Selective deuterium labeling of the sphingoid backbone: Facile syntheses of 3,4,5-trideuterio-d-erythro-sphingosine and 3-deuterio-d-erythro-sphingomyelin

Article abstract of DOI:10.1016/j.chemphyslip.2010.09.001

Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen-deuterium exchange reaction of a β-ketophosphonate intermediate catalyzed by ND₄Cl in D₂O/tetrahydrofuran. To install deuterium at C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction of an α,β-enone in perdeuteromethanol was used.

Full text of DOI:10.1016/j.chemphyslip.2010.09.001

Chemistry and Physics of Lipids 163 (2010) 809–813
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Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Short communication
Selective deuterium labeling of the sphingoid backbone: facile syntheses of 3,4,
5
-trideuterio-d-erythro-sphingosine and 3-deuterio-d-erythro-sphingomyelin
∗
Hoe-Sup Byun, Robert Bittman
Department of Chemistry and Biochemistry, Queens College of CUNY, 65-30 Kissena Blvd., Flushing, NY 11367-1597, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2010
Received in revised form 31 August 2010
Accepted 2 September 2010
Available online 17 September 2010
Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen–deuterium exchange reaction of
a ␤-ketophosphonate intermediate catalyzed by ND4Cl in D2O/tetrahydrofuran. To install deuterium at
C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction
of an ␣,␤-enone in perdeuteromethanol was used.
©
2010 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Lipid synthesis
Sphingolipid
2
H-labeled
3
1
. Introduction
gosine, sphinganine, and ceramide by sodium [ H]-borohydride
3
or lithium aluminum [ H]-hydride reduction of 3-keto analogues,
Stable isotope-labeled natural products are widely used in the
elucidation of the structures of metabolites, toxicity studies, and
investigations of metabolism in normal and diseased states in vivo
Mutlib, 2008; Magkos and Mittendorfer, 2009; Postle and Hunt,
009). There has been increased interest in lipids labeled with
which affords a mixture of the erythro and threo stereoisomers
of the C3-labeled compound (Hoffman and Tao, 1998; reviewed
by Bielawska et al., 2000). Regiospecific oxidation of the primary
hydroxy group was achieved by using C2- and C3-protected sphin-
golipids, giving a protected aldehyde that on reduction provided a
C1-tritiated sphingolipid. Alternatively, an enantioselective aldol
condensation of (E)-2-hexadecenal with a chiral iminoglycinate
provided a 1-carboxylic ester intermediate (Solladié-Cavallo and
(
2
stable isotopes (principally deuterium) because of the dramatic
success of lipidomics in identifying and quantifying individual
molecular species present in lipid extracts by tandem mass spec-
trometry (Han, 2010; Shevchenko and Simons, 2010). Therefore,
the development of efficient and facile synthetic methods to incor-
porate deuterium regioselectively into lipids is needed.
Deuterated sphingolipids have been used in deuterium NMR
studies to determine the C–D bond order in the N-acyl chain of
sphingomyelin bilayers (Mehnert et al., 2006; Bartels et al., 2008).
However, selective deuteration of the interfacial region of the long-
chain base of sphingomyelin has not yet been reported. In this
communication we report a convenient methodology for inserting
deuterium labels into the vinyl group and/or the C3 position of the
sphingoid backbone, thereby enabling analysis of the conforma-
tional and order parameters in the lipid–water interfacial region of
sphingolipid bilayers by solid-state 2H NMR spectroscopy.
3
Koessler, 1994); reduction of the 1-carboxylic ester with [ H]-
3
NaBH4 afforded [1,1- H]-sphingosine (Li et al., 1999).
The structures of the deuterium-labeled sphingolipids
we prepared are shown in Fig. 1. A key step in our syn-
thesis of 3,4,5-trideuterio-d-erythro-sphingosine (1) is
a
Horner–Wadsworth–Emmons (HWE) olefination reaction of
1-deuteromyristaldehyde with a dideutero-␤-ketophosphonate
derived from N-Boc-l-serine methyl ester. 3-Deuterio-d-erythro-
sphingomyelin (2) was prepared via a NaBD -mediated reduction
4
of an unlabeled enone, which was synthesized in a HWE reaction
of myristaldehyde with a ␤-ketophosphonate; in a subsequent
step, the phosphocholine head group was installed.
Many deuterium-labeled lipids can be prepared by employing
the routes for the synthesis of their radiolabeled counterparts. For
example, tritium was incorporated at the C3 positions of sphin-
2. Results and discussion
2.1. Synthesis of compound 1
1
-Deuteriotetradecanal (5) was prepared from methyl myris-
∗ Corresponding author. Tel.: +1 718 997 3279; fax: +1 718 997 3349.
E-mail address: robert.bittman@qc.cuny.edu (R. Bittman).
tate (3) via 2,2-dideuteromyristyl alcohol (4) in two steps, as shown
in Scheme 1. Lithium aluminum deuteride reduction of 3 afforded
0
009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2010.09.001

8
10
H.-S. Byun, R. Bittman / Chemistry and Physics of Lipids 163 (2010) 809–813
amount of tetradeuterio species may be enhanced by conversion
of CeCl ·7H O to CeCl ·7D O via multiple treatments of the salt
3
2
3
2
with D O.
2
Treatment of acetonide 12 with 1 M HCl in THF at room tem-
perature provided 3,4,5-trideuterio-N-Boc-sphingosine 13 in 78%
yield. At this stage, the traces of the threo isomer that had remained
were completely removed by column chromatography on silica
gel (elution with hexane/EtOAc 1:1). Finally, removal of the N-Boc
Fig. 1. Structures of 3,4,5-trideuterio-d-erythro-sphingosine (1) and 3-deutero-d-
erythro-N-palmitoylsphingosylphosphocholine (2).
◦
group with 3 M HCl in THF at 70 C furnished deuterium-labeled
sphingosine 1 as its hydrochloride salt in quantitative yield.
␣
,␣-dideuterated alcohol 4 in 91% yield after purification by col-
umn chromatography. Oxidation of alcohol 4 with PCC in CH Cl2
2
gave deuterated aldehyde 5 in 82% yield, which was spectroscop-
2
.2. Synthesis of compound 2
1
ically pure by H NMR without the need for chromatography. As
1
expected, the H NMR spectrum of 5 showed no CHO peak at ı
Scheme 3 outlines the preparation of 3-deuterio-N-palmitoyl-
1
0 ppm. The deuterium atom in 5 became incorporated into the 5
d-erythro-sphingosylphosphocholine (2). Enone 14 was prepared
by HWE reaction of 7 with unlabeled myristaldehyde. Reduc-
tion of 14 with NaBD4 in the presence of CeCl ·7H O in
position of sphingosine (1) as discussed below.
The synthesis of 3,4,5-trideuteriosphingosine (1) is depicted in
Scheme 2. Condensation of l-Garner methyl ester 6 (prepared from
N-Boc-l-serine methyl ester as described by Garner and Park, 1987)
with the lithium salt of dimethyl methylphosphonate in THF is
known to afford ␤-ketophosphonate 7 (Koskinen and Koskinen,
3
2
THF/CD OD (10:1) for 4 h at room temperature gave alcohol
3
1
5 together with traces of its threo isomer. After removal
of the isopropylidene group, the phosphocholine moiety was
inserted into N-Boc-3-deuterio-sphingosine 16 via a one-pot reac-
tion with ethylene chlorophosphite, oxidation with bromine,
and quaternization with trimethylamine (Byun et al., 1994;
Erukulla et al., 1994). After the N-Boc group was removed
by heating with 3 M HCl in methanol, the resulting pro-
tonated amine was treated with diisopropylethylamine. An
N-acylation reaction of C3-deuteriosphingosylphosphocholine
with p-nitrophenyl palmitate provided 3-deuterio-N-palmitoyl-d-
erythro-sphingosylphosphocholine (2). The extent of deuteration
was estimated to be approximately 95%, based on the relative inten-
2
000) via enolate intermediate 8. Since the H–D exchange reac-
tion is a straightforward method to introduce C–D bonds vicinal
to carbonyl and phosphonyl groups, we first attempted to carry
out a hydrogen–deuterium exchange reaction with 7 by using D O
2
in THF or MeCN and catalysis by either K CO3 or ND Cl. This was
2
4
unsuccessful. However, we succeeded in preparing the desired
dideuterio-␤-ketophosphonate by the following sequence of reac-
tions. First, lithium enolate 8 was converted to mono-deuterated
␤
-ketophosphonate 9 with a catalytic amount of ND Cl in D O/THF
4 2
(
1:1) at room temperature. The hydrogen–deuterium exchange
+
sities of the m/z peaks at 703.5752 ([M+H ] ion of nondeuterated
2
isotope peak of the [M+H ] ion of mono-deuterated 2).
reaction was repeated three times, affording ␣,␣-dideuterio-␤-
+
13
), 704.5812 ([M+H ] ion of mono-deuterated 2), and 705.5844 (
C
ketophosphonate 10. The product of the exchange reactions was
+
1
confirmed by H NMR (disappearance of the PCH CO multiplet
2
In summary, we developed simple procedures for the prepara-
tion of deuterium-labeled sphingosine (1) and sphingomyelin (2)
from l-Garner methyl ester 6. The methodology described here can
be extended to prepare a variety of other deuterated sphingolipids.
For example, other deuterium-labeled derivatives of sphingosine
are readily accessible by using multi-deuterated analogues of 5,
deuterated glyco- or phosphosphingolipids can be prepared from
intermediate 13, and deuterium-labeled ceramides can be con-
veniently obtained from 1. Compounds containing deuterium at
peaks around ı 3.15 ppm). A precedent for hydrogen–deuterium
exchange with ND Cl in D O/THF is the synthesis of 5-epi-[6-
4
2
2
H]-valiolone after reductive desulfurization of a bis(methylthio)
moiety alpha to a carbonyl group (Mahmud et al., 2001).
In the next step, HWE reaction of ␣,␣-dideuterio-␤-
ketophosphonate 10 with 1-deuterotetradecanal (5) in D O/THF
2
◦
(
1:1) containing 3 equiv. of K CO3 at 0 C for 4 h and then at
2
room temperature for 72 h provided ␣,␤-enone 11, which bears
deuterium atoms in the vinyl group, in 61% yield. To incorporate
deuterium at C-3, Luche reduction (Gemal and Luche, 1981)
of the carbonyl group of 11 with sodium borodeuteride was
employed. Thus, reaction of enone 11 with NaBD4 in the presence
of CeCl ·7H O (to suppress 1,4-hydride addition) in THF/CD OD
selected sites in the interfacial region of the sphingoid backbone
may be useful as probes for 2H NMR spectroscopic analysis and as
internal standards for analysis of sphingosine and sphingomyelin
in biological samples by electrospray ionization/tandem mass spec-
trometry.
3
2
3
(
10:1) for 4 h at room temperature gave alcohol 12 together with
traces of its threo isomer (Chun et al., 2002a,b, 2003). In order to
reduce amount of perdeuterated methanol, we used THF as the
co-solvent. Column chromatography on silica gel (elution with
hexane/EtOAc 10:1) gave erythro-enriched alcohol 12 in 89% yield.
High-resolution mass spectral (HRMS) analysis of the relative
intensities in the isotope pattern of 12 indicated that the isotopic
composition was approximately 16% dideuterio, 64% trideuterio,
and 20% tetradeuterio. The tetradeuterio species may arise from
3
. Experimental procedures
3.1. General methods
HRMS were obtained by electrospray ionization (ESI) on an
Agilent Technologies G1969A or G6520A Q-TOF mass spectrom-
eter, which have mass accuracies of <3 and <2 ppm, respectively.
1
13
Analyses were done using two reference ions. H,
C, and
1
,4-addition of deuteride ion to 11, followed by protonation of the
31
P NMR spectra were recorded at 400, 100, and 161.9 MHz,
enolate ion intermediate with the traces of water present in the
hydrated CeCl rather than deuteration with CD OD. Therefore, the
respectively, on a Bruker Avance I spectrometer, and were
3
3
1
referenced to the residual chloroform at ı 7.24 ( H) and ı
7.2 (13C) and to external 85% H PO4 at ı 0.00 ppm ( P).
31
7
3
TLC was carried out on aluminum-backed silica gel GF plates
250-␮m thickness), and the compounds were visualized by
(
charring with 10% sulfuric acid in ethanol and/or short wave-
length UV light. For flash chromatography, silica gel 60 (230–400
ASTM mesh) was used. THF was distilled from Na and ben-
Scheme 1. Synthesis of 1-deuteriomyristaldehyde (5) from methyl myristate (3).

H.-S. Byun, R. Bittman / Chemistry and Physics of Lipids 163 (2010) 809–813
811
Scheme 2. Synthesis of 3,4,5-trideuteriosphingosine (1).
zophenone before use. CH Cl2 and i-Pr NEt were dried over
passed through a pad of silica gel, which was washed 100 mL of
2
2
CaH2.
Et O. The filtrate was concentrated under reduced pressure. The
2
residue was dissolved in a minimal volume of Et O and passed again
2
through a pad of silica gel, which was washed with Et O. The filtrate
2
3
.2. 1,1-Dideuteriotetradecanol (4)
was concentrated under reduced pressure to give 795 mg (82%) of
aldehyde 5: 1H NMR (CDCl ) ı 0.88 (t, 3H, J = 6.8 Hz), 1.25 (s, 20H),
3
To a solution of methyl myristate (3, 2.50 g, 10.3 mmol) in
13
◦
1.62 (m, 2H), 2.41 (t, 2H, J = 7.2 Hz); C NMR (CDCl3) ı 14.1, 21.3,
2.5, 28.8, 28.9, 29.3, 29.60, 29.61, 29.7, 32.1, 43.73 (t, J = 4 Hz), 202.6
t, J = 25 Hz).
THF was added LiAlD4 (600 mg, 14.3 mmol) at −78 C. After 2 h,
the reaction mixture was gradually warmed to room temperature
and stirred overnight. After the reaction was quenched with solid
Na SO ·10H O (2.0 g, 6.2 mmol), the mixture was passed through
2
(
2
4
2
a pad of Celite, which was washed with 100 mL of EtOAc. The fil-
trate was concentrated under reduced pressure, and the residue
was purified by column chromatography on silica gel (elution with
ꢀ
ꢀ
ꢀ
3
.4. N-tert-Butoxycarbonyl (4S)-[2 ,2 -dideuterio-2 -
dimethylphosphonoacetyl]-2,2-dimethyl-1,3-oxazolidine (10)
hexane/EtOAc 4:1) to give 2.03 g (91%) of alcohol 4 (which is avail-
To
a
solution of dimethyl methylphosphonate (1.65 g,
1
able commercially from several vendors): H NMR (CDCl ) ı 0.88
3
1
2
3.3 mmol) in 50 mL of THF was added dropwise n-BuLi (5.2 mL of a
(
(
t, 3H, J = 6.4 Hz), 1.26 (m, 22H), 1.55 (t, 2H, J = 6.4 Hz); 1 C NMR
3
◦
.5 M solution in hexane, 13.0 mmol) at −78 C. After 2 h, a solution
CDCl ) ı 14.1, 22.7, 25.7, 29.4, 29.4, 29.59, 29.60, 29.7, 31.9, 32.6,
3
of l-Garner methyl ester 6 (1.65 g, 6.36 mmol) in 10 mL of THF
6
2.3 (pentet, J = 21 Hz).
◦
was added slowly. The mixture was stirred at −78 C for 2 h, then
allowed to warm to room temperature, and stirred overnight. After
3.3. 1-Deuteriotetradecanal (5)
the reaction was quenched by addition of solid ND Cl (770 mg,
4
1
3.4 mmol), it was concentrated under reduced pressure. The
To a mixture of PCC (2.16 g, 10.0 mmol) and silica gel (3 g)
residue was dissolved in EtOAc (300 mL) and washed with brine.
in 50 mL of CH Cl2 was added a solution of alcohol 4 (984 mg,
The organic layer was dried (MgSO ) and concentrated. Purifica-
2
4
4
.55 mmol) in 10 mL of CH Cl at room temperature. After the mix-
tion by chromatography on silica gel (elution with hexane/EtOAc
2
2
ture was stirred for 2 h, it was diluted with 300 mL of Et O and
1:1 and then EtOAc) gave 1.50 g (67%) of ␤-ketophosphonate 9
2
Scheme 3. Synthesis of 3-deuterio-N-palmitoyl-d-erythro-sphingosylphosphocholine (2).

8
12
H.-S. Byun, R. Bittman / Chemistry and Physics of Lipids 163 (2010) 809–813
1
as a colorless oil. To a solution of 9 in 5 mL of THF and 5 mL of
D O was added ND Cl (2.0 mg, 34.6 ␮mol) at room temperature.
responding d and d species as described above): H NMR (CDCl )
2 4 3
2
4
ı 0.88 (t, 3H, J = 6.8 Hz), 1.26 (m, 20H), 1.37 (m, 2H), 1.45 (s, 9H), 2.04
(q, 2H, J = 7.1 Hz), 3.07 (br s, 2H), 3.68 (m, 1H), 3.77 (m, 1H), 3.91
After the mixture was stirred overnight, it was concentrated under
reduced pressure to give a residue. This exchange reaction was
repeated three times. Dideuterated ␤-ketophosphonate 10 was
(m, 1H), 5.32 (br s, 1H); 13C NMR (CDCl ) ı 14.1, 22.7, 28.4, 29.04,
3
29.07, 29.3, 29.47, 29.59, 29.62, 29.7, 31.9, 32.1, 55.4, 62.5, 64.1,
74.1 (t, J = 18 Hz), 79.8, 128.4 (t, J = 16 Hz), 133.6 (t, J = 16 Hz), 156.2;
HRMS [M+Na]+ m/z calcd for C23H42D NNaO , 425.3429, found,
used without further purification; 1H NMR (CDCl ) ı 1.45–1.75 (m,
3
1
5H), 3.65–3.95 (m, 6H), 4.04–4.17 (m, 2H), 4.49–4.60 (m, 1H).
3
4
4
25.3424.
ꢀ ꢀ ꢀ ꢀ
.5. N-tert-Butoxycarbonyl (4S)-[1 -oxo-(2 E)-2 ,3 -
3
dideuteriohexadecenyl]-2,2-dimethyl-1,3-oxazolidine (11)
3.8. (2S,3R)-2-Amino-(4E)-3,4,5-trideuteriooctadecene-1,3-diol
hydrochloride salt (1)
To a mixture of ␤-ketophosphonate 10 (1.50 g, 4.25 mmol) and
K CO (1.80 g, 13.0 mmol) in 10 mL of D O was added a solution of
2
3
2
◦
A solution of N-Boc-sphingosine-d3 (13) (417 mg, 1.03 mmol)
in 2 mL of 3 M HCl and 10 mL of THF was heated at 70 C with stir-
ring overnight under nitrogen. The reaction mixture was cooled
aldehyde 5 (788 mg, 3.69 mmol) in 10 mL of THF at 0 C. After 4 h,
the reaction mixture was gradually raised to room temperature and
stirred for 72 h. After dilution with EtOAc (250 mL), the mixture
was washed with brine, dried (Na SO ), and concentrated under
◦
to room temperature, concentrated, and lyophilized from C H to
6
6
2
4
give 351 mg (100%) of sphingosine-d3 HCl salt 1: 1H NMR (CDCl3)
ı 0.85 (t, 3H, J = 6.8 Hz), 1.26 (m, 20H), 1.36 (m, 2H), 2.08 (q, 2H,
J = 7.1 Hz), 3.07 (br s, 2H), 3.68 (m, 1H), 3.77 (m, 1H), 3.91 (m,
reduced pressure. Purification of the residue by column chromatog-
raphy on silica gel (elution with a gradient of hexane, hexane/EtOAc
0:1 and 20:1) gave 991 mg (61%) of enone 11: 1H NMR (CDCl ) ı
.88 (t, 3H, J = 7.0 Hz), 1.09–1.31 (m, 20 H), 1.48 (s, 9H), 1.54 (s, 3H),
5
0
1
3
1
2
H), 5.32 (br s, 1H); 13C NMR (CDCl /CD OD) ı 14.2, 22.6, 28.4,
3 3
9.06, 29.08, 29.3, 29.48, 29.58, 29.63, 29.7, 31.9, 32.1, 55.4, 62.5,
.83 (s, 3H), 1.95 (q, 2H, J = 6.5 Hz), 3.82 (m, 2H), 5.85 (m, 1H), 5.98
m, 1H), 6.23 (d, 1H, J = 15.5 Hz), 7.30 (m, 1H); 1 C NMR (CDCl ) ı
3
64.1, 74.2 (t, J = 18 Hz), 128.3 (t, J = 18 Hz), 133.6 (t, J = 18 Hz); HRMS
(
3
[
M+H]+ m/z calcd. for C18H35D NO , 303.3085, found, 303.3089;
3
2
1
6
1
4.1, 20.1, 28.4, 29.0, 29.1, 29.4, 29.5, 29.7, 32.2, 33.2, 33.3, (64.7),
5.0, (66.0), 66.3, (79.9), 80.0, (94.4), 95.3, 123.3, (124.4), (128.2),
29.2, (144.0), 144.2, (145.8), 146.4, 151.7, (152.2), (195.5), 196.0.
+
HRMS [M+Na] m/z calcd for C23H42D NNaO , 325.2905, found,
3
2
3
25.2905.
13
The C NMR chemical shifts in parentheses indicate the small
peaks that arise from minor rotamers in the oxazolidine system,
which undergoes a slow dynamic equilibrium at room temperature.
ꢀ ꢀ ꢀ
.9. N-tert-Butoxycarbonyl (4S)-4-[1 -hydroxy-1 -deutero-(2 E)-
hexadecanyl]-2,2-dimethyl-1,3-oxazolidine (15)
3
ꢀ
ꢀ
ꢀ
ꢀ
3
.6. N-tert-Butoxycarbonyl (4S)-[(1 S)-hydroxy-1 ,2 ,3 -
To the mixture of enone 14 (298 mg, 0.68 mmol) and CeCl ·7H O
3
2
ꢀ
trideuterio-(2 E)-hexadecenyl]-2,2-dimethyl-1,3-oxazolidine (12)
(
56 mg, 0.15 mmol) in 20 mL of THF and 2 mL of CD OD was
3
◦
added NaBD (37 mg, 0.89 mmol) at −78 C. The mixture was grad-
4
To a mixture of enone 11 (985 mg, 2.24 mmol) and CeCl ·7H O
3
2
ually raised to room temperature. After 10 h, the mixture was
filtered through a pad of Celite, which was washed with 150 mL
(
186 mg, 0.50 mmol) in 40 mL of THF and 4 mL of CD OD was added
3
◦
NaBD4 (123 mg, 2.94 mmol) at −78 C. The mixture was gradually
raised to room temperature. After 4 h, the mixture was filtered
through a pad of Celite, which was washed with 150 mL of EtOAc.
The filtrate washed with brine, dried (Na SO ), and concentrated
under reduced pressure. The residue was purified by column chro-
matography on silica gel (elution with hexane/EtOAc 9:1) to give
82 mg (89%) of alcohol 12: H NMR (CDCl ) ı 0.89 (t, 3H, J = 6.6 Hz),
.27 (m, 20H), 1.48 (s, 9H), 1.53 (s, 3H), 1.57 (s, 3H), 1.98 (br s,
H), 2.08 (q, 2H, J = 6.8 Hz), 4.03 (m, 1H), 4.08 (br s, 1H), 4.16 (br
m, 1H), 4.31 (br s, 1H), 6.28 (m, 1H); C NMR (CDCl ) ı 14.1,
0.0, 22.9, 24.4, 26.8, 28.1, 28.4, 29.5, 29.67, 29.72, 29.89, 29.99,
0.0, 32.3, 32.9, 33.1. 62.0, 73.6, 80.2, 94.6, 128.4, 128.7, 133.8,
34.1, 161.1; HRMS [M+H] m/z calcd. for C26H47D NO , 443.3923,
found, 443.3924. Additional [M+H] ions were also present: m/z
calcd. for C26H46D NO , 444.3985, found, 443.3972; m/z calcd. for
of EtOAc. The filtrate washed with brine, dried (Na SO ), and
2
4
concentrated under reduced pressure. A residue was purified by
column chromatography on silica gel (elution with hexane/EtOAc
2
4
9
:1) to give 240 mg (80%) of 3-deuterio-alcohol 15 as a color-
1
less oil: H NMR ı 0.88 (t, 3H, J = 6.6 Hz), 1.25 (m, 20H), 1.49 (s,
9
(
2
3
H), 1.30–1.70 (m, 10H), 2.05 (m, 2H), 3.75–4.25 (m, 4H), 5.44
1
8
1
1
13
3
d, 1H, J = 15.2 Hz), 5.73 (m, 1H);
C NMR ı 14.1, 10.0, 22.9,
4.4, 26.8, 28.1, 28.4, 29.5, 29.67, 29.72, 29.89, 29.99, 30.0, 32.3,
2.9, 33.1, 59.4, 61.3, 62.3, 64.8, 65.2, 66.1, 66.4, 73.4, 81.0 (t,
13
3
J = 24 Hz), 94.4, (95.0), 128.0, (129.5), 131.8, 133.4, (135.4), 171.3,
170.92).
1
3
1
(
+
3
4
+
3.10. (2S,3R)-2-(N-tert-Butoxycarbonylamido)-3-deutero-4(E)-
octadecene-1,3-diol (16)
4
4
C26H48D NO , 442.3860, found, 442.3858. The relative intensities
2
4
13
of these peaks, after correction for the natural abundance of C,
are 0.64, 0.20, and 0.16, respectively.
A solution of alcohol 15 (233 mg, 0.53 mmol) in 10 mL of 1 M
HCl and 50 mL of THF was stirred at room temperature until the
reaction was completed. The mixture was neutralized with satu-
rated aqueous NaHCO3 solution, and the product was extracted
with EtOAc (100 mL ×3). The combined organic layers were washed
3
.7. (2S,3R)-2-(N-tert-Butoxycarbonylamido)-4(E)-3,4,5-
trideuteriooctadecene-1,3-diol (13)
with brine, dried (Na SO ), and concentrated under reduced pres-
2
4
A solution of acetonide 12 (874 mg, 1.97 mmmol) in 1 M HCl
10 mL) and THF (20 mL) was stirred at room temperature until the
sure. The residue was purified by column chromatography on
silica gel (elution with hexane/EtOAc 2:1 then 1:1) to give 180 mg
(85%) of 3-deuterio-N-Boc-sphingosine 16: 1H NMR ı 0.88 (t, 3H,
J = 6.8 Hz), 1.25 (m, 20H), 1.45 (s, 9H), 2.05 (q, 2H, J = 7.1 Hz), 2.45
(br s, 2H), 3.61 (br s, 1H), 3.71 (m, 1H), 3.95 (m, 1H), 5.81 (br
s, 1H), 5.53 (d, 1H, J = 15.3 Hz), 5.78 (m, 1H); 13C NMR ı 14.1,
22.6, 22.7, 25.8, 29.0, 29.2, 29.3, 29.59, 29.62, 29.65, 31.7, 31.9,
32.7, 36.8, 55.3, 62.6, 64.2, 74.1 (t, J = 18 Hz), 79.8, 134.0, 138.0,
154.6.
(
reaction was completed. The mixture was neutralized with satu-
rated aqueous NaHCO3 solution, and the product was extracted
with EtOAc (3× 100 mL). The combined organic layers were washed
with brine, dried (Na SO ), and concentrated under reduced pres-
2
4
sure. The residue was purified by column chromatography on silica
gel (elution with hexane/EtOAc 2:1 then 1:1) to give 621 mg (78%)
of N-Boc-sphingosine-d 13 (containing minor amounts of the cor-
3

H.-S. Byun, R. Bittman / Chemistry and Physics of Lipids 163 (2010) 809–813
813
3.11. 3-Deuterio-N-palmitoyl-d-erythro-
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1
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Acknowledgments
We are grateful to Dr. Cliff Soll for HRMS analyses and to the
National Institutes of Health, Grant HL-083187, for financial sup-
port.