A new approach to the stereoselective total synthesis of isotopically labeled D-ribo-phytosphingosine

Article abstract of DOI:10.1016/S0957-4166(99)00161-5

We describe a novel stereoselective total synthesis of D-ribo-[1,1-²H- 1,2-¹³C]phytosphingosine (12). Chirality at the incipient C-4 position was derived from asymmetric dihydroxylation of 1-hexadecene. The remaining chiral centers were formed by Sharpless epoxidation of an allylic alcohol, followed by benzoylcarbamate cyclization to furnish a 2-amino-1,3,4-triol derivative with the desired stereochemistry. The ²H and ¹³C labels were introduced by Horner-Emmons condensation of ¹³C-labeled triethyl phosphonoacetate, followed by reduction of the resulting carboxylic ester with AlCl₃/LiAlD₄. Mass spectral results indicated the suitability of 12 as an internal standard for analyses by the isotope dilution method.

Full text of DOI:10.1016/S0957-4166(99)00161-5

Pergamon
Tetrahedron: Asymmetry 10 (1999) 1697–1707
A new approach to the stereoselective total synthesis of
isotopically labeled D-ribo-phytosphingosine
Shengrong Li, Jihai Pang, William K. Wilson ∗ and George J. Schroepfer Jr. †
Departments of Biochemistry and Cell Biology and of Chemistry, Rice University, 6100 Main Street, Houston, Texas
77005-1892, USA
Received 14 March 1999; accepted 19 April 1999
Abstract
We describe a novel stereoselective total synthesis of D-ribo-[1,1-2H-1,2-13C]phytosphingosine (12). Chirality
at the incipient C-4 position was derived from asymmetric dihydroxylation of 1-hexadecene. The remaining chiral
centers were formed by Sharpless epoxidation of an allylic alcohol, followed by benzoylcarbamate cyclization to
2
furnish a 2-amino-1,3,4-triol derivative with the desired stereochemistry. The H and 13C labels were introduced
by Horner–Emmons condensation of 13C-labeled triethyl phosphonoacetate, followed by reduction of the resulting
carboxylic ester with AlCl3/LiAlD4. Mass spectral results indicated the suitability of 12 as an internal standard for
analyses by the isotope dilution method. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
Sphingolipids are important structural constituents of eukaryotic cell membranes. Complex sphingo-
lipids and their metabolites also have diverse roles as receptors and as second messengers in the regulation
of a broad range of cellular processes, including growth and apoptosis.1–3 Among the metabolites
showing potent biological activities are the simple long-chain bases, such as sphingosine, dihydro-
sphingosine, and phytosphingosine. Assessing the physiological relevance of various in vitro activities
of the long-chain bases requires the reliable determination of their normal range of concentration in
tissues of healthy and diseased subjects. However, the most common approaches4–7 for measuring levels
of the long-chain bases provide little or no structural evidence for the authenticity of the analyte and
consequently are susceptible to significant errors. More dependable measurements can be expected
from LC–MS8 or GC–MS analysis in conjunction with mass fragmentography and the isotope-dilution
technique,9 which involves quantitation against an isotopically labeled internal standard.
∗
†
Corresponding author. Tel: +1 713-527-4865; fax: +1 713-285-5154; e-mail: billw@bioc.rice.edu
Deceased 11 December 1998.
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00161-5

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We have recently described the synthesis of [1,1,3-2H]D-erythro-sphingosine10 as an internal standard
for isotope-dilution GC–MS analysis, and minor synthetic modifications would also provide [1,1,3-2H]D-
erythro-dihydrosphingosine. In designing a similar isotopomer of phytosphingosine, we first considered
the mass spectral fragmentation of its volatile tetra-TMS derivative, which is readily formed in a single
step and produces distinctive fragment ions under electron-impact conditions. Apart from the (CH3)3Si
ion (m/z 73), the two most abundant ions (m/z 132 and 204) contain C-1, C-2, and the C-1 hydrogens.
For isotope-dilution analyses, it would be desirable to place isotopic labels (13C and 2H) at each of these
positions to furnish an isotopomer of 4 added mass units.
In designing a route to [1,1-2H-1,2-13C]-C18-phytosphingosine, we investigated the possibility of
adapting an existing synthesis for the introduction of the isotopic labels. In most syntheses of phyto-
sphingosine, the stereochemistry at the critical C-2 position is derived from sugars,11–16 L-serine,17–20
sphingosine,21,22 or other natural materials23–25 that are either costly or unavailable with 13C labels.
Other reported syntheses26–28 are also unsuitable for introducing the isotopic labels at the appropriate
positions. This situation prompted us to develop a new total synthesis of D-ribo-phytosphingosine. Our
2
synthesis uses relatively inexpensive 13C and H reagents to provide a facile introduction of 13C labels
at C-1 and C-2 and 2H labels at C-1. Furthermore, the final product is obtained in good overall yield and
shows high stereochemical homogeneity.
2. Results and discussion
Our synthesis of D-ribo-C18-phytosphingosine began with the asymmetric dihydroxylation29 of 1-
hexadecene with AD-mix-β. A 9:1 mixture of 2R and 2S diols (2) was obtained, a respectable enan-
tioselectivity for the dihydroxylation of a terminal olefin in view of similar results for 1-heptene.30
Purification of the 9:1 mixture was delayed until the introduction of the remaining two chiral centers. By
use of standard methodology,31 the 1,2-diol 2 was selectively protected as the 2-benzyl ether 3, followed
by mild oxidation to aldehyde 4 with Dess–Martin periodinane (Scheme 1).32 The remaining two carbons
of the phytosphingosine chain were introduced by Horner–Emmons condensation of aldehyde 4 with
labeled triethyl phosphonoacetate, a reagent that is readily available at reasonable cost with 13C labels at
the incipient C-1 and C-2 carbons. Reduction of the resulting α,β-unsaturated ester 5 with AlCl3/LiAlD4
gave the allylic alcohol 6 and completed the introduction of the isotopic labels.
The two remaining chiral centers were introduced by Sharpless epoxidation of 6. Owing to the presence
of the minor (10%) enantiomer of 6, epoxide 7 was obtained as a 9:1 mixture of C-4 epimers, which were
not readily separable on silica gel. Following an elegant strategy for the stereospecific transformation of
epoxy alcohols into amino alcohols,33 we converted 7 into its benzoylcarbamate derivative 8, which
was cyclized with NaH to furnish a 2:3:2 mixture of starting material (8), oxazolidinone 9, and the
debenzoylated oxazolidinone 10. The benzoylcarbamate cyclization involved backside attack by the
amido nitrogen anion on C-2 of the epoxide, accompanied in part by the anticipated33 transfer of the
benzoyl group to the C-3 oxygen, to produce the desired stereochemistry for D-ribo-phytosphingosine.
MPLC purification furnished homogenous samples of 9 and 10, free of the minor C-4 epimers derived
ultimately from incomplete enantioselectivity in the dihydroxylation of 1.
Stepwise removal of the protecting groups afforded D-ribo-phytosphingosine, which showed a single
component by NMR as the free base 12 and its tetraacetate 13. After consideration of the effects of the
isotopic labels, NMR spectra of 12 and 13 were essentially superimposable with those34 of the unlabeled
analogs. Comparison of the 1H and 13C NMR spectra for 13 with detailed NMR data reported for the four

S. Li et al. / Tetrahedron: Asymmetry 10 (1999) 1697–1707
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Scheme 1.
phytosphingosine tetraacetate diastereomers35 also confirmed the stereochemistry as ribo and indicated
the absence of the lyxo, arabino, and xylo isomers at a detection limit of 0.5%.
The locations of the isotopic labels were confirmed by NMR. The 2H NMR spectrum of tetraacetate 13
showed two signals with chemical shifts matching those of the C-1 protons of unlabeled 13.34 In the 13
C
NMR spectrum of 13, intense signals for C-1 and C-2 and a weak doublet for C-3 showed the anticipated
1
shieldings and coupling patterns for the 1,1-2H-1,2-13C isotopic substitution. The H NMR spectrum
showed signals for H-2, H-3, and H-4 with the expected chemical shifts and coupling constants (Fig. 1).
Essentially no signal was observed for the C-1 protons, and a very minor signal corresponding to H-2
bonded to 12C indicated 99% 13C at C-2. These high isotopic purities were confirmed by the FAB mass
spectrum of 12, which showed m/z 322 (M+H) as the base peak and 0.1–0.9% relative abundances for
m/z 318–321. The EI mass spectra of tetraacetate 13 and the tetra-TMS derivative of 12 were essentially
identical to those of their unlabeled counterparts except for shifts in mass of certain ions due to isotopic
substitution. These shifts supported the ion assignments shown in Fig. 2 for the tetra-TMS derivative
of D-ribo-phytosphingosine and confirmed that the abundant ions at m/z 132 and 204 are suitable for
isotope-dilution GC–MS analyses.
The present synthesis of phytosphingosine is unique in furnishing isotopic labels at C-1, C-2, and
the C-1 protons. We have demonstrated that these labels produce the desired mass shifts in the EI
mass spectrum. Consequently, the [1,1-2H-1,2-13C]phytosphingosine should be valuable as an internal
standard for measuring levels of phytosphingosine in biological matrices and to compensate for losses
of this labile sphingolipid base during sample processing. Another feature of our synthesis of 12 is its
comparability with other total syntheses of unlabeled phytosphingosine26–28 in terms of diastereo- and
enantioselectivity, length, and overall yield (9% from 1-hexadecene).

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Figure 1. 1H NMR spectrum of D-ribo-[1,1-2H-1,2-13C]phytosphingosine tetraacetate (13). Integration of the triplets at δ 4.32
and 4.60 relative to the low-intensity triplet at δ 4.46 indicated a 99:1 ratio of 13C:12C isotopes at C-2. A similarly high
incorporation of 13C and 2H labels at C-1 is indicated by the absence of signals corresponding to the chemical shifts for the C-1
protons (δ 4.01 and 4.29) or their 13C satellites (indicated by arrows in the figure)
Figure 2. Electron-impact mass spectrum of the tetra-TMS derivative of D-ribo-phytosphingosine (FW 605). Masses of
corresponding ions observed in the spectrum of the 1,1-2H-1,2-13C labeled derivative are given in parentheses. Suggested
assignments are presented for selected ions; 13C labeled atoms are indicated by an asterisk (*). No significant ions were observed
above m/z 519
3. Experimental
3.1. Materials and methods
Melting points (mps) were measured with a Thomas–Hoover apparatus in sealed, evacuated capillary
tubes. IR spectra were obtained from KBr pellets on a Mattson Galaxy 6020 Fourier-transform infrared
spectrometer. Optical rotations were measured on a JASCO DIP-4 digital polarimeter. TLC was carried
out on aluminum-backed, silica gel 60 F254 plates (EM Science, Gibbstown, NJ). Components of the
plates were charred by spraying with 5% ammonium molybdate in 10% sulfuric acid followed by heating.
MPLC was done on glass columns dry-packed with silica gel (230–400 mesh; EM Science); fraction
volumes were 20 mL. Direct-inlet electron-impact (EI) mass spectra and fast atom bombardment (FAB,
positive ion) mass spectra were acquired with a ZAB-HF reverse-geometry double-focusing instrument
in the positive-ion mode and are reported as m/z (relative abundance, suggested assignment). NMR

S. Li et al. / Tetrahedron: Asymmetry 10 (1999) 1697–1707
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1
spectra were measured on a Bruker AMX500 instrument (500 MHz for H) at 25°C and referenced
to tetramethylsilane or to CD3OD at 3.30 ppm (1H) or CDCl3 at 77.0 ppm (13C). Signal assignments
were made from HSQC, COSY-DQF, and 1D spectra as described previously.36 Identified 13C–1H
1
‡
couplings in H spectra and 13C signals corresponding to 13C-labeled positions are denoted by , and
multiplets with line positions distorted by second-order effects at 500 MHz are designated with §. NMR
data and stereochemistry of carbamates 8–11 are described using the usual carbon numbering for the
phytosphingosine backbone.
1-Hexadecene, AD-mix-β [K3Fe(CN)6, K2CO3, K2OsO4·2H2O, hydroquinidine 1,4-
phthalazinediyl diether ((DHQ)2PHAL)], diisobutylaluminum hydride (DIBAL), (S)-α-methoxy-
α-(trifluoromethyl)phenylacetyl chloride (98% ee), silver cyanate, (−)-diisopropyl-D-tartrate (DIPT),
titanium tetraisopropoxide, tert-butyl hydroperoxide, 10% palladium on carbon, lithium hydroxide, and
triethyl phosphonoacetate-13 C2 ((Et2O)2P(O)13CH213CO2Et) were purchased from Aldrich Chemical
Co. (Milwaukee, WI), Dess–Martin periodinane and benzaldehyde dimethyl acetal from Lancaster
Synthesis Inc. (Windham, NH), and lithium aluminum deuteride (99% D) from Isotech (Miamisburg,
OH). α-Methoxy-α-(trifluoromethyl)phenylacetate (MTPA) derivatives were prepared by reaction of
the alcohol (2 mg) with (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (30 µL) in pyridine
(100 µL) as described previously.37
3.2. (2R)-Hexadecane-1,2-diol (2)
To a solution of 1-hexadecene (2.0 g, 8.1 mmol, 92% purity) in tert-butanol:water (1:1, 72 mL) was
added AD-mix-β (10 g) at 0°C. The resulting solution was stirred at 4°C for 40 h, followed by addition
of solid sodium sulfite (9 g). The solution was warmed to room temperature, stirred for 1 h, and extracted
with dichloromethane (3×100 mL). The combined extracts were washed with water (100 mL) and brine
(40 mL), dried (Na2SO4), and evaporated to give a white solid, which was subjected to MPLC (500×25
mm i.d. column, ethyl acetate:hexane, 1:3). Evaporation of fractions 35–50 gave 2 as a white solid (1.6
g, 76% yield): mp, 78–79°C; single component by TLC (Rf 0.38, ethyl acetate:hexane, 1:1); [α]D23 −1.6
(c 1.6, CHCl3); IR, 3477, 1084, 989, 719 cm−1; 1H NMR, δ 3.71 (m, 1H), 3.66 (ddd, 11.0, 5.4, 3.1 Hz,
1H), 3.44 (ddd, 11.0, 7.7, 3.8 Hz, 1H), 2.08 (br s, 0.7H), 1.97 (br s, 0.7H), 1.63 (br s, 0.6H), 1.44 (m,
3H), 1.26 (m, ∼23H), 0.88 (t, 7 Hz, 3H); 13C NMR, δ 72.32 (C-2), 66.85 (C-1), 33.21, 31.91, 29.68,
29.67, 29.65, 29.64 (2C), 29.63, 29.58, 29.54, 29.35, 25.53, 22.68, 14.11; MS (EI), 241 (2), 227 (100),
208 (5), 153 (13), 139 (24), 125 (45), 111 (66), 97 (88), 83 (90), 71 (55); high-resolution MS (EI), calcd
for C15H31O (M−CH2OH) 227.2375, found 227.2373. 1H NMR of the MTPA derivative of 2 indicated a
9:1 ratio of 2R and 2S isomers.
3.3. (−)-(2R)-2-Benzyloxyhexadecan-1-ol (3)
To a solution of diol 2 (2.2 g, 8.5 mmol) in benzene (150 mL) was added benzaldehyde dimethyl
acetal (2.6 g, 17 mmol) and p-toluenesulfonic acid monohydrate (0.1 g). The resulting solution was
stirred at room temperature for 18 h, followed by concentration to dryness under vacuum. The residue
was dissolved in dichloromethane (100 mL) and cooled to −78°C, followed by dropwise addition of
diisobutylaluminum hydride (38 mL, 1.0 M in toluene). The reaction mixture was stirred at −78°C for
30 min, gradually warmed to room temperature, and quenched with methanol (4 mL). Diethyl ether (300
mL) and saturated Rochelle’s salt (100 mL) were added, and the mixture was stirred until the two phases
became clear. The separated organic phase was washed with water (100 mL) and brine (40 mL), dried
(Na2SO4), and evaporated to a yellow oil, which was subjected to MPLC (500×25 mm i.d. column, ethyl

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acetate:hexane, 15:85). Evaporation of fractions 71–98 gave 3 as a white solid (2.5 g, 84% yield): mp,
23
36–37°C; single component by TLC (Rf 0.70, ethyl acetate:hexane, 3:7); [α]D −13.0 (c 1.9, CHCl3);
IR, 3447, 1057, 749, 696 cm−1; 1H NMR, δ 7.34 (m, 4H), 7.29 (m, 1H), 4.61 (d, 11.5 Hz, 1H), 4.54 (d,
11.5 Hz, 1H), 3.68 (ddd§, 11, 7, 2.5 Hz, 1H), 3.52 (ddd, 11.1, 6.3, 5.2 Hz, 1H), 3.49 (m, 1H), 1.62 (m,
1H), 1.49 (m, 1H), 1.27 (m, ∼24H), 0.88 (t, 7 Hz, 3H); 13C NMR, δ 138.49, 128.45 (2C), 127.77 (2C),
127.72, 79.82 (C-2), 71.48, 64.29 (C-1), 31.91, 30.78, 29.78, 29.68, 29.67, 29.65, 29.63 (2C), 29.57,
29.53, 29.34, 25.36, 22.67, 14.10; MS (FAB), 387 (3, M+K), 371 (5, M+Na), 347 (2, M−H), 107 (20),
91 (100); MS (EI), 348 (4, M+), 317 (12, M−CH2OH), 225 (18), 107 (13), 91 (100); high-resolution MS
(EI), calcd for C23H40O2 348.3028, found 348.3022. 1H NMR of the MTPA derivative of 3 showed a 9:1
ratio of 2R and 2S isomers.
3.4. (+)-(2R)-2-Benzyloxyhexadecan-1-al (4)
To a solution of Dess–Martin periodinane (3.4 g) in dichloromethane (40 mL) was added pyridine
(1.3 mL). The resulting slurry was stirred until the reagents dissolved (∼5 min), followed by dropwise
addition of a solution of 3 (2.4 g, 6.9 mmol) in dichloromethane (15 mL). The reaction mixture was
stirred at room temperature for 3 h, quenched with brine (20 mL) and saturated sodium sulfide solution
(20 mL), and extracted with dichloromethane (3×50 mL). The extracts were washed with water (100
mL) and brine (40 mL), dried (Na2SO4), and evaporated to give a residue, which was subjected to flash
chromatography (150×10 mm i.d. column, ethyl acetate:hexane, 15:85). Evaporation of fractions 3–7
gave 4 as an oil (2.2 g, 92% yield): single component by TLC (Rf 0.70, ethyl acetate:hexane, 15:85);
23
[α]D +9.6 (c 0.8, CHCl3); IR, 1738, 1111, 737, 698 cm−1; 1H NMR, δ 9.65 (d, 2.2 Hz, 1H), 7.35 (m,
4H), 7.31 (m, 1H), 4.67 (d, 11.7 Hz, 1H), 4.53 (d, 11.7 Hz, 1H), 3.75 (ddd§, 6.5, 5.6, 2.2 Hz, 1H), 1.67
(m, 2H), 1.41 (m, 2H), 1.26 (m, ∼22H), 0.88 (t, 7 Hz, 3H); 13C NMR, δ 203.89 (C-1), 137.37, 128.49
(2C), 128.03, 127.98 (2C), 83.47, 72.48 (C-2), 31.90, 30.02, 29.67, 29.66, 29.63 (2C), 29.59, 29.51,
29.37 (2C), 29.34, 24.72, 22.67, 14.10; MS (FAB), 345 (2, M−H), 181 (7), 91 (100); MS (EI), 317 (11,
M−CHO), 248 (7), 225 (25), 208 (12), 91 (100); high-resolution MS (EI), calcd for C22H37O (M−CHO)
317.2827, found 317.2831.
3.5. (2E,4R)-[1,2-13C]Ethyl 4-benzyloxy-2-octadecenoate (5)
Triethyl phosphonoacetate-13C2 (1.0 g, 4.4 mmol) was added dropwise at 0°C to a suspension of NaH
(0.11 g, 4.6 mmol) in anhydrous THF (30 mL), followed by stirring at 0°C for 0.5 h and addition of 4
(1.7 g, 4.9 mmol) in one portion. The reaction mixture was allowed to warm to room temperature and
extracted with dichloromethane (3×50 mL). The organic extracts were dried (Na2SO4) and evaporated
to give a residue, which was purified by MPLC (500×25 mm i.d. column; ethyl acetate:hexane, 15:85).
Evaporation of fractions 40–57 gave a colorless oil (1.65 g, 90% yield), which was characterized as an
8:1 mixture of 5 and 4 (as judged by 1H NMR): single component by TLC (Rf 0.80; diethyl ether:hexane,
5:95); IR, 1680, 1149, 732, 698 cm−1; 1H NMR, δ 7.33 (m, 4H), 7.28 (m, 1H), 6.86 (dtd, 15.8, 6.6, 2.5
Hz, 1H, H-3), 6.00 (dddd, 163.2‡, 15.8, 3.4, 1.2 Hz, 1H, H-2), 4.59 (d, 11.9 Hz, 1H), 4.37 (d, 11.9 Hz,
1H), 4.22 (qd, 7.1, 3.0‡ Hz, 2H), 1.66 (m, 1H), 1.55 (m, 1H), 1.38 (m, 1H), 1.31 (t, 7.1 Hz, 3H), 1.25
(m, ∼23H), 0.88 (t, 7 Hz, 3H); 13C NMR, δ 166.30‡ (d, 74.5 Hz, C-1), 148.50 (dd, 70.7, 1.3 Hz, C-3),
138.19, 128.37 (2C), 127.69 (2C), 127.62, 121.93‡ (d, 74.8 Hz, C-2), 78.07 (dd, 6.5, 1.4 Hz, C-4), 70.94,
60.46 (dd, 2.3, 1.5 Hz), 34.91, 31.91, 29.68, 29.67, 29.65, 29.64, 29.62, 29.57, 29.49, 29.47, 29.35, 25.13,
22.68, 14.23 (d, 2.3 Hz), 14.10; MS (FAB), 419 (2, M+H), 373 (10, M−EtO), 311 (16, M−PhCH2O), 91

S. Li et al. / Tetrahedron: Asymmetry 10 (1999) 1697–1707
1703
(100); MS (EI), 418 (1, M+), 373 (7, M−EtO), 327 (14, M−PhCH2), 298 (9), 281 (6), 225 (12), 221 (16),
91 (100); high-resolution MS (EI), calcd for C2313C2H39O2 (M−EtO) 373.3017, found 373.2988.
3.6. (2E,4R)-[1,1-2H-1,2-13C]4-Benzyloxy-2-octadecen-1-ol (6)
Alane-d3 (AlD3) was formed by stirring AlCl3 (0.36 g, 2.7 mmol) and LiAlD4 (0.27 g, 6.4 mmol) in
dry diethyl ether (20 mL) for 15 min at 0°C. To this solution was added dropwise a solution of 5 (1.60 g,
3.8 mmol, containing 12% 4) in dry diethyl ether (10 mL). After the addition was complete, the mixture
was stirred at 0°C for 40 min. The reaction was quenched by addition of water (0.2 mL), followed by
filtration of the resulting inorganic precipitate and evaporation of the filtrate to dryness. The residue was
subjected to MPLC purification (500 mm×25 mm i.d. column; ethyl acetate:hexane, 5:95). Evaporation
of fractions 63–83 gave 6 as a colorless oil (1.1 g, 76%); single component by TLC (Rf 0.62, diethyl
ether:hexane, 3:7); [α]D +22.6 (c 0.9, CHCl3); IR, 3377, 2181, 2085, 1068, 748, 696 cm−1; 1H NMR,
23
δ 7.33 (m, 4H), 7.27 (m, 1H), 5.79 (ddd§, ∼164‡, 15.6, 4.5 Hz, 1H, H-2), 5.62 (m, 1H, H-3), 4.57 (d,
11.9 Hz, 1H), 4.37 (d, 11.9 Hz, 1H), 3.76 (m, 1H, H-4), 1.64 (m, 1H), 1.49 (m, 1H), 1.25 (m, ∼24H),
0.88 (t, 7 Hz, 3H); 13C NMR, δ 138.83, ∼132 (C-3), 131.56‡ (d, 45.8 Hz, C-2), 128.29 (2C), 127.70
(2C), 127.39, 79.46 (dd, 5.9, 1.5 Hz, C-4), 70.17, 62.36‡ (d of quintet, 45.8, 21.7 Hz, C-1), 36.63 (d, 2.9
Hz, C-5), 31.91, 29.69, 29.68, 29.66, 29.65, 29.64, 29.61, 29.57, 29.55, 29.35, 25.38, 22.68, 14.10; MS
(FAB), 417 (5, M+K), 401 (3, M+Na), 377 (1, M−H), 271 (3, M−PhCH2O), 253 (4), 91 (100); MS (EI),
378 (1, M+), 344 (3, M−13CD2OH), 287 (4, M−PhCH2), 225 (6), 181 (49), 91 (100); high-resolution MS
(EI), calcd for C2313C2H40D2O2 378.3377, found 378.3377.
3.7. (2R,3S,4R)-[1,1-2H-1,2-13C]-2,3-Epoxy-4-benzyloxyoctadecan-1-ol (7)
To a solution of D-(−)-DIPT (0.72 mL, 3.4 mmol) containing oven-dried molecular sieve (2 g; 3 Å) in
dichloromethane (50 mL) was added titanium tetraisopropoxide (0.87 mL, 2.9 mmol) at −30°C, followed
by stirring for 15 min at −30°C and dropwise addition of tert-butyl hydroperoxide (2 mL, 5–6 M) in
decane. After further stirring at −30°C for 15 min, a solution of 6 (1.1 g, 2.9 mmol) in dichloromethane
(10 mL) was added dropwise. The reaction mixture was stirred for an additional 5 min at −30°C and
stored in a −20°C freezer for 3 days. TLC analysis indicated conversion of nearly all the starting material
to give a polar product. The reaction mixture was washed vigorously for 15 min with 30% sodium
hydroxide solution (15 mL), and the aqueous washings were extracted with dichloromethane (2×100
mL). The combined organic phase was dried (Na2SO4) and evaporated to give an oil, which was purified
by MPLC (500 mm×25 mm i.d. column, 15% ethyl acetate–hexane). Fractions 46–70 gave 7 as a white
solid (0.8 g, 70% yield): mp, 37–40°C; single component by TLC (Rf 0.50, ethyl acetate:hexane, 3:7);
IR, 3443, 2170, 2085, 1107, 729, 692 cm−1; 1H NMR, δ 7.33 (m, 4H), 7.28 (m, 1H), 4.61 (d, 11.9 Hz,
1H), 4.55 (d, 11.9 Hz, 1H), 3.32 (dtd, 7.1, 5.2, 3.7, 1H, H-4), 3.10 (ddd, 173.4‡, 7.7‡, 2.3 Hz, 1H, H-2),
2.94 (dddd, 5.3, 2.0, 2.0‡, 2.0‡ Hz, 1H, H-3), 1.62 (m, 2H), 1.49 (m, 1H), 1.38 (m, 1H), 1.26 (m, ∼22H),
0.88 (t, 7 Hz, 3H); 13C NMR, δ 138.62, 128.37 (2C), 127.66, 127.63 (2C), 77.58 (C-4), 72.46, 60.69 (d
of quintet, 46.0, 21.7 Hz, C-1), ∼57 (C-3), 56.71 (d, 46.0 Hz, C-2), 32.79 (d, 2.0 Hz, C-5), 31.91, 29.68,
29.67, 29.66, 29.64 (2C), 29.62, 29.59, 29.54, 29.34, 25.10, 22.67, 14.10; 2H NMR, δ 3.83 (d, 21.7 Hz),
3.55 (d, 21.7 Hz); MS (FAB), 433 (1, M+K), 417 (1, M+Na), 393 (1, M−H), 287 (4, M−PhCH2O), 91
(100); MS (EI), 394 (1, M+), 329 (3), 317 (4), 287 (2, M−PhCH2O), 254 (27), 225 (13), 107 (38), 91
(100); high-resolution MS (EI), calcd for C2313C2H40D2O3 394.3327, found 394.3315. 1H NMR analysis
indicated a 85:15 mixture of 4R and 4S isomers; NMR data for minor (4S) epimer (originating from the
minor enantiomer of 4, which ultimately was derived from incomplete stereoselectivity in the asymmetric

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S. Li et al. / Tetrahedron: Asymmetry 10 (1999) 1697–1707
dihydroxylation of 1): δH 4.80 (d, 11.8 Hz), 4.58 (d, 11.8 Hz), 3.15 (dddd, 8.0, 6.8, 5.3, 1.3 Hz, H-4),
3.07 (dq, 7.0, 2.1 Hz, H-3), 2.94 (ddd, 172.0‡, 7.3‡, 2.4 Hz, H-2); δC 54.60‡ (d, 46.3 Hz).
3.8. (2R,3S,4R)-[1,1-2H-1,2-13C]-Carbamate (8)
Benzoyl chloride (0.46 mL, 4.0 mmol) was added to a suspension of silver cyanate (0.66 g, 4.4 mmol)
in CCl4 (10 mL) at room temperature, followed by refluxing for 12 h, cooling, and filtration under
nitrogen. To the filtrate was added 7 (0.80 g, 2.0 mmol) in CCl4 (10 mL), and the reaction mixture was
stirred at room temperature for 4 h. Dichloromethane (100 mL) was added, and the solution was washed
with saturated sodium bicarbonate and brine and evaporated to give a residue, which was subjected to
MPLC (500 mm×25 mm i.d. column, ethyl acetate:hexane, 15:85). Evaporation of fractions 46–76 gave
8 as a white solid (0.94 g, 86% yield; 10:1 mixture of 8 and its 4S epimer by 1H NMR): mp, 50–52°C;
single component by TLC (Rf 0.47, ethyl acetate:hexane, 3:7); IR, 3288, 2158, 1772, 1753, 1207, 694
cm−1; 1H NMR, δ 8.22 (br s, 1H, NH), 7.82 (dd§, 8.4, 1.3 Hz, 2H), 7.59 (tt§, 7.5, 1.3 Hz, 1H), 7.48 (tt§, 8,
1.5 Hz, 2H), 7.33 (m, 4H), 7.28 (m, 1H), 4.61 (d, 11.9 Hz, 1H), 4.54 (d, 11.9 Hz, 1H), 3.23 (ddd, 178.0‡,
5.6, 2.2 Hz, 1H, H-2), 3.33 (dtd, 7.2, 5.1, 4.0 Hz, 1H, H-4), 2.91 (dq, 5.2, 1.9 Hz, 1H, H-3), 1.62 (m, 2H),
1.49 (m, 1H), 1.38 (m, 1H), 1.26 (m, ∼22H), 0.88 (t, 7 Hz, 3H); 13C NMR, δ 164.60, 150.48, 138.49,
133.11, 132.74, 128.90 (2C), 128.40 (2C), 127.71, 127.63 (2C), 127.60 (2C), 77.18 (C-4), 72.59, 65.28‡
(d of quintet, 49, 22 Hz, C-1), 53.34‡ (d, 49.7 Hz, C-2), 32.73 (d, 2.1 Hz), 31.90, 29.68, 29.67, 29.65,
29.63 (2C), 29.60, 29.59, 29.53, 29.34, 25.06, 22.67, 14.10; 2H NMR, δ 4.49 (d, 22.4 Hz), 3.99 (d, 22.7
Hz); MS (FAB), 580 (13, M+K), 564 (16, M+Na), 542 (1, M+H), 105 (38), 91 (100); high-resolution
MS (FAB), calcd for C3113C2H45D2NO5Na (M+Na) 564.3545, found 564.3553. NMR data for the minor
(4S) isomer: δH 4.79 (d, 11.8 Hz), 4.57 (d, 11.9 Hz), 3.14 (dddd, 8.0, 6.6, 5.1, 1.4 Hz, H-4), 3.08 (ddd,
176.6‡, 5.5, 2.2 Hz, H-2), 2.95 (dq§, 5.3, 1.9 Hz, H-3); δC 51.46‡ (d, 49.8 Hz, C-2).
3.9. (2S,3S,4R)-[1,1-2H-1,2-13C]-Oxazolidinone-benzoate (9)
Crude 8 (0.80 g, 1.5 mmol) was added to a suspension of NaH (14 mg, 0.6 mmol) in freshly distilled
THF (30 mL), followed by refluxing for 6 h, cooling to room temperature, addition of water (100 mL),
and extraction with dichloromethane (2×200 mL). The organic extracts were washed with brine, dried
(Na2SO4), and evaporated to give a residue, which was subjected to MPLC (500 mm×25 mm i.d. column,
10–35% ethyl acetate in hexane). Fractions 35–48 gave 8 as a white solid (200 mg, 25% recovery).
1
Evaporation of fractions 58–82 provided 9 as a colorless oil (300 mg, 38% yield, ≥98% purity by H
NMR): single component by TLC (Rf 0.40, ethyl acetate:hexane, 3:7); IR, 3420, 2240, 1759, 1722, 1271,
1111, 712 cm−1; 1H NMR, δ 7.99 (dd§, 8.4, 1.3 Hz, 2H), 7.59 (tt, 7.5, 1.3 Hz, 1H), 7.46 (ddt§, 8.3, 7.5,
1.6 Hz, 2H), 7.31 (m, 5H), 5.35 (dd§, 5.5, 4.0 Hz, 1H, NH), 5.25 (ddt, 5.4, 4.3, 3.6 Hz, 1H, H-3), 4.60
(11.4, 1H), 4.52 (11.5, 1H), 4.31 (dddd, 146.0‡, 3.6, 2.7, 1.0, 1H, H-2), 3.71 (tt, 5.9, 4.2 Hz, 1H, H-4),
1.61 (m, 2H), 1.43 (m, 1H), 1.38 (m, 1H), 1.25 (m, ∼22H), 0.88 (t, 7 Hz, 3H); 13C NMR, δ 165.85 (d,
1.5 Hz), 158.95, 137.40, 133.60, 129.75 (2C), 129.14, 128.61 (2C), 128.57 (2C), 128.08, 127.99 (2C),
78.81 (d, 1.1 Hz), 74.61 (d, 41.6 Hz), 72.79, 65.84‡ (d of quintet, 32, 24 Hz), 52.31‡ (d, 32.8 Hz), 31.90,
31.30, 29.67, 29.66, 29.63 (2C), 29.59, 29.57, 29.50, 29.41, 29.34, 24.95, 22.66, 14.10; 2H NMR, δ 4.56
(d, 23 Hz), 4.40 (d, 23 Hz); MS (FAB), 580 (2, M+K), 564 (2, M+Na), 542 (7, M+H), 105 (47), 91 (100);
high-resolution MS (FAB), calcd for C3113C2H46D2NO5 (M+H) 542.3725, found 542.3727. Evaporation
of fractions 146–169 gave 10 (150 mg, 23% yield, 98% purity) as a white solid.

S. Li et al. / Tetrahedron: Asymmetry 10 (1999) 1697–1707
1705
3.10. (2S,3S,4R)-[1,1-2H-1,2-13C]-Oxazolidinone-alcohol (10)
Lithium hydroxide (2 mL, 2.25 M in water) was added to a solution of 9 (300 mg, 0.55 mmol) in THF
(20 mL), followed by stirring at room temperature for 12 h and extraction with ethyl acetate (2×30 mL).
The organic extracts were washed with water (20 mL) and brine (20 mL), dried (Na2SO4), and evaporated
to give a residue, which was subjected to MPLC (1 m×10 mm i.d. column; ethyl acetate:hexane, 3:7).
Evaporation of fractions 32–55 gave 10 as a white solid (0.21 g, 87% yield): mp, 73–74°C; single
23
component by TLC (Rf 0.40; ethyl acetate:hexane, 1:1); [α]D −16.6 (c 0.7, CHCl3); IR, 3487, 3360,
1
2246, 2168, 1786, 1099, 748, 698 cm−1; H NMR δ 7.36 (m, 2H), 7.31 (m, 3H), ∼5.9 (ddd§, 5.8, 4.0,
0.8 Hz, 1H, NH), 4.60 (d, 11.4 Hz, 1H), 4.47 (d, 11.4 Hz, 1H), 4.00 (dddd, 145.6‡, 4.6, 2.2‡, 1.0‡ Hz,
1H, H-2), 3.72 (dqd, 5.6, 4.6, 3.1 Hz, 1H, H-3), 3.45 (dddd, 6.3, 5.4, 4.7, 3.1 Hz, 1H, H-4), ∼3.0 (dd,
4.7, 2.9 Hz, 1H, 3-OH), 1.59 (m, 2H), 1.42 (m, 1H), 1.35 (m, 1H), 1.25 (m, ∼22H), 0.88 (t, 7 Hz, 3H);
13C NMR, δ 160.56, 137.72, 128.59 (2C), 128.04, 128.01 (2C), 79.73 (d, 2.0 Hz, C-4), 72.69 (d, 40.8
Hz, C-3), 72.26, 65.93‡ (d of quintet, 32, 23 Hz, C-1), 53.75‡ (d, 33.1 Hz, C-2), 31.91, 30.12, 29.82,
2
29.70, 29.69, 29.68, 29.66, 29.65, 29.61, 29.58, 29.35, 24.72, 22.68, 14.11; H NMR, δ ∼4.45 (d, 23
Hz), ∼4.26 (d, 23 Hz); MS (FAB), 476 (11, M+K), 460 (18, M+Na), 438 (2, M+H), 394 (7, M−CO2+H),
91 (100); high-resolution MS (FAB), calcd for C2413C2H42D2NO4 (M+H) 438.3463, found 438.3459.
3.11. (2S,3S,4R)-[1,1-2H-1,2-13C]-Oxazolidinone-diol (11)
To a solution of 10 (300 mg, 0.68 mmol) in THF (30 mL) was added 10% palladium on carbon (30
mg). The solution was stirred under a balloon of hydrogen at room temperature for 12 h. The catalyst was
removed by filtration on filter paper, and the filtrate was evaporated to a residue, which was subjected to
MPLC (1 m×10 mm i.d. column; methanol:chloroform, 5:95). Fractions 41–54 gave 11 as a white solid
(160 mg, 67% yield): mp, 77–78°C; single component by TLC (Rf 0.35, methanol:chloroform, 1:9); IR,
3406, 2255, 2160, 1747, 1261, 1074 cm−1; 1H NMR (CDCl3–CD3OD; referenced to tetramethylsilane;
the CD3OD quintet appeared at δ 3.37) δ 3.96 (ddd, 145.3‡, 4.9, 2.2 Hz, 1H, H-2), 3.74 (br s, 2H), 3.41
(ddt, 9.2, 6.3, 3.0 Hz, 1H, H-4), 3.35 (dtd, 6.5, 4.6, 2.9, 1H, H-3), 1.58 (m, 1H), 1.45 (m, 1H), 1.19 (m,
∼24H), 0.81 (t, 7 Hz, 3H); 13C NMR (CDCl3–CD3OD) δ 160.8, 74.1 (dd, 41.2, 1.2 Hz, C-3), 73.0 (br
s, C-4), 66.0 (d of quintet, 33, 23 Hz, C-1), 54.2 (d, 33.1 Hz, C-2), 33.4, 31.7, 29.5–29.4 (8C), 29.1,
2
25.3, 22.4, 13.8; H NMR (CHCl3–CH3OH), δ 4.24 (d, 24 Hz); MS (FAB), 733 (4, 2M+K), 717 (6,
2M+Na), 695 (8, 2M+H), 386 (93, M+K), 370 (52, M+Na), 348 (70, M+H), 304 (51, M−CO2+H), 192
(45); high-resolution MS (FAB), calcd for C1713C2H36D2NO4 (M+H) 348.2993, found 348.2980.
3.12. (2S,3S,4R)-[1,1-2H-1,2-13C]-2-Aminooctadecane-1,3,4-triol (D-ribo-[1,1-2H-1,2-13C]phyto-
sphingosine) (12)
To a solution of 11 (120 mg, 0.35 mmol) in ethanol (10 mL) was added 2.0 M sodium hydroxide (10
mL), followed by refluxing under nitrogen for 12 h. The product was extracted with chloroform:methanol
(9:1; 2×30 mL) and washed with water (20 mL) and brine (20 mL). The organic extracts were dried
(Na2SO4) and evaporated to give a residue, which was subjected to MPLC (1 m×10 mm i.d. column,
ammonia:methanol:chloroform, 1.5:15:85). Evaporation of fractions 30–37 gave 12 as a white solid
(78 mg, 70% yield): mp, 78–80°C; single component by TLC (Rf 0.40, ammonia:methanol:chloroform,
1.5:15:85); [α]D +6.8 (c 1.1, pyridine); IR, 3373, 2199, 2097, 1093, 721 cm−1; H NMR (CD3OD),
δ 3.51 (tt§, 8, 2.5‡ Hz, 1H, H-4), 3.34 (dddd, 7.8, 5.5, 4.3‡, 3.1‡ Hz, 1H, H-3), 2.95 (ddd, 135.7‡, 5.5,
3.2‡ Hz, 1H, H-2), 1.73 (m, 1H), 1.55 (m, 1H), 1.28 (m, ∼24H), 0.89 (t, 7 Hz, 3H); 13C NMR (CD3OD),
23
1

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S. Li et al. / Tetrahedron: Asymmetry 10 (1999) 1697–1707
δ 76.4 (d, 40.4 Hz, C-3), 74.4 (d, 1.3 Hz, C-4), 63.3 (d of quintet, 39, 21 Hz, C-1), 55.7 (d, 38.8 Hz,
2
C-2), 34.8, 33.1, 31.0, 30.8 (7C), 30.5, 26.6, 23.7, 14.4; H NMR (CD3OD), δ 3.72 (d, 22 Hz), 3.53 (d,
22 Hz); MS (FAB), 344 (5, M+Na), 322 (100, M+H), 304 (5, M−H2O+H); high-resolution MS (FAB),
calcd for C1613C2H38D2NO3 (M+H) 322.3201, found 322.3192. GC–MS of the tetra-TMS derivative of
12, 522 (3), 431 (2), 341 (30), 312 (23), 299 (25), 297 (15), 222 (9), 208 (44), 136 (100), 73 (97). The
overall yield of 12 from 1 was 9% (after compensation in the two-step conversion of 8 to 10 for recovered
starting material and for the direct formation of 10 in 23% yield from 8).
3.13. D-ribo-[1,1-2H-1,2-13C]-Phytosphingosine tetraacetate (13)
A solution of 12 (4 mg) in pyridine (100 µL), acetic anhydride (100 µL), and dichloromethane (5
mL) was stirred at room temperature for 12 h, followed by addition of water (2 mL) and extraction with
dichloromethane (2×5 mL). The organic extracts were washed with water (2 mL) and brine (2 mL), dried
(Na2SO4), and evaporated under nitrogen to give a residue, which was purified by HPLC (250×10 mm
i.d. Sphereclone ODS-2 C18 column; Phenomenex, Torrance, CA; elution with acetonitrile). Evaporation
of the eluate corresponding to the peak at 10.5 min gave 13 as a white solid (2.5 mg): single component
1
by TLC (Rf 0.5, methanol:chloroform, 1:9); H NMR, δ 5.97 (d, ∼9 Hz, 1H), 5.099 (dddd, 8.3, 4.5‡,
3.2‡, 3.2 Hz, 1H, H-3), 4.934 (dddd, 9.9, 3.3‡, 3.3, 1.9‡, 1H, H-4), 4.459 (dddd, 139.2‡, 9.5, 8.3, 1.6‡ Hz,
1H, H-2), 2.080 (s, 3H), 2.049 (s, 3H), 2.047 (s, 3H), 2.026 (s, 3H), 1.65 (m, 2H), 1.25 (m, ∼24H), 0.880
(t, 7 Hz, 3H); 13C NMR, δ 171.17, 170.87, 170.09 (d, 1.8 Hz), 169.71, 72.97, 71.97 (d, 43 Hz), 62.23‡
(d of quintet, 44, 22 Hz), 47.49‡ (d, 40.8 Hz), 31.91, 29.69, 29.68, 29.67, 29.65, 29.62, 29.57, 29.49,
29.35, 29.29, 28.16, 25.50, 23.30, 22.68, 21.04, 20.77, 20.74, 14.11; 2H NMR, 4.29 (d, 23 Hz), 4.00 (d,
23 Hz); MS (EI), 490 (1, M+H), 429 (2, M−AcOH), 413 (9, M−13CD2OAc), 387 (10), 370 (20), 353
(30), 327 (17), 311 (31), 309 (36), 293 (34), 284 (11), 269 (21), 222 (16), 205 (14), 190 (13), 149 (68),
148 (90), 106 (48), 88 (100); high-resolution MS (EI), calcd for C2413C2H46D2NO7 (M+H) 490.3530,
found 490.3565.
Acknowledgements
We gratefully acknowledge the support of the National Institutes of Health (HL-49122) and the Robert
A. Welch Foundation (C-583).
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