6,7=7.2
J
Hz, 6), 1.36 (m, 2H, 7), 1.26 (br s, 20H, 8–17),
0.88 (t, 3H, J17,18=6.9 Hz, 18).
2.13.5. (hS,2R,3R)-2-[h-Methoxy-h-
(trifluoromethyl)phenylacetamido]-4-octadecene-
1,3-diol ((hS)-MTPA-D-threo-Sph)
((hS,2R,3R)-13)
2.15. (hS,2S,3R)-1,3-Diacetyl-2-[h-methoxy-h-
(trifluoromethyl)phenylacetamido]-4-octadecene-
1
TLC (95:5:0.5 CHCl3/MeOH/TEA) Rf 0.30; H
NMR (CDCl3) l 3.43 (s, 3H, OMe), 7.35–7.60
(m, 6H, Ar, 2-NH), 3.85 (dd, 1H, J1a,1b=11,
1,3-diol ((hS)-MTPA-D-erythro-Sph-Ac2)
((hS,2S,3R)-15)
J
1a,2=4 Hz, 1a), 3.88 (dd, 1H, J1a,1b=11, J1b,2
=
=
4 Hz, 1b), 3.99 (m, 1H, 2), 4.44 (dd, 1H, J2,3
To a stirred solution of diol (aS,2S,3R)-13 and
5 mol% of freshly recrystallized (Patel and Spar-
row, 1979) 4-pyrrolidinopyridine in pyridine was
added 700 mol% Ac2O. After 3 h, the solvent was
removed and the mixture chromatographed (80:20
hex/EtOAc) to give the homogeneous product
2.8, J3,4=6.3 Hz, 3), 5.35 (dd, 1H, J3,4=6.3,
4,5=15.5 Hz, 4), 5.70 (dt, 1H, J4,5=15.5, J5,6
6.6 Hz, 5), 1.95 (dt, 2H, J5,6=6.6, J6,7=6.9 Hz,
J
=
6), 1.25–1.35 (m, 22H, 7–17), 0.88 (t, 3H, J17,18
=
6.9 Hz, 18).

122
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
(aS,2S,3R)-15 as a viscous liquid: TLC (90:10
orless liquid that was homogeneous: TLC (87:12:1
hex/EtOAc/TEA) Rf 0.21; 1H NMR (CDCl3) l
7.20–7.48 (m, 15H, Ar), 3.30 (d, 2H, J1,2=5.4
Hz, 1), 3.52 (dt, 1H, J1,2=5.4, J2,3=5 Hz, 2),
4.18 (dd, 1H, J2,3=5, J3,4=7.1, Hz, 3), 5.31 (dd,
1H, J3,4=7.1 J4,5=15.4 Hz, 4), 5.65 (dt, 1H,
1
CH2Cl2/EtOAc) Rf 0.52; H NMR (CDCl3) l 1.94
(s, 3H, Ac), 2.06 (s, 3H, Ac), 3.43 (s, 3H, OMe),
7.30–7.60 (m, 5H, Ar), 4.09 (dd, 1H, J1a,1b=11.6,
J
J
J
J
1a,2=4.2 Hz, 1a), 4.19 (dd, 1H, J1a,1b=11.6,
1b,2=7.2 Hz, 1b), 4.52 (m, 1H, 2), 6.89 (d, 1H,
2,2-NH=9.4 Hz, 2-NH), 5.34 (dd, 1H, J2,3=5.6,
J4,5=15.4, J5,6=6.6 Hz, 5), 1.95 (dt, 2H, J5,6=
3,4=7.4 Hz, 3), 5.40 (dd, 1H, J3,4=7.4, J4,5
=
6.6, J6,7=6 Hz, 6), 1.20–1.40 (m, 22H, 7–17),
15.1 Hz, 4), 5.84 (dt, 1H, J4,5=15.1, J5,6=6.7 Hz,
5), 2.04 (dt, 2H, J5,6=6.7, J6,7=7 Hz, 6),
1.36 (m, 2H, 7), 1.26 (br s, 20H, 8–17), 0.88 (t,
3H, J17,18=6.9 Hz, 18); 19F NMR (CDCl3) see
Fig. 2.
0.88 (t, 3H, J17,18=6.3 Hz, 18); 13C NMR
(CDCl3) l 87.50 (C6 Ph3); 143.59 (×3), 128.66
(×6), 127.96 (×6), 127.22 (×3) [Ar×18]; Sph,
63.46 (1), 65.93 (2), 73.09 (3), 127.73 (4), 135.33
(5), 31.94 (6), 28.94–29.70 (7–15), 32.24 (16),
25
22.70 (17), 14.09 (18); [a]D 0.0° (c 2.0, CHCl3)
20
2.15.1. (hR,2S,3R)-1,3-Diacetyl-2-[h-methoxy-h-
(trifluoromethyl)phenylacetamido]-4-octadecene-
(Lit. (Zimmermann and Schmidt, 1988) [a]D
−
25
2.4° (c 2, CHCl3); Lit. (Mori et al., 1990) [a]D
1,3-diol ((hR)-MTPA-D-erythro-Sph-Ac2)
+6.6° (c 1.5, CHCl3)).
((hR,2S,3R)-15)
The diol (aR,2S,3R)-13 was acylated by the
method previously detailed and chromatographed
(90:10 CH2Cl2/EtOAc) to give the homogeneous
product (aR,2S,3R)-15 as a viscous liquid: TLC
2.17. (2S,3R,4E)-2-Azido-3-benzoyl-1-
(triphenylmethyl)-4-octadecene-1,3-diol (17)
To a stirred solution of 1-tritylazidosphingosine
16 (0.684 g, 1.20 mmol, 100 mol%), diisopropy-
lethylamine (1.04 ml, 0.772 g, 5.97 mmol, 500
mol%) and freshly recrystallized (Patel and Spar-
row, 1979) 4-pyrrolidinopyridine (0.178 g, 1.20
mmol, 100 mol%) in 10 ml of anhydrous PhMe
was added benzoyl chloride (0.278 ml, 0.337 g,
2.40 mmol, 200 mol%) dropwise over 1 min. After
16 h, the reaction mixture was concentrated and
filtered through a short column (95:5 hex/EtOAc).
The crude product was re-chromatographed to
give 3-benzoyl-1-tritylazidosphingosine 17 (0.635
g, 0.945 mmol, 79%) (Schmidt and Zimmermann,
1986b; Zimmermann and Schmidt, 1988) as a
clear, colorless, viscous liquid which was homoge-
1
(90:10 CH2Cl2/EtOAc) Rf 0.52; H NMR (CDCl3)
l 1.99 (s, 3H, Ac), 2.06 (s, 3H, Ac), 3.39 (s, 3H,
OMe), 7.30–7.60 (m, 5H, Ar), 4.13 (dd, 1H,
J
J
1a,1b=11.6, J1a,2=4.1 Hz, 1a), 4.27 (dd, 1H,
1a,1b=11.6, J1b,2=7.4 Hz, 1b), 4.51 (m, 1H, 2),
6.94 (d, 1H, J2,2-NH=9.4 Hz, 2-NH), 5.30–5.36
(m, 2H, 3, 4), 5.76 (m, 1H, 5), 1.98 (dt, 2H,
J
5,6=7, J6,7=7 Hz, 6), 1.30 (m, 2H, 7), 1.26 (br s,
20H, 8–17), 0.88 (t, 3H, J17,18=6.9 Hz, 18); 19F
NMR (CDCl3) see Fig. 2
2.16. (2S,3R,4E)-2-Azido-1-(triphenylmethyl)-4-
octadecene-1,3-diol (16)
1
To a mixture of azidosphingosine 8 (0.430 g,
1.32 mmol, 100 mol%) and diisopropylethylamine
(0.35 ml, 0.26 g, 2.0 mmol, 150 mol%) in 4 ml of
dry CH2Cl2 was added trityl chloride (0.393 g,
1.41 mmol, 107 mol%) and the reaction mixture
stirred for 26 h. The reaction mixture was concen-
trated and filtered through a short column
(87:12:1 hex/EtOAc/TEA). The crude product
was re-chromatographed to give 1-tritylazidosph-
ingosine 16 (0.684 g, 1.20 mmol, 91%) (Mori et
al., 1990; Schmidt and Zimmermann, 1986b; Zim-
mermann and Schmidt, 1988) as a clear and col-
neous: TLC (90:10 hex/EtOAc) Rf 0.41; H NMR
(CDCl3) l 7.95 (d, 2H, J=7.9 Hz, Ar), 7.55 (t,
1H, J=7.1 Hz, Ar), 7.19–7.45 (m, 17H, Ar); Sph,
3.20 (dd, 1H, J1a,1b=9.7, J1a,2=5.3 Hz, 1a), 3.29
(dd, 1H, J1a,1b=9.7, J1b,2=6.7 Hz, 1b), 3.86
(ddd, 1H, J1a,2=5.3, J1b,2=6.7, J2,3=4.8 Hz, 2),
5.63 (dd, 1H, J2,3=4.8, J3,4=7.8 Hz, 3), 5.42 (dd,
1H, J3,4=7.8, J4,5=15.3 Hz, 4), 5.82 (dt, 1H,
J4,5=15.3, J5,6=6.7 Hz, 5), 1.96 (dt, 2H, J5,6=
6.7, J6,7=6.2 Hz, 6), 1.20–1.40 (m, 22H, 7–17),
0.88 (t, 3H, J17,18=6.3 Hz, 18); 13C NMR
(CDCl3) l 165.16 (OCꢀO), 87.25 (C6 Ph3); 143.48

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
123
(×3), 133.08, 130.00, 129.74 (×2), 128.60 (×6),
128.38 (×2), 127.90 (×6), 127.14 (×3) [Ar×
24]; Sph, 62.83 (1), 64.48 (2), 74.74 (3), 123.13 (4),
138.32 (5), 32.30 (6), 28.68–29.68 (7–15), 31.94
(16), 22.71 (17), 14.14 (18); IR (NaCl, cm−1) 2095
D-glucose (3.75 g, 10.8 mmol, 100 mol%) (Ex-
coffier et al., 1975) and Cl3CCN (4.33 ml, 6.24 g,
43.2 mmol, 400 mol%, distilled from CaH2) in 20
ml of dry CH2Cl2 was added NaH (0.518 g, 0.216
mol, 200 mol%) over 1 min. Many related subse-
quent experiments have demonstrated 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU) to be an equally
effective base. The vigorous reaction was stirred
for an additional 25 min, then suction filtered
through silica gel on fritted glass and was washed
with additional CH2Cl2. The filtrates were com-
bined, concentrated, and chromatographed
(Et2O). The crude product was carefully re-chro-
matographed to give a-trichloroacetimidate a-19
(2.88 g, 5.84 mmol, 54%) (Cook et al., 1984;
Schmidt and Kla¨ger, 1985; Schmidt and Michel,
1980; Schmidt et al., 1984; Schmidt and Stumpp,
1983; Schmidt and Zimmermann, 1988) as a col-
orless viscous semi-solid which contained no trace
of the b-anomer and was homogeneous: TLC
(Et2O) Rf 0.52, (1:1 hex/EtOAc) Rf 0.28; 1H NMR
(CDCl3) l 8.70 (s, 1H, NH); 2.08, 2.05, 2.03, 2.02
[(s, 3H, CH3)×4]; Glc, 6.56 (d, 1H, J1%,2%=3.7 Hz,
1%), 5.13 (dd, 1H, J1%,2%=3.7, J2%,3%=10.2 Hz, 2%),
5.57 (dd, 1H, J2%,3%=10.2, J3%,4%=9.5 Hz, 3%), 5.18
(dd, 1H, J3%,4%=9.5, J4%,5%=10.3 Hz, 4%), 4.21 (ddd,
1H, J4%,5%=10.3, J5%,6a%=1.9, J5%,6b%=4.2 Hz, 5%),
4.13 (dd, 1H, J5%,6a%=1.9, J6a%,6b%=12.4 Hz, 6a%),
4.27 (dd, 1H, J5%,6b%=4.2, J6a%,6b%=12.4 Hz, 6b%);
13C NMR (CDCl3) l 170.53, 169.97, 169.12,
169.47 [CꢀO×4]; 20.64 (×2), 20.55, 20.41
[CH3×4]; 160.77 (CꢀN), 90.66 (CCl3); Glc, 92.88
(1%), 69.98, 69.84, 69.70 (2%, 3%, 5%), 67.76 (4%), 61.35
(6%).
25
(N3), 1723 (CꢀO); [a]D −17.4° (c 6.99, CHCl3)
20
(Lit. (Zimmermann and Schmidt, 1988) [a]D
16.2° (c 7, CHCl3)).
−
2.18. (2S,3R,4E)-2-Azido-3-benzoyl-4-
octadecene-1,3-diol (18)
To a stirred solution of 17 (0.585 g, 0.871
mmol, 100 mol%) in 4.5 ml of 3:2 PhMe/MeOH
was added F3B–OEt2 (0.117 ml, 0.135 g, 9.51
mmol, 109 mol%) dropwise over 5 min. After 5 h,
the reaction was quenched by the addition of 10
ml of aq NaHCO3 (0.55 g, 6.5 mmol, 750 mol%)
followed by 35 ml of CH2Cl2. The organic phase
was washed with 20 ml of H2O, dried, concen-
trated, and filtered through a short column (80:20
hex/EtOAc). The crude product was carefully re-
chromatographed (gradient of 86:14 hex/EtOAC
to 50:50 hex/EtOAc). The rearrangement
byproduct eluted just ahead of the 3-benzoylazi-
dosphingosine 18 (0.346 g, 0.805 mmol, 92%) (Ito
et al., 1988, 1989; Schmidt and Zimmermann,
1986b; Zimmermann and Schmidt, 1988), which
was a homogeneous clear and colorless liquid, but
prone to rearrangement, and was used promptly
for glycosidic coupling reactions: TLC (80:20 hex/
1
EtOAc) Rf 0.24; H NMR (CDCl3) l 8.06 (br d,
2H, J=7.9 Hz, Ar), 7.55 (br t, 1H, J=7.1 Hz,
Ar), 7.45 (br dd, 2H, J=7.9, 7.1 Hz, Ar); Sph,
3.55–3.90 (m, 3H, 1a, 1b, 2), 5.54–5.67 (m, 2H, 3,
4), 5.95 (m, 1H, 5), 2.07 (dt, 2H, J5,6=7.2, J6,7
6.6 Hz, 6), 1.20–1.40 (m, 22H, 7–17), 0.88 (t, 3H,
17,18=6.4 Hz, 18); 13C NMR (CDCl3) l 165.52
=
2.20. (2S,3R,4E)-2%,3%,4%,6%-Tetraacetyl-i-D-
glucopyranosyl-(1%l1)-2-azido-3-benzoyl-4-
octadecene-1,3-diol (20)
J
(OCꢀO); 133.37, 129.84 (×3), 128.53 (×2) [Ar×
6]; Sph, 62.05 (1), 66.26 (2), 74.62 (3), 123.38 (4),
138.83 (5), 32.39 (6), 28.72–29.68 (7–15), 31.95
(16), 22.71 (17), 14.13 (18); IR (NaCl, cm−1) 3450
(br, OH), 2100 (N3), 1721 (CꢀO).
A solution of 3-benzoylazidosphingosine 18
(0.346 g, 0.805 mol, 100 mol%), trichloroacetimi-
date a-19 (0.596 g, 1.21 mmol, 150 mol%), and
,
0.15 g of dry 4 A molecular sieves in 2.5 ml of
CH2Cl2 and 10 ml of hexanes was stirred for 30
min. A solution of F3B-OEt2 (30 ml, 35 mg, 0.25
mmol, 31 mol%) in 2.4 ml of CH2Cl2 was then
added dropwise over 10 min. After 6 h, the reac-
tion mixture was quenched with 13 ml of satd aq
2.19. 2,3,4,6-Tetraacetyl-h-D-
glucopyranosyltrichloroacetimidate (h-19)
To a stirred solution of 2,3,4,6-tetraacetyl-a/b-

124
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
2.21. (2S,3R,4E)-i-D-Glucopyranosyl-
(1%l1)-2-azido-4-octadecene-1,3-diol (21)
NaHCO3 and an additional 20 ml of CH2Cl2.
The organic phase was combined with addi-
tional CH2Cl2 extracts (3×20 ml), dried, con-
centrated, and filtered through a short column
(70:30 hex/EtOAc). The crude product was re-
chromatographed (70:30 hex/EtOAc) to recover
starting material as 1-acetyl-3-benzoylazidosphin-
gosine [0.109 g, 0.239 mmol, TLC (60:40 hex/
EtOAc) Rf 0.65, which was hydrolyzed to give
60 mg, 0.18 mmol, 22 mol% of pure azidosphin-
To a stirred solution of fully protected azi-
docerebroside 20 (56 mg, 7.4×10−5 mol, 100
mol%) in 0.20 ml of 85:15 CH2Cl2/MeOH was
added a solution of catalytic NaOMe (1.8×
10−5 mol, 25 mol%) in 0.14 ml of MeOH. The
reaction was followed by TLC (85:15 CH2Cl2/
MeOH, Rf of 20 is 0.79, Rf of (2S,3R,4E)-b-D-
gosine 8], followed by
a small amount of
glucopyranosyl-(1%l1)-2-azido-3-benzoyl-4-octa-
decene-1,3-diol is 0.45, Rf of 21 is 0.25). The
acetate ester methanolyses were rapid, and the
benzoate ester methanolysis was complete after
6 h. The reaction mixture was neutralized with
20 mg of Amberlite IRC-50 resin which was re-
moved by filtration through a plug of glass
wool. The filtrate was concentrated and chro-
matographed (82.5:17.5 CH2Cl2/MeOH) to give
azidocerebroside 21 (34 mg, 7.0×10−5 mol,
95%) (Schmidt and Zimmermann, 1986b, 1988;
Zimmermann et al., 1988) as a white solid that
was homogeneous: TLC (82.5:17.5 CH2Cl2/
MeOH) Rf 0.38; 1H NMR (DMSO-d6) l Glc,
4.12 (d, 1H, J1%,2%=7.8 Hz, 1%), 2.95 (ddd, 1H,
starting material 18 [TLC (60:40 hex/EtOAc)
Rf 0.53], followed by the nearly pure product
20. Trace amounts of a slightly higher Rf
impurity and a slightly lower Rf impurity were
removed by a single injection onto a prep
HPLC column (60:40 hex/EtOAc) to give 20
(0.343 g, 0.451 mmol, 56%) (Zimmermann et al.,
1988) as an amorphous waxy white solid that
was homogeneous: TLC (60:40 hex/EtOAc) Rf
0.35; m.p. 44–46°C (Lit. (Zimmermann et al.,
1
1988) m.p. 45°C); H NMR (CDCl3) l 8.05 (br
d, 2H, J=8.3 Hz, Ar), 7.57 (t, 1H, J=7.4 Hz,
Ar), 7.45 (dd, 2H, J=8.3, 7.4 Hz, Ar); 2.09,
2.04, 2.02, 2.01 [(s, 3H, CH3)×4]; Glc, 4.55 (d,
1H, J1%,2%=7.9 Hz, 1%), 5.26 (dd, 1H, J1%,2%=7.9,
J2%,3%=9.6 Hz, 2%), 5.20 (dd, 1H, J2%,3%=9.6,
J3%,4%=9.3 Hz, 3%), 5.09 (dd, 1H, J3%,4%=9.3,
J4%,5%=10.1 Hz, 4%), 3.69 (ddd, 1H, J4%,5%=10.1,
J5%,6a%=2.3, J5%,6b%=4.7 Hz, 5%), 4.12 (dd, 1H,
J5%,6a%=2.3, J6a%,6b%=12.3 Hz, 6a%), 4.21 (dd, 1H,
J5%,6b%=4.7, J6a%,6b%=12.3 Hz, 6b%); Sph, 3.60 (dd,
1H, J1a,1b=9.7, J1a,2=5.2 Hz, 1a), 3.90 (dd,
1H, J1a,1b=9.7, J1b,2=6.6 Hz, 1b), 3.92–3.96
(m, 1H, 2), 5.59 (dd, 1H, J2,3=3.7, J3,4=8.1
Hz, 3), 5.55 (dd, 1H, J3,4=8.1, J4,5=14.7 Hz,
4), 5.93 (dt, 1H, J4,5=14.7, J5,6=6.7 Hz, 5),
2.00–2.10 (m, 2H, 6), 1.38 (m, 2H, 7), 1.19–
1.35 (m, 20H, 8–17), 0.88 (t, 3H, J17,18=6.9
Hz, 18); 13C NMR (CDCl3) l 133.18, 129.96,
129.74 (×2), 128.44 (×2) [Ar×6]; 170.55,
170.23, 169.30, 169.21, 165.07 [CꢀO×5]; 20.61
(×2), 20.55 (×2) [CH3×4]; Glc, 100.53 (1%),
71.08 (2%), 72.78 (3%), 68.33 (4%), 72.02 (5%), 61.84
(6%); Sph, 68.15 (1), 63.49 (2), 74.66 (3), 122.75
(4), 139.03 (5), 32.36 (6), 28.75–29.63 (7–15),
31.90 (16), 22.66 (17), 14.07 (18).
J1%,2%=7.8, J2%,2%-OH, =4.8 J2%,3%=9.5 Hz, 2%),
3.00–3.18 (m, 3H, 3%, 4%, 5%), 3.38–3.68 (m, 2H,
6a%, 6b%); Sph, 3.38–3.68 (m, 2H, 1a, 2), 3.71
(dd, 1H, J1a,1b=10, J1b,2=8 Hz, 1b), 4.10 (ddd,
1H, J2,3=6, J3,3-OH=5, J3,4=6.9 Hz, 3), 5.43
(dd, 1H, J3,4=6.9, J4,5=15.4 Hz, 4), 5.66 (dt,
1H, J4,5=15.4, J5,6=6.6 Hz, 5), 1.99 (dt, 2H,
J5,6=6.6, J6,7=6.7 Hz, 6), 1.18–1.35 (m, 22H,
7–17), 0.85 (t, 3H, J17,18=6.7 Hz, 18); 1H
NMR (2:1 CDCl3/CD3OD) l Glc, 4.31 (d, 1H,
J1%,2%=7.5 Hz, 1%), 3.20–3.50 (m, 4H, 2%, 3%, 4%,
5%), 3.49–3.80 (m, 1H, 6a%), 3.87 (dd, 1H,
J5%,6b%=2.2, J6a%,6b%=12.1 Hz, 6b%); Sph, 3.49–
3.80 (m, 2H, 1a, 2), 3.98 (dd, 1H, J1a,1b
=10.4, J1b,2=6.5 Hz, 1b), 4.20 (dd, 1H, J2,3
6.2, J3,4=7.3 Hz, 3), 5.51 (dd, 1H, J3,4=7.3,
=
J
J
4,5=15.4 Hz, 4), 5.80 (dt, 1H, J4,5=15.4,
5,6=6.6 Hz, 5), 2.08 (dt, 2H, J5,6=6.6, J6,7
=
6.7 Hz, 6), 1.15–1.50 (m, 22H, 7–17), 0.88 (t,
3H, J17,18=6.1 Hz, 18); ESIMS (neg) m/z 486
(M–H)−.

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
125
2.22. (2S,3R,4E)-i-
(1%l1)-2-amino-4-octadecene-1,3-diol (glucosyl-
sphingosine, GlcSph) (22)
D
-Glucopyranosyl-
re-chromatographed (80:20 CH2Cl2/MeOH) to
give pure cerebroside 23 (37 mg, 5.3×10−5 mol,
91%) (Schmidt and Kla¨ger, 1985; Schmidt and
Zimmermann, 1986b, 1988; Shibuya et al., 1992;
Zimmermann et al., 1988) as a white solid that was
homogeneous: TLC (80:20 CH2Cl2/MeOH) Rf
0.52; m.p. 147–150°C (Lit. (Shibuya et al., 1992)
m.p. 179–180.5°C (needles from MeOH/H2O)); 1H
Through a stirred solution of azide 21 (32 mg,
6.6×10−5 mol, 100 mol%) in 1:1 pyridine/water
was bubbled H2S for 2 d. The solvent was removed
and the residue chromatographed (60:30:5 CHCl3/
MeOH/aq NH4OH). The crude product was re-
chromatographed (60:30:5 CHCl3/MeOH/aq
NH4OH) to give amine 22 (27 mg, 5.8×10−5 mol,
88%) (Schmidt and Zimmermann, 1986b, 1988;
Zimmermann et al., 1988) as a white solid which
was homogeneous: TLC (60:30:5 CHCl3/MeOH/aq
NMR (DMSO-d6, 32°C) l Glc, 4.08 (d, 1H, J1%,2%
=
7.7 Hz, 1%), 2.98 (ddd, 1H, J1%,2%=7.7, J2%,2%-OH=3.7,
J2%,3%=9.1, Hz, 2%), 4.96 (d, 1H, J2%,2%-OH=3.7 Hz,
2%-OH), 3.14 (ddd, 1H, J2%,3%=9.1, J3%,3%-OH=4.8,
J3%,4%=8.2 Hz, 3%), 4.86 (d, 1H, J3%,3%-OH=4.8 Hz,
3%-OH), 3.04 (ddd, 1H, J3%,4%=8.2, J4%,4%-OH=4.9,
J4%,5%=9.6 Hz, 4%), 4.84 (d, 1H, J4%,4%-OH=4.9 Hz,
4%-OH), 3.09 (ddd, 1H, J4%,5%=9.6, J5%,6a%=5,
J5%,6b%=1.7 Hz, 5%), 3.44 (ddd, 1H, J5%,6a%=5, J6a%,6%-
OH=5.8, J6a%,6b%=11.6 Hz, 6a%), 3.67 (ddd, 1H,
J5%,6b%=1.7, J6b%,6%-OH=5.8, J6a%,6b%=11.6 Hz, 6b%),
4.44 (t, 1H, J6a%,6%-OH=J6b%,6%-OH=5.8 Hz, 6%-OH);
Sph, 3.42 (dd, 1H, J1a,1b=10.1, J1a,2=3.6 Hz, 1a),
3.96 (dd, 1H, J1a,1b=10.1, J1b,2=4.9 Hz, 1b), 3.78
(dddd, 1H, J1a,2=3.6, J1b,2=4.9, J2,2-NH=9.1,
1
NH4OH) Rf 0.29; H NMR (2:1 CDCl3/CD3OD)
l Glc, 4.30 (d, 1H, J1%,2%=7.5 Hz, 1%), 3.22–3.48 (m,
4H, 2%, 3%, 4%, 5%), 3.71 (dd, 1H, J5%,6a%=4, J6a%,6b%=12
Hz, 6a%), 3.87 (m, 1H, 6b%); Sph, 3.82 (dd, 1H,
J1a,1b=10, J1a,2=3 Hz, 1a), 3.92 (dd, 1H, J1a,1b
=
10, J1b,2=7 Hz, 1b), 3.10 (m, 1H, 2), 4.14 (dd, 1H,
J
J
2,3=5.5, J3,4=7.2 Hz, 3), 5.44 (dd, 1H, J3,4=7.2,
4,5=15.3 Hz, 4), 5.81 (dt, 1H, J4,5=15.3, J5,6
=
6.6 Hz, 5), 2.08 (dt, 2H, J5,6=6.6, J6,7=6.7 Hz, 6),
1.15–1.50 (m, 22H, 7–17), 0.88 (t, 3H, J17,18=6.3
Hz, 18).
J2,3=8.1 Hz, 2), 7.44 (d, 1H, J2,2-NH=9.1, 2-NH),
3.89 (ddd, 1H, J2,3=8.1, J3,3-OH=5.6, J3,4=7.1,
Hz, 3), 4.82 (d, 1H, J3,3-OH=5.6 Hz, 3-OH), 5.36
(dd, 1H, J3,4=7.1, J4,5=15.4 Hz, 4), 5.54 (dt, 1H,
2.23. (2S,3R,4E)-i-D-Glucopyranosyl-
(1%l1)-2-(hexadecanoylamido)-4-octadecene-1,3-
diol (cerebroside, glucosylceramide, GlcCer) (23)
J
4,5=15.4, J5,6=6.6 Hz, 5), 1.94 (m, 2H, 6), 1.28
(m, 2H, 7), 1.18–1.27 (m, 20H, 8–17), 0.85 (t, 3H,
17,18=6.9 Hz, 18); FA, 2.03 (t, 2H, J2,3=7.4 Hz,
J
2), 1.45 (m, 2H, 3), 1.18–1.27 (m, 24H, 4–15), 0.85
(t, 3H, J15,16=6.9 Hz, 16); 13C NMR (DMSO-d6,
32°C) l Glc, 103.76 (1%), 73.45 (2%), 76.32 (3%), 70.01
(4%), 76.79 (5%), 61.01 (6%); Sph, 68.97 (1), 53.03 (2),
70.70 (3), 131.26 (4), 131.34 (5), 31.66 (6), 28.62–
29.00 (7–15), 31.21 (16), 21.99 (17), 13.79 (18); FA,
171.78 (NCꢀO), 35.55 (2), 25.30 (3), 28.62–29.00
(4–13), 31.21 (14), 21.99 (15), 13.79 (16); 1H NMR
(2:1 CDCl3/CD3OD) l Glc, 4.27 (d, 1H, J1%,2%=7.8
Hz, 1%), 3.26 (dd, 1H, J1%,2%=7.8, J2%,3%=8.9, Hz, 2%),
4.42 (dd, 1H, J2%,3%=8.9, J3%,4%=8.8 Hz, 3%), 3.37 (dd,
1H, J3%,4%=8.8, J4%,5%=9.0 Hz, 4%), 3.29 (ddd, 1H,
J4%,5%=9.0, J5%,6a%=5.3, J5%,6b%=2.6 Hz, 5%), 3.72 (dd,
1H, J5%,6a%=5.3, J6a%,6b%=12.0 Hz, 6a%), 3.87 (dd, 1H,
J5%,6b%=2.6, J6a%,6b%=12.0 Hz, 6b%); Sph, 3.59 (dd,
1H, J1a,1b=10.1, J1a,2=3.2 Hz, 1a), 4.16 (dd, 1H,
To a vigorously stirred solution of glucosylsphin-
gosine 22 (27 mg, 5.8×10−5 mol, 100 mol%) in 0.9
ml of pure THF was added 0.9 ml of 1:1 NaOAc–
3H2O/H2O. A solution of palmitoyl chloride (18
mg, 65×10−5 mol, 110 mol%, prepared from
palmitic acid, a sample of which had been methy-
lated and determined to be 0.04% myristic acid,
99.81% palmitic acid, and 0.15% stearic acid by gas
chromatography using 5% DEGS-PS on 100/120
supelcoport at 160°C) in 0.17 ml of THF was added
over 1 min, and the heterogeneous mixture stirred
vigorously for 2 h. The mixture was concentrated,
then partitioned between 5 ml of H2O and 10 ml
of 3:1 CH2Cl2/MeOH. The organic phase was
combined with additional 3:1 CH2Cl2/MeOH ex-
tracts (2×10 ml), dried briefly, and concentrated.
The crude product was filtered through a short
column (80:20 CH2Cl2/MeOH). The product was
J
J
1a,1b=10.1, J1b,2=4.6 Hz, 1b), 4.00 (ddd, 1H,
1a,2=3.2, J1b,2=4.6, J2,3=7.4 Hz, 2), 4.10 (dd,
1H,
J2,3=7.4, J3,4=7.5 Hz, 3), 5.46 (dd,

126
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
1H, J3,4=7.5, J4,5=15.3 Hz, 4), 5.70 (dt, 1H,
4,5=15.3, J5,6=6.7 Hz, 5), 2.03 (dt, 2H, J5,6
3. Results and discussion
J
=
3.1. Syntheses of sphingosine 9 and ceramide 10
6.7, J6,7=7.3 Hz, 6), 1.35 (m, 2H, 7), 1.20–1.34
(m, 20H, 8–17), 0.89 (t, 3H, J17,18=6.9 Hz, 18);
FA, 2.18 (t, 2H, J2,3=7.7 Hz, 2), 1.59 (m, 2H, 3),
1.20–1.34 (m, 24H, 4–15), 0.89 (t, 3H, J15,16 =6.9
Hz, 16); 13C NMR (2:1 CDCl3/CD3OD) l Glc,
102.72 (1%), 73.21 (2%), 75.85 (3%), 69.76 (4%), 76.05
(5%), 61.07 (6%); Sph, 68.21 (1), 52.98 (2), 71.69 (3),
128.85 (4), 133.92 (5), 31.93 (6), 28.83–29.24 (7-
15), 31.47 (16), 22.19 (17), 13.41 (18); FA, 174.29
(NCꢀO), 36.04 (2), 25.52 (3), 28.83–29.24 (4–13),
D
-Galactose (1) was converted to 4,6-benzyli-
dene-a/b- -galactose (a/b-2) (Byun and Bittman,
D
1993; Dong, 1990; Gros and Deulofeu, 1962,
ˇ
1964; Kiso et al., 1986a; Paca´k and Cerny´, 1963;
Schmidt and Zimmermann, 1986a, 1988; Zimmer-
mann and Schmidt, 1988) in 13.4% yield (see
Scheme 1). A mixture of predominantly the a-
anomer crystallized from EtOH/EtOAc. The ben-
zylidene derivative 2 was demonstrated to be an
85:15 mixture of a and b anomers, which rapidly
epimerized to an equilibrium containing nearly
half b-anomer in D2O, or which slowly reached
this equilibrium in the polar nonprotic solvent
DMSO-d6. The benzylidene protecting group
would seem to be more useful than the corre-
sponding isopropylidene protecting group (Kiso
et al., 1986a,b; Zimmermann and Schmidt, 1988)
and as it imparted crystallinity useful for large
31.47 (14), 21.19 (15), 13.41 (16); [a]2D4 −9° (c 1.0,
20
598
pyridine) (Lit. (Zimmermann et al., 1988) [a]
−9.5° (c 1, pyridine); Lit. (Shibuya et al., 1992)
[a]2D3 +3.8° (c 1.1, MeOH)); MALDIMS m/z
722.5 (M+Na)+; FABMS (pos) m/z 700.7 (M+
H)+, 682.7 (M+H%)+, 520.6 (Z°)+, 264.2 (W¦)+;
HRFABMS (pos) calculated for C40H78NO8
(M+H)+ m/z 700.5727, found m/z 700.5692.
Anal. Calcd. for C40H77NO8: C, 68.63; H, 11.09;
N, 2.00. Found: C, 68.99; H, 11.08; N, 2.02.
Scheme 1. Syntheses of
D-erythro-sphingosine 9 and ceramide 10.

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
127
scale preparations and provided an easily de-
tectable derivative for many subsequent synthetic
steps.
1988b; Schmidt and Zimmermann, 1986a), or the
corresponding tosylate (Schmidt and Zimmer-
mann, 1986a; Yadav et al., 1993). (2S,3R,4E)-2-
Azido-1,3-benzylidene-4-octadecene-1,3-diol (7)
was first cautiously prepared from alcohol 5 via
the corresponding triflate intermediate by the re-
ported (Byun and Bittman, 1993; Jung and
Schmidt, 2000; Zimmermann and Schmidt, 1988)
method using freshly purified anhydrous solvents.
Although no difficulties were ever encountered, it
should be noted that the extraction and concen-
tration of reaction mixtures containing both
dichloromethane and sodium azide has been re-
ported (Hruby et al., 1993; Peet and Weintraub,
1993, 1994) to be potentially explosive and that
this procedure should be performed with caution.
The crude product 7 was demonstrated to contain
byproducts from double bond isomerization and
from the loss of the benzylidene protecting group,
which was consistent with a related report (Kiso
et al., 1986b). Nearly pure product 7 was obtained
after a lengthy and tedious series of chromatogra-
phy columns including final chromatography with
CH2Cl2/hexanes to remove most of the cis-olefin
impurity, which has a slightly lower Rf. Yields
from several attempts ranged from 35 to 60%
yield. An alternative synthesis of azide 7 utilizing
zinc azide under Mitsunobu conditions (Viaud
and Rollin, 1990) also resulted in a product con-
taining a large amount of double bond isomeriza-
tion, although there was less degradation of the
benzylidene protecting group. A small amount of
the cis-7 byproduct was purified nearly to homo-
geneity from each of these two methods and this
cis-7 was characterized by NMR. The conversion
of the alcohol 5 to the azide 7 with inversion of
configuration was best performed using the inter-
mediate mesylate 6 (Byun and Bittman, 1993;
Ohashi et al., 1989, 1988b; Schmidt and Zimmer-
mann, 1986a) or the corresponding tosylate
(Schmidt and Zimmermann, 1986a; Yadav et al.,
1993) which have been previously reported. The
intermediate mesylate 6 was easily prepared on
many scales in high yield in either neat pyridine or
with excess triethylamine in CH2Cl2. The mesylate
6 was chromatographically purified to homogene-
ity and was highly crystalline. The reaction of
mesylate 6 with NaN3 proceeded slightly faster in
(2R,3R,4E)-1,3-Benzylidene-4-octadecene-1,2,3-
triol (5) (Byun and Bittman, 1993; Dong, 1990;
Jung and Schmidt, 2000; Kiso et al., 1986a; Ku-
mar and Schmidt, 1998; Ohashi et al., 1989,
1988b; Schmidt and Zimmermann, 1986a, 1988;
Yadav et al., 1993; Yamanoi et al., 1989; Zimmer-
mann and Schmidt, 1988) was prepared several
times on a variety of scales. Results agreed with
the original (Schmidt and Zimmermann, 1986a)
and subsequently detailed (Byun and Bittman,
1993; Jung and Schmidt, 2000; Zimmermann and
Schmidt, 1988) reports that trans-olefin 5 was
obtained after chromatography in a nearly pure
form which contained very little cis-olefin. No
specific rotation was observable in CHCl3. De-
composition in CDCl3 was demonstrated, and this
may account for the variations in reported (Kiso
et al., 1986a; Zimmermann et al., 1988) specific
1
rotations. Thus, only H and 13C NMR data in
DMSO-d6, which are reported here for the first
time, are the most useful. The presence of the
cis-olefin was always observed to be well under
5% as a minor impurity characterized by reso-
nances slightly downfield of C6-H and C1-Hb as
well as an olefinic resonance slightly upfield of the
C4-H for the trans-olefin 5. It is possible that
improper handling of the acid-sensitive 5 accounts
for the reports (Kiso et al., 1986a; Ohashi et al.,
1989, 1989) of double bond isomerization and
possible protecting group rearrangement.
(2S,3R,4E)-2-Azido-1,3-benzylidene-4-octadec-
ene-1,3-diol (7) (Byun and Bittman, 1993; Dong,
1990; Jung and Schmidt, 2000; Ohashi et al.,
1989, 1988b; Schmidt and Zimmermann, 1986a,
1988; Yadav et al., 1993; Zimmermann and
Schmidt, 1988) should be recognized as a key
synthetic intermediate from which sphingosine 9,
ceramide 10, cerebroside 23, and other glycosph-
ingolipids can be synthesized. There has been
some question of whether the synthesis of azide 7
should proceed via the corresponding triflate,
(Byun and Bittman, 1993; Jung and Schmidt,
2000; Schmidt and Zimmermann, 1986a, 1988;
Zimmermann and Schmidt, 1988) mesylate 6
(Byun and Bittman, 1993; Ohashi et al., 1989,

128
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
DMSO than in DMF at 90°C, and the disappear-
ance of starting material was followed over 3 days
by TLC. No cis-7 byproduct with its characteris-
tic lower Rf was observed to be formed under
these reaction conditions, and this is the primary
reason that the mesylate 6 intermediate was used
in spite of the lower overall yield for conversion
to the azide 7. Purification of azide 7 by prepara-
tive HPLC was performed to easily remove a very
small amount of a higher Rf impurity. This was a
convenient stage in the synthesis to demonstrate
the removal of all cis byproduct, which was
slightly more polar. The separation of cis and
trans isomers was previously demonstrated for a
related diastereomer (Fujita et al., 1991). The
product azide 7 was demonstrated to be com-
pletely stable in DMSO at 90°C for 3 days or by
resubmitting the azide 7 to the reaction conditions
in DMF followed by 1H NMR analyses. The
azide 7 was relatively stable in CDCl3. More
decomposition was seen by two-dimensional TLC
with hexanes/CH2Cl2 than with hexanes/EtOAc,
although no separation of the desired trans-azide
trans-7 from the byproduct cis-azide cis-7 was
possible unless the hexanes/CH2Cl2 elution was
used, and this may account for the reports (Byun
and Bittman, 1993; Dong, 1990; Jung and
Schmidt, 2000; Ohashi et al., 1989; Zimmermann
and Schmidt, 1988) of low melting material.
Epimerization of the benzylidene center and
byproducts from rearrangement of the benzyli-
dene protecting group, in addition to isomeriza-
tion of the olefinic bond, may also account for the
higher reported (Byun and Bittman, 1993; Jung
and Schmidt, 2000; Zimmermann and Schmidt,
1988) specific rotation for azide 7. Variations of
reaction conditions (Cvetovich et al., 1997; He et
al., 1999; Hirata et al., 1991; Kiso et al., 1986a,b;
Somfai and Olsson, 1993) are currently under
investigation.
a TEA quench and non-aqueous workup to give a
significantly higher yield conversion to azidosph-
ingosine 8 (Dong, 1990; Ito et al., 1988; Jung and
Schmidt, 2000; Kiso et al., 1986a,b; Kumar and
Schmidt, 1998; Ohashi et al., 1989; Schmidt and
Zimmermann, 1986a,b, 1988; Somfai and Olsson,
1993; Yadav et al., 1993; Zimmermann and
Schmidt, 1988) by preventing re-equilibration to
starting material. The NMR spectra of azidosph-
ingosine 8 were obtained in both DMSO-d6 and
CDCl3 solutions. The azidosphingosine 8 was rel-
atively stable in chloroform, and the optical rota-
tion agreed with the reported (Jung and Schmidt,
2000; Zimmermann and Schmidt, 1988) value in
chloroform solution. Reduction of azide 8 with
H2S gave pure
D
-erythro-sphingosine 9 (Dong,
1990; Hirata et al., 1991; Jung and Schmidt, 2000;
Kiso et al., 1986a,b; Li and Wu, 1996; Schmidt
and Zimmermann, 1986a; Shibuya et al., 1992;
Yadav et al., 1993; Zimmermann and Schmidt,
1988). Sphingosine 9 was readily acylated to give
ceramide 10 which was chromatographed in the
presence of TEA. The observed melting point
(100.5–101.5°C) of synthetic ceramide 10 was
higher than that of ceramide (Weiss and Raizman,
1958) (89–91°C) prepared from sphingosine
derived from a natural source, which has some
heterogeneity in the chain length of the sphin-
gosine base. The NMR data and specific rotation
data were obtained in chloroform solutions,
though it should be noted that the olefinic bond is
quite labile in moist chloroform, and that the
rotation result was only included for comparison
with literature reports. Optical rotation values
may vary somewhat in pure chloroform at the
same concentration where some aggregation
should be expected, and chloroform/methanol
mixtures should be used (Ohashi et al., 1988a,
1989; Shibuya et al., 1992), preferably a chloro-
form/methanol 2:1 mixture. NMR characteriza-
tions were performed for the first time in
DMSO-d6, though ceramide 10 was nearly
insoluble.
Azide 7 was converted to ceramide 10 both via
sphingosine 9 and also by a variation that avoids
sphingosine 9 as a synthetic intermediate, which
may be useful on larger scales. Synthetic
thro-sphingosine 9 was first prepared from the key
synthetic intermediate 1,3-benzylidene-2-azi-
dosphingosine 7 in two steps. Deprotection of
azide 7 by acid-catalyzed transacetalation utilized
D
-ery-
Ceramide 10 was also prepared via an alterna-
tive pathway from azide 7 by azide reduction in
the presence of a benzylidene protected diol. Re-
duction of the azide 7 by NaBH4 was found to be
preferable to the reported (Byun and Bittman,

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
129
Scheme 2. Determination of sphingosine 9 optical purity.
1993; Ohashi et al., 1989, 1988b) reduction
with Ph3P, which proceeded through a reactive
phosphinimine intermediate. An aqueous workup
of the amine 11 was avoided. Acylation of the
amine 11 gave the 1,3-benzylidene derivative 12
(Ohashi et al., 1989, 1988b) of the ceramide 10.
The deprotection of 12 utilized a novel TEA
quench and a non-aqueous workup to minimize
re-equilibration to starting material, and gave ce-
ramide 10 (Dong, 1990; Kanemitsu and Sweeley,
1986; Koike et al., 1986; Ohashi et al., 1988a,
1989; Shah et al., 1995a; Shibuya et al., 1992)
which was determined to be identical to ceramide
10 prepared from the alternative pathway via
sphingosine 9.
useful for 19F NMR analysis. Each of these three
methods of analysis were investigated primarily to
differentiate the MTPA derivative of
sphingosine from the derivative of the same
MTPA reagent with -erythro-sphingosine, or the
equivalent separation of -erythro-sphingosine
D-erythro-
L
D
derivatives from each of the two enantiomeric
MTPA reagents. The (aS)-MTPA-derivative 13
was prepared from synthetic sphingosine 9. Also,
the (aS)-MTPA-derivatives 13 from commercially
available samples of each of the four possible
sphingosine diastereomers were prepared to give
the four corresponding amides, (aS)-MTPA-
D-
erythro-Sph (aS,2S,3R)-13, (aS)-MTPA- -ery-
L
thro-Sph (aS,2R,3S)-13, (aS)-MTPA-
(aS,2S,3S)-13, and (aS)-MTPA-
L
-threo-Sph
-threo-Sph
D
3.2. Determination of the optical purity of
(aS,2R,3R)-13. In addition, (aR)-MTPA-
D-ery-
D
-erythro-sphingosine 9
thro-Sph (aR,2S,3R)-13 was prepared and found
to be identical to its enantiomer (aS)-MTPA-L-
New methodologies for determination of the
erythro-Sph (aS,2R,3S)-13 by TLC, HPLC, 1H
NMR, 19F NMR, and these enantiomers were the
only crystalline isomers of the four possible enan-
optical purity (de, ee) of sphingosine 9 were inves-
tigated. The two erythro-sphingosine enantiomers
were chromatographically separable from the two
more polar threo-sphingosine enantiomers. The
synthetic sphingosine 9 was easily demonstrated
tiomeric pairs of amides 13. (aS)-MTPA-
D-ery-
thro-Sph (aS,2S,3R)-13 and (aS)-MTPA-
L
-
erythro-Sph (aS,2R,3S)-13 were not separable by
TLC. Some separation of these two erythro-sphin-
gosine derivatives was possible by analytical
HPLC (4.6×250 mm, 10 mm Si-60, 97.5:2.5
CH2Cl2/MeOH at 1.0 ml/min, u 254 nm) eluting
to be free of both
L-threo-sphingosine and D-
threo-sphingosine by TLC (80:20:2 CHCl3/
MeOH/2 M aq NH4OH) and subsequently by
spectroscopic methods. The problem of the deter-
mination of percent enantiomeric excess [100 (
D
-
-
(aS)-MTPA-
30 s, co-eluting (aS)-MTPA-
(aS,2S,3R)-13 and (aS)-MTPA-
(aS,2R,3R)-13) at 9 min, and eluting (aS)-
MTPA- -erythro-Sph (aS,2R,3S)-13 at 9 min 40
L
-threo-Sph (aS,2S,3S)-13) at 8 min
-erythro-Sph
-threo-Sph
erythro-Sph- -erythro-Sph)/( -erythro-Sph+
L
D
L
D
erythro-Sph)] of the synthetic sphingosine 9 was
then investigated by the preparation of the corre-
sponding Mosher’s (Dale et al., 1969) amide
derivative 13 (see Scheme 2). The a-methoxy-a-
(trifluoromethyl)phenylacetyl (MTPA) derivative
contained a chromophore useful for HPLC analy-
sis with UV detection, a methoxy group useful for
1H NMR analysis, and a trifluoromethyl group
D
L
s. In spite of the high sensitivity of UV detection,
the HPLC separation method was most useful for
detecting impurities of (aS)-MTPA-
D-erythro-
sphingosine in (aS)-MTPA- -erythro-sphingosine
L

130
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
at levels of under 0.5% corresponding to greater
than 99% ee. For the more important assay for
the presence of any impurity (aS)-MTPA-D-ery-
13, the synthetic product was again demonstrated
to be \98% ee by this third method. All of the
19F spectra contained the presence of about 1% of
an apparent doublet JF,F of 290 Hz, possibly
indicating that the Mosher’s acid derivative con-
tained some difluoromethyl impurity.
The 1,3-di-(trimethylsilyl) derivatives 14 were
prepared from the diols 13 in accordance with a
related report (Boutin and Rapoport, 1986). The
trimethylsilyl groups were demonstrated to be too
unstable for an HPLC analysis by two-dimen-
sional TLC. Since the chemical conversion yields
were often below 80%, this derivative was also
unsatisfactory to determine optical purity by
NMR spectroscopy.
The diacetate analogs 15 provided the necessary
derivatization for the determination of optical
purity of the erythro enantiomers of sphingosine 9
at a greater sensitivity. For this analysis, the
2
thro-Sph (aS,2R,3S)-13 in the (aS)-MTPA-D-
erythro-Sph (aS,2S,3R)-13 derivative of synthetic
sphingosine 9, no impurity peak was detected in
the tailing after the main peak. However, the limit
of detection for any impurity present in the peak
tailing was only demonstrated to be above 1% by
doping, corresponding to an optical purity of
greater than 98% ee by this HPLC method. The
1H NMR and 19F NMR methods were much less
time-consuming and both subsequently confirmed
1
this HPLC result. The H NMR spectra of the
four diastereomeric sphingosine (aS)-MTPA
derivatives 13 were examined primarily to develop
a methodology to determine the optical purity of
the erythro-sphingosines. The methoxy resonances
were not sufficiently resolved to be useful. How-
ever, the two highest field peaks from the C5-H
doublet of triplets, the two highest field peaks of
the C4-H doublet of doublets, the completely
resolved C3-H multiplet at l 4.32, and the C1-Ha
synthetic
D
-erythro-sphingosine (2S,3R)-9 was
separately treated with both enantiomers of
Mosher’s acid chloride and subsequently diacy-
lated in yields above 90%. Nearly all of the reso-
nances in the 1H NMR spectra of the
diastereomeric Mosher’s amide diacetates, (aS)-
doublet of doublets at l 3.76 of the
L-erythro
impurity of 13 were demonstrated to be detectable
in (aS)-MTPA-
D
-erythro-Sph (aS,2S,3R)-13 by
MTPA-
(aR)-MTPA-
D
-erythro-Sph-Ac2 (aS,2S,3R)-15 and
-erythro-Sph-Ac2 (aR,2S,3R)-15,
doping above 1%. These characteristic resonances
D
were not observable in the synthetic 13 spectrum,
and thus the synthetic (aS)-MTPA-D-erythro-Sph
were resolvable. Of particular importance, the
characteristic methoxy resonances of the two
diastereomers were nearly baseline separated with
resonances at l 3.43 and 3.38, respectively, as
shown in Fig. 1. With much greater sensitivity
now possible, each derivative of the synthetic
sphingosine was easily observed to contain less
than 0.5% of the corresponding diastereomeric
(aS,2S,3R)-13 was again demonstrated to be \
98% ee by this second method. This spectroscopic
result was also re-confirmed by an 19F NMR
method where the trifluoromethyl group of (aS)-
MTPA-
the highest field, with (aS)-MTPA-
(aS,2R,3S)-13 downfield at l 0.12, and with both
D
-threo-Sph (aS,2R,3R)-13) resonated at
L
-erythro-Sph
impurity.
The
methoxy
peaks
of
the
(aS)-MTPA-
(aS)-MTPA-
D
-erythro-Sph (aS,2S,3R)-13 and
diastereomeric impurities in each of the diacetate
derivatives from the two Mosher’s reagents was
slightly smaller than the nearest 13C satellite peak
(natural abundance 1.1%+2=0.55%). The sensi-
tivity of the method appeared to now be limited
by the purity of the commercial MTPA chloride
reagents which were \99.5% a single enantiomer
for each of the reagents, and this was confirmed
by the 19F NMR experiments. The 19F NMR
spectra also contained a trace amount of the
apparent difluoromethyl impurity already dis-
cussed. The trifluoromethyl group of (aS)-
L
-threo-Sph (aS,2S,3S)-13) further
downfield at l 0.16. The trifluoromethyl groups of
the erythro diastereomers of 13 were not baseline
resolved, although the resolution was better than
the separation of the corresponding methoxy
groups in the 1H NMR experiments.
A
diastereomeric impurity could only be detected
with this 19F NMR method by doping at 1%.
Since an upfield impurity peak for any
diastereomeric impurity was not observed in the
synthetic (aS)-MTPA- -erythro-Sph (aS,2S,3R)-
L-erythro-
D

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
131
nient 1H and 19F NMR methods can each be
considered as easy alternatives to the chiral HPLC
method (Kawamura et al., 1996). The diacetate
15, prepared in two easy steps from sphingosine 9,
was the preferred derivative to establish optical
purity as enantiomeric excess and diastereomeric
MTPA-
baseline resolved l 0.30 downfield from the corre-
sponding (aR)-MTPA- -erythro-Sph-Ac2
(aR,2S,3R)-15 trifluoromethyl resonance as
demonstrated in Fig. 2. The derivatives 15 of
synthetic sphingosine 9 were confirmed to be opti-
cally pure as \99% ee and \99% de by this
second spectroscopic method. These new conve-
D
-erythro-Sph-Ac2 (aS,2S,3R)-15 was
D
1
excess. Both H NMR and 19F NMR were suffi-
ciently sensitivity methods, at 500 and 471 MHz,
Fig. 1. 1HNMR (CDCl3) determination of the optical purity of synthetic D-erythro-sphingosine 9: (A) (aS, 2S, 3R)-1,3-diacetyl-2-[a-
methoxy-a-(trifluoromeyhyl)phenylacetamido]-4-octadecene-1,3-diol ((aS)-MTPA-D-erythro-Sph-Ac2) ((aS, 2S, 3R)-15) contained
less than 0.5% the corresponding (aR)-derivative; (B) (aR, 2R, 3R)-1,3-diacetyl-2-[a-methoxy-a-(trifluormethyl)phenylacetamido]-4-
octadecene-1,3-diol ((aR)-MTPA-D-erythro-Sph-Ac2) ((aR,2R,3R)-15) contained less than 0.5% the corresponding (aS)-derivative.

132
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
Fig. 2. Baseline resolution was demonstrated in this 19F NMR (CDCl3) spectrum of a 2:1 mixture of (aS)-MTPA-D-erythro-Sph-Ac2
((aS,2S,3R)-15) and (aR)-MTPA-D-erythro-Sph-Ac2 ((aR,2S,3R)-15), the derivatives used to determine the optical purity of
synthetic D-erythro-sphingosine 9.
respectively, limited only by the optical purity of
the Mosher’s reagents.
10 was recently demonstrated to be more useful
than an azido-analog for the synthesis of gan-
glioside GM3 (Duclos, 2000), due to the problem-
atic azide reduction of an acidic azidoganglioside
analog. However, the use of the azidosphingosine
analog 8 for the synthesis of cerebroside 23, a
neutral glycosphingolipid, was repeated and
found to be perfectly satisfactory (see Scheme 3).
The trityl derivative 16 (Mori et al., 1990; Schmidt
3.3. Synthesis of cerebroside 23
Ceramide 10 can be a synthetic intermediate for
a variety of classes of sphingolipids including
sphingomyelin, cerebroside 23, and other gly-
cosphingolipids including gangliosides. Ceramide

R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
133
and Zimmermann, 1986b; Zimmermann et al.,
1988) of azidosphingosine 8 was relatively un-
stable in chloroform. While no rotation was ob-
served in chloroform, this compound has been
reported to have both a negative (Zimmermann
and Schmidt, 1988) and a positive (Mori et al.,
1990) specific rotation in CHCl3 solution. The
esterification with benzoyl chloride gave ester 17
(Schmidt and Zimmermann, 1986b; Zimmermann
and Schmidt, 1988) in yields as high as 92% with
the use of 4-pyrrolidinopyridine catalyst. The de-
protection gave 3-benzoylazidosphingosine 18 (Ito
et al., 1989, 1988; Schmidt and Zimmermann,
1986b; Zimmermann and Schmidt, 1988), which
was used within a day or two due to slow partial
acyl rearrangement to 2-azido-1-benzoyl-4-oc-
tadecene-1,3-diol. The trityl group was easily re-
moved under mild conditions to avoid double
bond isomerization with the same Lewis acid that
would be used to catalyze the subsequent glycosy-
lation reaction. Glycosyl coupling of acceptor 18
(Zimmermann et al., 1988) in 56% yield, although
the yield was 72% based on the recovery of start-
ing material. Treatment of ester 20 with catalytic
NaOMe in MeOH resulted in rapid methanolysis
of the acetate groups, and the slower methanolysis
of the benzoate group was followed by TLC. The
azidocerebroside 21 (Schmidt and Zimmermann,
1986b, 1988; Zimmermann et al., 1988) was iso-
lated in 95% yield.
The reduction of azide 21 with Ph3P/pyridine/
water gave a number of decomposition byprod-
ucts. However, the reduction of the azide 21 with
H2S/pyridine/water gave clean amine 22 (Schmidt
and Zimmermann, 1986b, 1988; Zimmermann et
al., 1988) that was compared to material derived
from a natural source (Hara and Taketomi, 1986).
The glucosylsphingosine 22 was converted to glu-
cosylceramide (GlcCer, cerebroside) 23 (Schmidt
and Kla¨ger, 1985; Schmidt and Zimmermann,
1986b, 1988; Shibuya et al., 1992; Zimmermann et
al., 1988) with palmitoyl chloride and was fully
characterized. A higher melting form of glucosyl-
ceramide 23 was reported (Shibuya et al., 1992);
however, their reported (Shibuya et al., 1992)
specific rotation data in methanol at 23°C also
could not be duplicated. Synthetic glucosylce-
ramide 23 was found to be insoluble in methanol
with donor 2,3,4,6-tetraacetyl-a-D-glucopyranosyl
trichloroacetimidate a-19 (Cook et al., 1984;
Schmidt and Kla¨ger, 1985; Schmidt and Michel,
1980; Schmidt et al., 1984; Schmidt and Stumpp,
1983; Schmidt and Zimmermann, 1988) gave the
fully protected azidosphingosine glycoside 20
Scheme 3. Synthesis of cerebroside 23.

134
R.I. Duclos, Jr / Chemistry and Physics of Lipids 111 (2001) 111–138
on cooling to ambient temperature. Synthetic
glucosylceramide 23 was found to have a nega-
tive optical rotation in pyridine corresponding
closely to the original literature report (Zimmer-
mann et al., 1988).
Acknowledgements
This research was supported by Research
Grants HL-57405 (to GGS) and HL-26335 (to
DMS and to GGS) from the National Institutes
of Health, and the author is grateful to G.G.
Shipley for mentorship. The Shipley Group and
the Department of Physiology and Biophysics
are gratefully acknowledged, especially C.J.
McKnight and J.M. Vural for assistance with
NMR, and M.S. Gigliotti for GC and other as-
sistance. FAB, MALDI, and ESI mass spectral
data were obtained by C.E. Costello, H. Per-
reault, S. Ye, and S.Y. Chen here at the Boston
University School of Medicine Mass Spectrome-
try Resource (CEC, HP, SY, SYC) which is
supported by NIH Grant RR 10888 (to CEC),
and also at the Department of Chemistry, Uni-
versity of Manitoba, Winnipeg, Canada (HP).
Special thanks to S.-L. Wu, D. M. Small, Z.X.
Dong and S. Lin.
3.4. Summary of synthetic methodology and
biophysical studies
Research quantities of optically pure and
highly homogeneous sphingosine 9 as well as the
sphingolipids N-palmitoylsphingosine (ceramide)
10 and glucosylceramide (cerebroside) 23 were
synthesized. Apparent discrepancies in literature
procedures and characterizations have been re-
solved. These lipids were prepared at the highest
possible levels of homogeneties and in optically
pure forms appropriate for biologically relevant
studies. The sphingosine base 9 was demon-
strated to be completely free of threo
diastereomers and to be above 99% ee, which
reflected the optical purity of the Mosher’s acid
reagents used in the optical purity determina-
tion, and therefore was between 99.0–99.98%
de. There was no detectable cis unsaturation in
the sphingosine base, which was greater than
99.9% sphing-4-enine (C18:1). The acyl
groups of ceramide 10 and cerebroside (GlcCer)
23 were greater than 99.8% palmitoyl (C16:0).
The biophysical properties of these optically
pure and highly homogeneous lipids were re-
cently investigated in this laboratory. The struc-
tural and thermotropic properties of both the
synthetic ceramide 10 (Shah et al., 1995a) and
the synthetic glucosylceramide (GlcCer) 23 (Sax-
ena et al., 1999) as a function of hydration were
studied by differential scanning calorimetry
(DSC) and X-ray diffraction. The biophysical
properties of hydrated lactosylceramide were
also recently detailed (Saxena et al., 2000). We
have consistently chosen to study the N-palmi-
toyl analogs in this series of sphingolipids, and
ceramide 10 was recently used in the chemical
total synthesis of highly homogeneous gan-
glioside GM3 (Duclos, 2000). The biophysical
properties of that next glycolipid in this series
are currently under investigation in this labora-
tory.
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