Synthesis and absolute configuration of 6-hydroxylated new ceramides in human skin, ceramides B, 4, 7 and 8

Article abstract of DOI:10.1002/ejoc.200500357

6R-Configuration was assigned to three new ceramides isolated from human skin such as ceramide B [1, (2S,3R,4E,6R)-6-hydroxy-N-(30′- hydroxytriacontanoyl)-4-sphingenine], ceramide 8 [2, (2S,3R,4E,6R)-6-hydroxy-N- (tetracosanoyl)-4-sphingenine] and ceramide 4 {3, (2S,3R,4E,6R)-6-hydroxy-N- [(30′-linoleoyloxy)triacontanoyl]-4-sphingenine}. (6R,2′ R)-Configuration was given to another ceramide in human skin, ceramide 7 [4, (2S,3R,4E,6R,2′ R)-6-hydroxy-N-(2′-hydroxytetracosanoyl)-4- sphingenine]. These assignments were made possible by enzymatic preparation of the enantiomers of 1-pentadecyn-3-ol and those of 2-hydroxytetracosanoic acid. Wiley-VCH Verlag GmbH & Co. KGaA, 2005.

Full text of DOI:10.1002/ejoc.200500357

FULL PAPER
Synthesis and Absolute Configuration of 6-Hydroxylated New Ceramides in
Human Skin, Ceramides B, 4, 7 and 8[‡]
Yui Masuda[a] and Kenji Mori*[a]
Keywords: Amino alcohols / Ceramides / Configuration determination / Sphingolipids
6R-Configuration was assigned to three new ceramides iso-
lated from human skin such as ceramide B [1, (2S,3R,4E,6R)-
ceramide 7 [4, (2S,3R,4E,6R,2ЈR)-6-hydroxy-N-(2Ј-hydroxy-
tetracosanoyl)-4-sphingenine]. These assignments were
6-hydroxy-N-(30Ј-hydroxytriacontanoyl)-4-sphingenine], cer- made possible by enzymatic preparation of the enantiomers
amide
sphingenine] and ceramide 4 {3, (2S,3R,4E,6R)-6-hydroxy-N-
[(30Ј-linoleoyloxy)triacontanoyl]-4-sphingenine}. (6R,2ЈR)-
8
[2, (2S,3R,4E,6R)-6-hydroxy-N-(tetracosanoyl)-4-
of 1-pentadecyn-3-ol and those of 2-hydroxytetracosanoic
acid.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
Configuration was given to another ceramide in human skin,
Introduction
Ceramides are predominant lipids of human epidermal
stratum corneum, acting as the water barrier to prevent loss
of body water.[1,2] They are classified into two groups, free
ceramides and protein-bound ones.[3,4] In the former case,
the acyl side-chain of ceramides do not possess an ω-hy-
droxy group, while in the latter it is present to enable the
binding of ceramides with proteins. In 1994, Downing and
co-workers reported the isolation and identification of se-
veral new ceramides with a hydroxy group at C-6 of its
sphingosine part as shown in Figure 1.[3] Some of them are
free ceramides such as ceramide 8 (2), ceramide 4 (3) and
ceramide 7 (4), while ceramide B (1) is a protein-bound cer-
amide.[3,4] Downing assigned the structures 1–4 to these
new 6-hydroxy-4-sphingenine-based ceramides by extensive
1H NMR studies.
As to the stereochemistry of 1–4, they almost certainly
possess (2S,3R,4E)-configuration, since all the known
mammalian sphingosines possess that configuration.[5]
However, Downing was unable to determine the absolute
configuration at C-6 of these 6-hydroxylated ceramides.
This aroused interests among synthetic chemists, and three
syntheses of 6-hydroxy-4-sphingenines were reported to
date.[6–8] We were the first to determine the 6R-configura-
tion of ceramide B as reported in our preliminary com-
munication.[8] In the case of ceramide 7 (4), it possesses an
additional hydroxy group at C-2Ј of the acyl side-chain,
Figure 1. Structures of 6-hydroxylated new ceramides of human
skin.
[‡] Synthesis of Sphingosine Relatives, XXVII. Part XXVI: Y. Ma-
suda, K. Mori, J. Indian Chem. Soc. 2003, 80, 1081–1083.
[a] Glycosphingolipid Synthesis Group, Laboratory for Immune
Regulation, RIKEN, Research Center for Allergy and Immu-
nology, c/o Seikagaku Corporation,
whose absolute configuration must also be determined.
Some marine sphingolipids are known to possess 2ЈR-hy-
droxy group in its acyl side-chain, as in the case of the sex
pheromone [(2S,3S,4R,2ЈR)-2-(2Ј-hydroxy-21Ј-methyldoco-
sanoylamino)-1,3,4-pentadecanetriol] of the female hair
Tateno 3-1253, Higashiyamato-shi, Tokyo 207-0021, Japan
Fax: +81-42-565-9906
E-mail: mori-riken@crocus.ocn.ne.jp
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crab, Erimacrus isenbeckii,[9,10] and also in the case of glyco- from 15-pentadecanolide (5) by employing the Wittig reac-
sphingolipids from marine sponge, Agelas clathrodes.[11] tion between 6 and 7 as the key step according to Mori and
Nevertheless, the absolute configuration at C-2Ј of ceramide Matsuda.[15] Acid 8 was converted into p-nitrophenyl 30-
7 (4) must be elucidated beyond doubt. This paper describes linoleoyloxytriacontanoate (9
= E) as reported pre-
our synthesis of 1–4, which resulted in the determination of viously.[15]
their stereochemistry as shown in Figure 1.
The first step of the synthesis of (R)-2-acetoxytetracos-
anoic acid (13 = G) was bromination of tetracosanoic acid
(10) with bromine in the presence of phosphorus. The re-
sulting ( )-2-bromo acid 11 was treated with aqueous so-
dium hydroxide to give the ( )-2-hydroxy acid 12. Its enzy-
matic kinetic resolution was achieved with lipase PS (Am-
ano) and vinyl acetate at 65 °C in the presence of a small
amount of butylated hydroxytoluene (BHT) according to
Sugai and Ohta,[16][10] to give (S)-2-acetoxy acid 13 and the
recovered (R)-2-hydroxy acid 12. Their enantiomeric purit-
ies were determined by HPLC analysis of the corresponding
(R)-2-methoxy-2-phenyl-2-(trifluoromethyl)acetates
(Mosher esters), (R)- and (S)-14, and found to be 95% ee
for both. The hydroxy acid (R)-12[17] was acetylated to give
the (R)-2-acetoxy acid 13.[17–19] Thus, lipase-catalyzed
asymmetric acetylation proved to be a very practical
method to make both the enantiomers of 13 available.
Results and Discussion
Retrosynthetic Analysis of the Ceramides 1–4
Because all of the target ceramides 1–4 are structurally
related, our plan was to synthesize them according to the
general retrosynthetic analysis as shown in Scheme 1. All
the ceramides 1–4 can be prepared from the protected 6-
hydroxy-4-sphingenine A, which is to be synthesized by tre-
ating Garner’s aldehyde B[12–14] with either the dianion de-
rived from acetylenic alcohol C or the anion derived from
D. For the synthesis of ceramide 4 (3), the protected 6-hy-
droxy-4-sphingenine A is to be acylated with the activated
ester E, which is a known compound prepared from F.[15]
Acylation of A with F eventually gives ceramide B (1). For
the synthesis of ceramide 7 (4), A must be acylated with
(R)-2-acetoxy acid G, which is to be prepared from com-
mercially available acid H. Tetracosanoic acid H and A
yields ceramide 8 (2). It is therefore evident that the most
important point in the present synthesis is how to prepare
pure acetylenic alcohol C and its enantiomer.[8]
Synthesis of the Enantiomers of Acetylenic Alcohol 18 (=
C)
Several asymmetric reactions were reported to be useful
in preparing optically active 1-alkyn-3-ols. Asymmetric ad-
Synthesis of the Acid Parts E, F and G
As shown in Scheme 2, the known 30-tert-butyldiphenyl- dition of a zinc acetylide to aldehydes in the presence of
silyl(TBDPS)oxytriacontanoic acid (8 = F) was synthesized (+)-N-methylephedrine was recently reported by Carreira to
Scheme 1. Retrosynthetic analysis of the target ceramides 1–4.
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Scheme 2. Synthesis of the acid parts 8, 9 and (R)-13; reagents (a) Br2, P (77%); (b) NaOH, H2O (97%); (c) lipase PS (Amano),
CH2=CHOAc, BHT, THF [38% for (R)-12, 38% for (S)-13]; (d) K2CO3, MeOH (quant.); (e) CH2N2, Et2O; (f) (S)-MTPACl, C5H5N;
(g) Ac2O, C5H5N (99%).
give optically active 1-alkyn-3-ols (91-98% ee).[20] Simulta- 16 with vinyl acetate in the presence of lipase PS (Amano)
neously with our present work, asymmetric acetylation of on Celite in diisopropyl ether for 10 days at room tempera-
( )-1-alkyn-3-ols with vinyl acetate and lipases was found ture was followed by chromatographic purification to give
to give (S)-1-alkyn-3-ols and the corresponding (R)-acetates the acetylated (R)-17 [99% ee as determined by the HPLC
(ca. 90% ee).[21–23] It must be added that asymmetric hy- analysis of the corresponding (R)-MTPA ester 19] in 49%
drolysis of the acetates of ( )-1-alkyn-3-ols with Bacillus
subtilis esterase gave optically active acetates and alcohols
of only mediocre enantiomeric purity.[24]
Because substantical amounts of the enantiomers of 1-
pentadecyn-3-ol (C = 18) were necessary to synthesize 1–4,
we searched for a simple method which could afford both
the enantiomers of 18 efficiently. Our choice was to adopt
the method reported by Anastasia, that is, lipase-catalyzed
enantiomer separtion of ( )-1-trimethylsilyl(TMS)-1-alkyn-
3-ols.[25] By attaching a TMS group at C-1, the enantio-
selectivity of asymmetric acetylation with lipase could be
improved to give long-chain 1-alkyn-3-ols of Ͼ90% ee. In-
deed, lipase PS and vinyl acetate converted ( )-1-trimethyl-
silyl-1-alkyn-3-ols to (R)-acetates and (S)-alcohols in a sin-
gle step without repeating the enzymatic kinetic resolu-
tion.[22]
Scheme 3. Synthesis of the enantiomers of 1-pentadecyn-3-ol (18);
reagents: (a) TMSCϵCH, nBuLi, THF (85%); (b) lipase PS-C,
Scheme 3 summarizes our results. Treatment of tride-
canal (15) with lithium trimethylsilylacetylide in THF fur-
nished the alkynol ( )-16. Asymmetric acetylation of ( )- C5H5N; (e) TBSCl, imidazole, DMF (quant.).
CH2=CHOAc, (iPr)2O [49% for (R)-17, 48% for (S)-16]; (c)
K2CO3, MeOH [92% for (R)-18, 95% for (S)-18]; (d) (S)-MTPACl,
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yield based on ( )-16, and the recovered (S)-16 [98% ee as bond to an (E)-double bond, and also to remove the protec-
determined by the HPLC analysis of the corresponding (R)- tive group, affording free 6-hydroxy-4-sphingenine. Protec-
MTPA ester 19] in 48% yield based on ( )-16. Both (R)-17 tion of its hydroxy groups as TBS ethers gave
and (S)-16 were converted into the parent acetylenic (2S,3R,4E,6R)-23, which was acylated with carboxylic acid
alcohols, (R)-18, m.p. 40.5–42.0 °C, [α]2D5 = +2.49 (c = 1.20, 8 in the presence of 3-(3-dimethylaminopropyl)-1-ethylcar-
CHCl3), and (S)-18, m.p. 41.0–42.0 °C, [α]2D5 = –2.30 (c = bodiimide hydrochloride (EDC) and 1-hydroxybenzotri-
1.00, CHCl3). These two enantiomers of 18 were silylated azole (HOBt) to give (2S,3R,4E,6R)-24. At this stage chro-
to the corresponding tert-butyldimethylsilyl (TBS) ethers matographic purification was possible over silica gel to give
(R)- and (S)-20. The overall yield of (R)- and (S)-18 was pure protected ceramide (2S,3R,4E,6R)-24 as the major
ca. 38% based on 15 (3 steps).
product in 15% yield based on (S)-21. The diastereoiso-
meric ratio of the products was (2S,3R,4E,6R)-24/
(2S,3S,4E,6R)-24, 4.2:1. Deprotection of the TBS protective
groups of (2S,3R,4E,6R)-24 with tetra(n-butyl)ammonium
fluoride (TBAF) gave (2S,3R,4E,6R)-1 as colorless powder,
m.p. 113.5–117.0 °C. The overall yield of (2S,3R,4E,6R)-1
was 8.3% based on (S)-Garner’s aldehyde (21, 5 steps).
Similarly, by employing (S)-alkynol 18, (2S,3R,4E,6S)-1
was also synthesized.
In order to improve the yield and diastereoselectivity at
the coupling step of (S)-21 with alkynol (R)-18, coupling of
(S)-21 with the TBS-protected reactant (R)-20 was at-
tempted as shown in Scheme 5. The product (4S,1ЈR,4ЈR)-
25 was stable enough to be purified by silica gel chromatog-
raphy, and obtained in 81% yield.
Synthesis of Ceramide B (1)
Scheme 4 illustrates further conversion of the acetylenic
alcohol (R)-18 to ceramide B (1). The alkynol (R)-18 was
treated with 2.2 equiv. of n-butyllithium in THF to afford
the corresponding dianion, to which was added (S)-Gar-
ner’s aldehyde (21) at –40 °C under argon. The product
(4S,1ЈR,4ЈR)-22 was rather unstable to preclude chromato-
graphic purification on silica gel. It was therefore immedi-
ately treated with lithium in ethylamine to reduce the triple
Scheme 5. Alternative synthesis of (2S,3R,4E,6R)-23; reagents: (a)
nBuLi, THF, HMPA (81%); (b) TBAF, THF; (c) Li, EtNH2, THF;
(d) TBSOTf, 2,6-lutidine, CH2Cl2 (3 steps, 37%).
The diastereoselectivity at this coupling step was im-
proved [(4S,1ЈR,4ЈR)-25/(4S,1ЈS,4ЈR)-25, 19:1]. The TBS
protective group of (4S,1ЈR,4ЈR)-25 was then removed with
TBAF, and the resulting acetylenic diol (4S,1ЈR,4ЈR)-22 was
converted to (2S,3R,4E,6R)-23 by reduction with lithium in
ethylamine followed by silylation. The overall yield of 23
could not be improved so much, and remained as 23% in
comparison to 15% in the previous case of employing the
dianion (R)-18.
Fortunately, it was available to us the detailed 600 MHz
1H NMR spectroscopic data including the spectral chart
itself of ceramide B tetraacetate.[3] We measured the
500 MHz 1H NMR spectra of our synthetic two dia-
stereomers of ceramide B tetraacetate, (2S,3R,4E,6R)-1Ј
and (2S,3R,4E,6S)-1Ј. As shown in Figure 2, the two spec-
Scheme 4. Synthesis of ceramide B (1); ragents: (a) nBuLi, THF;
(b) Li, EtNH2, THF; (c) TBSOTf, 2,6-lutidine, CH2Cl2 (3 steps,
15%); (d) EDC, HOBt, 8, CH2Cl2 (72%); (e) TBAF, THF (64%);
(f) Ac2O, C5H5N (85%).
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1
Figure 2. H NMR spectra at 500 MHz of (a) (2S,3R,4E,6R)-1Ј and (b) (2S,3R,4E,6S)-1Ј as CDCl3 solutions.
tra were clearly different with regard to the chemical shifts
of the protons at C-4,5 and C-6, and the spectrum of
(2S,3R,4E,6R)-1Ј was identical to that of ceramide B tetra-
acetate. The stereochemistry of ceramide B at C-6 must be
R, assuming that it shares in common the (2S,3R,4E)-ste-
reochemistry of the mammalian sphingolipids.
Synthesis of Ceramide 8 (2) and Ceramide 4 (3)
Acylation of the protected sphingosine (2S,3R,4E,6R)-23
with tetracosanoic acid (10) under the conventional condi-
tions was followed by deprotection to afford ceramide 8
[(2S,3R,4E,6R)-2] as shown in Scheme 6. The 1H NMR
spectroscopic data (500 MHz) of synthetic ceramide 8 tri-
acetate was in good accord with those reported for the tri-
acetate of the naturally occurring ceramide 8.[26]
For the synthesis of ceramide 4, the protected sphingo-
sine (2S,3R,4E,6R)-23 was acylated with the long-chain ac-
tivated ester 9. After deprotection, ceramide
4
[(2S,3R,4E,6R)-3] was obtained as a colorless solid. Its tri-
acetate showed 1H NMR spectroscopic data identical to
those of the triacetate of the naturally occurring ceramide
4.[26] Accordingly, both ceramide 8 (2) and ceramide 4 (3)
were shown to possess R-configuration at C-6, assuming
their common (2S,3R,4E)-stereochemistry.
Scheme 6. Synthesis of ceramide 8 (2) and ceramide 4 (3): reagents:
(a) EDC, HOBt, CH2Cl2 (96%); (b) TBAF, THF (71% for 2; 73%
for 3); (c) C5H5N (76%).
Synthesis of Ceramide 7 (4) and Its Diastereomer at C-2Ј
In the case of ceramide 7 (4), too, we assumed its com-
mon (2S,3R,4E,6R)-configuration, and planned to synthe-
size its two diastereomers at C-2Ј, (2S,3R,4E,6R,2ЈR)-4 and
(2S,3R,4E,6R,2ЈS)-4, one of which must be the natural
product.
Similarly, acylation of 23 with (S)-13 gave the protected
ceramide 7 (2S,3R,4E,6R,2ЈS)-26, which was deprotected to
As shown in Scheme 7, acylation of the protected sphin- give (2S,3R,4E,6R,2ЈS)-4. The corresponding tetraacetate
gosine (2S,3R,4E,6R)-23 with (R)-2-acetoxy acid 13 yielded (2S,3R,4E,6R,2ЈS)-4Ј was prepared, and the two tetraace-
1
totally protected ceramide 7 (2S,3R,4E,6R,2ЈR)-26. De- tates were compared by H NMR analysis.
1
acetylation followed by desilylation of 26 gave
Figure 3 shows H NMR spectra (500 MHz) of the two
(2S,3R,4E,6R,2ЈR)-4, which was converted into the corre- diastereomers of 4Ј. They are definitely different from each
sponding tetraacetate (2S,3R,4E,6R,2ЈR)-4Ј. other, especially with regard to the signals due to two pro-
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tons at C-1. The 1H NMR spectrum (600 MHz) of naturally
occurring ceramide tetraacetate[3] was identical with that of
(2S,3R,4E,6R,2ЈR)-4Ј. Accordingly, the stereochemistry of
ceramide 7 (4) was determined as 2S,3R,4E,6R,2ЈR.
It must be noted that the R-configuration at C-2Ј of cera-
mide 7 is same as that of the known marine sphingolip-
ids.[10,11]
Conclusions
Synthesis of all of the four 6-hydroxylated ceramides in
human skin was accomplished, and their hitherto unknown
stereochemistries at C-6 and C-2Ј were determined as
shown in Figure 1, with 6R [ceramide B (1), ceramide 8 (2),
and ceramide 4 (3)] and 6R,2ЈR [ceramide 7 (4)] configura-
tion.
The overall yield of ceramides based on tridecanal (15)
were 5.4% (10 steps) for ceramide B (1), 33% (6 steps) for
ceramide 8 (2), 27% (6 steps) for ceramide 4 (3) and 5.0%
(11 steps) for ceramide 7 (4). The overall yield of ceramide
7 (4) was 12.1% (7 steps) based on tetracosanoic acid (10).
Enzymatic asymmetric acetylation of 1-trimethylsilyl-1-al-
kyn-3-ol was demonstrated to be a useful method for the
preparation of the enantiomers of 1-alkyn-3-ol.
Experimental Section
Melting point (Yanaco MP-S3) are uncorrected. IR spectra were
1
measured with a Jasco FT/IR-460 spectrometer. H NMR spectra
were recorded at 90 MHz by a Jeol JNM-EX 90A spectrometer, at
400 MHz by a Jeol JNM-LA400 spectrometer, and at 500 MHz by
a Jeol JNM-LA500 spectrometer. The peak for TMS, or CHCl3 in
CDCl3 (at δ = 7.26 ppm), was used as the internal standard. 13C
NMR spectra were recorded at 100 MHz by a Jeol JNM-LA400
spectrometer and at 126 MHz by a Jeol JNM-LA500 spectrometer.
The peak for CDCl3 (at δ = 77.0 ppm) was used as the internal
Scheme 7. Synthesis of ceramide 7 (4); reagents: (a) EDC, HOBt,
CH2Cl2 (73%); (b) K2CO3, MeOH (78%); (c) TBAF, THF (75%);
(d) Ac2O, C5H5N (92%).
1
Figure 3. H NMR spectra at 500 MHz of (a) (2S,3R,4E,6R,2ЈR)-4Ј and (b) (2S,3R,4E,6R,2ЈS)-4Ј as CDCl3 solutions.
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standard. Optical rotation values were measured with a Jasco P-
1010 polarimeter, and mass spectra were measured with a Jeol
JMS-SX102A spectrometer. Column chromatography was carried
tR = 15.64 min (97.5%); (R)-MTPA ester of (S)-12 methyl ester: tR
= 19.64 min (2.5%). The enantiomeric purity of (R)-12 was there-
fore 95% ee.
(S)-2-Acetoxytetracosanoic Acid [(S)-13]: Lipase PS (960 mg) was
suspended in a solution of ( )-12 (1.00 g, 2.6 mmol), and BHT
(9 mg) in vinyl acetate (10 mL) and THF (10 mL), and the mixture
was stirred at 65 °C for 20 h. After filtration and concentration, the
residue was chromatographed on silica gel (20 g, CHCl3/MeOH,
30:1) to give (S)-13 (422 mg, 38%) as colorless powder, m.p. 72.5–
73.5 °C. [α]2D3 = –8.9 (c = 1.0, CHCl3). ref.[18]: m.p. 81–82 °C, [α]2D0
out on Merck Kieselgel 60 Art 1.07734, and TLC analyses were
performed on Merck 60F–254 silica gel plates.
( )-2-Bromotetracosanoic Acid [( )-11]: Red phosphorus (1.24 g,
40 mmol) was added to tetracosanoic acid (10) (11.4 g, 31 mmol),
and the mixture was heated at 95 °C. Bromine (19.7 g, 6.4 mL,
124 mmol) was added dropwise to the heated mixture, and heating
and stirring were continued at 95 °C for 6 h. Water was then added,
and the mixture was stirred for 20 min. The separated acid was
extracted with diethyl ether, and the extract was successively
washed with water and brine, dried with MgSO4, and concentrated
under reduced pressure. The residue was recrystallized from hexane
= –7.65 (c = 0.98, CHCl ). IR (KBr): ν = 3185 cm–1 (br. m, CO2H),
˜
3
1745 (m, C=O), 1690 (m, CO2H), 1240 (m, C–OAc) cm–1. 1H NMR
(500 MHz, CDCl3): δ = 0.88 (t, J = 7.0 Hz, 3 H, 24-H3), 1.10–1.31
(m, 41 H, 4–23-H2, CO2H), 1.34–1.45 (m, 2 H, 3-H2), 2.17 (s, 3 H,
OAc), 5.00–5.06 (m, 1 H, 2-H) ppm. HRMS: m/z [M + H]+: calcd.
to give ( )-11 (10.7 g, 77%) as a colorless solid, m.p. 71.0–74.0 °C. for C26H51O4, 427.3787; found, 427.3785.
IR (KBr): ν = 3000 cm–1 (br., O–H), 1700 (s, C=O), 900 (m), 660
˜
Determination of the Enantiomeric Purity of (S)-13: (S)-2-Methoxy-
2-phenyl-2-(trifluoromethyl)acetyl chloride (MTPACl; 50 mg,
0.2 mmol) was added to a solution of the hydroxylated methyl ester
derived from (S)-13 [50 mg, 0.13 mmol; prepared by treating (S)-
13 with diazomethane after deacetylation] in dry pyridine (2 mL),
and the mixture was stirred at 0 °C for 12 h. The reaction was
1
(w) cm–1. H NMR (90 MHz, CDCl3): δ = 0.68–1.10 (m, 3 H, 24-
H3), 1.11–1.70 (m, 41 H, 4–23-H2, CO2H), 1.81–2.21 (m, 2 H, 3-
H2), 4.24 (t, J = 7.3 Hz, 1 H, 2-H) ppm. C24H47BrO2 (447.5): calcd.
C 64.41, H 10.59; found C 64.18, H 10.69.
( )-2-Hydroxytetracosanoic Acid [( )-12]: 2 m NaOH aq. (200 mL)
was added to ( )-11 (19.0 g, 42.3 mmol), and the mixture was quenched by adding water, and the mixture was diluted with diethyl
stirred and heated at 85 °C for 30 h. After cooling, the mixture was
acidified with diluted aq. HCl and extracted with diethyl ether. The
extract was successively washed with water and brine, dried with
MgSO4, and concentrated under reduced pressure. The residue was
recrystallized from acetone to give ( )-12 (15.7 g, 97%) as a color-
ether. The extract was successively washed with saturated aq.
CuSO4, saturated aq. NaHCO3, water and brine, dried with
Na2SO4, and concentrated under reduced pressure. The residue was
analyzed by HPLC (Pegasil-Senshu column, 25 cm×4.6 mm; elu-
ent n-hexane/ethyl acetate, 60:1; flow rate, 1 mL /min). (R)-MTPA
less solid, m.p. 100.0–103.0 °C. IR (KBr): ν = 3445 cm–1 (br. m, O– ester of the (R)-13 methyl ester: tR = 15.78 min (2.5%); (R)-MTPA
˜
H), 1745 (m, C=O) cm–1. 1H NMR (90 MHz, CDCl3/CD3OD, 1:1):
ester of the (S)-13 methyl ester: tR = 19.82 min (97.5%). The enan-
δ = 0.58–0.75 (m, 3 H, 24-H3), 1.04–1.24 (m, 42 H, 4–23-H2, OH, tiomeric purity of (S)-13 was therefore 95% ee
CO2H), 1.26–1.44 (m, 2 H, 3-H2), 3.92 (t, J = 5.8 Hz, 1 H, 2-H)
(R)-2-Acetoxytetracosanoic Acid [(R)-13]: To a stirred solution of
(R)-12 (250 mg, 0.64 mmol) in dry pyridine (10 mL) was added
Ac2O (20 mL). The reaction mixture was stirred for 4 h at room
ppm. C24H48O3 (384.6): calcd. C 74.94, H 12.58; found C 74.76, H
12.32.
(R)-2-Hydroxytetracosanoic Acid [(R)-12]: Lipase PS (Amano En- temperature. It was then poured into water and extracted with ethyl
zyme, Inc.; 4.54 g) was suspended in a solution of ( )-12 (5.00 g,
13.0 mmol), and BHT (butylated hydroxytoluene, 200 mg) in vinyl
acetate (48 mL) and THF (48 mL), and the mixture was stirred at
65 °C for 30 h. After cooling, the mixture was filtered through Ce-
lite, and the Celite layer was washed with diethyl ether. The com-
bined filtrate and washings were concentrated under reduced pres-
sure, and the residue was dissolved in vinyl acetate (48 mL) and
THF (48 mL) containing BHT (200 mg). Lipase PS (4.54 g) was
added to the solution, and the mixture was stirred at 65 °C for 3 h.
After filtration and concentration, the residue was recrystallized
from acetone to give (R)-12 (950 mg, 38%) as colorless powder,
m.p. 101.0–102.0 °C. [α]2D3 = –2.7 (c = 0.1, CHCl3/MeOH, 1:1).
ref[17]: m.p. 104.5–105.5 °C, [α]2D5 = +3.4 (c = 1.6, pyridine). Its IR
and 1H NMR spectra were identical with those of ( )-12. C24H48O3
(384.6): calcd. C 74.94, H 12.58; found C 74.74, H 12.65.
acetate. The extract was successively washed with water and brine,
dried with MgSO4, and concentrated under reduced pressure. The
residue was chromatographed on silica gel (5 g, CHCl3/MeOH,
30:1) to give (R)-13 as colorless powder, m.p. 72.5–75.5 °C. [α]2D3
=
+8.8 (c = 1.0, CHCl3). ref.[17]: m.p. 65–67 °C, [α]2D6 = +2.6 (c = 1.0,
CHCl3); ref.[18] m.p. 81.5–82.5 °C, [α]2D0 = –8.46 (c = 1.01, CHCl3).
This negative [α]D value must be in error, because the same sample
was reported to be: [α]2D0 = +8.5 (c = 1.0, CHCl3).[19] Sugai and
Ohta reported the specific rotation of (S)-13 as: [α]2D0 = –7.65 (c =
1
0.98, CHCl3).[18] Its IR and H NMR spectra were identical with
those of (S)-13. HRMS: m/z [M + H]+: calcd. for C26H51O4,
427.3787; found, 427.3790.
( )-1-Trimethylsilyl-1-pentadecyn-3-ol [( )-16]: To a stirred solu-
tion of trimethylsilylacetylene (14.9 g, 151 mmol) in dry THF
(500 mL) was added n-butyllithium (1.59 m in hexane; 100 mL,
159 mmol) dropwise at –78 °C under Ar. After the reaction mixture
Determination of the Enantiomeric Purity of (R)-12: (S)-2-Methoxy-
2-phenyl-2-(trifluoromethyl)acetyl chloride (MTPACl; 50 mg, had been stirred at –78 °C for 1 h, a solution of tridecanal (15)
0.2 mmol) was added to a solution of the methyl ester derived from
(R)-12 [50 mg, 0.13 mmol; prepared by treating (R)-12 with ethe-
real diazomethane] in dry pyridine (2 mL), and the mixture was
stirred at 0 °C for 12 h. The reaction was quenched by adding
water, and the mixture was diluted with diethyl ether. The extract
was successively washed with saturated aq. CuSO4, saturated aq.
NaHCO3, water and brine, dried with Na2SO4, and concentrated
under reduced pressure. The residue was analyzed by HPLC (Pega-
sil-Senshu column, 25 cm×4.6 mm; eluent n-hexane/ethyl acetate,
60:1; flow rate, 1 mL/min). (R)-MTPA ester of (R)-12 methyl ester:
(20 g, 101 mmol) in dry THF (500 mL) was added dropwise at
–78 °C. The mixture was then stirred for 5 h while being warmed
to room temperature, before being quenched with NH4Cl aq. and
extracted with ethyl acetate. The extract was successively washed
with water and brine, dried with MgSO4, and concentrated under
reduced pressure. The residue was chromatographed on silica gel
(400 g, hexane/ethyl acetate, 30:1) to give ( )-16 (25.4 g, 85%) as a
colorless oil, n2D6 = 1.4546. IR (film): ν = 3315 cm–1 (w, OH), 2170
˜
1
(w, CϵC), 1250 (s, Si–Me) cm–1. H NMR (90 MHz, CDCl3): δ =
0.17 (m, 9 H, Si–Me), 0.88 (m, 3 H, 15-H3), 1.26–1.41 (m, 22 H,
Eur. J. Org. Chem. 2005, 4789–4800
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4795

Y. Masuda, K. Mori
FULL PAPER
4–14-H2), 1.72 (d, J = 5.5 Hz, 1 H, OH), 4.35 (dt, J = 5.5, 6.4 Hz,
1 H, 3-H) ppm. C18H36OSi (296.6): calcd. C 72.90, H 12.24; found
C 72.63, H 12.30.
and analyzed under the same conditions. (R)-MTPA ester of (R)-
18: tR = 9.77 min (1.0%); (R)-MTPA ester of (S)-18: tR = 10.94 min
(99.0%). The enantiomeric purity of (S)-18 was therefore 98% ee
(S)-1-Trimethylsilyl-1-pentadecyn-3-ol [(S)-16] and (R)-3-Acetoxy- (R)-3-tert-Butyldimethylsilyloxy-1-pentadecyne [(R)-20]: To
a
1-trimethylsilyl-1-pentadecyne [(R)-17]: Lipase PS-C (Amano En-
zyme, Inc.; 12.2 g) was suspended in a solution of ( )-16 (14.5 g,
49.0 mmol) in vinyl acetate (290 mL) and (iPr)2O (580 mL), and
the mixture stirred at room temperature for 10 d. The mixture was
filtered through Celite, and the Celite layer was washed with diethyl
ether. The combined filtrate and washings were concentrated under
reduced pressure. The residue was chromatographed on silica gel
(900 g, hexane/ethyl acetate, 60:1) to give (S)-16 (7.27 g, 48%) and
(R)-17 (8.12 g, 49%) as a colorless oil.
stirred solution of (R)-18 (2.0 g, 8.91 mmol) in DMF (50 mL) was
added imidazole (1.29 g, 18.7 mmol) and TBSCl (1.47 g,
9.74 mmol). The reaction mixture was stirred for 3 h at room tem-
perature. It was then poured into water and extracted with diethyl
ether. The extract was successively washed with water, diluted HCl
aq. and brine, dried with MgSO4, and concentrated under reduced
pressure. The residue was chromatographed on silica gel (60 g, hex-
ane) to give (R)-20 (3.0 g, quant.) as a colorless oil, n2D2 = 1.4442.
[α]2D3 = +32.3 (c = 1.0, CHCl ). IR (film): ν 3310 (m, HCϵC–),
˜
3
2115 (w, HCϵC–), 1250 (s, Si–Me) cm–1. 1H NMR (500 MHz,
CDCl3): δ = 0.11, 0.13 (each s, 6 H, Si–Me), 0.86 (t, J = 6.9 Hz, 3
H, 15-H3), 0.91 (s, 9 H, tBu), 1.10–1.27 (m, 20 H, 5–14-H2), 1.63–
1.70 (m, 2 H, 4-H2), 2.37 (d, J = 2.1 Hz, 1 H, 1-H), 4.33 (dt, J =
2.1, 6.6 Hz, 1 H, 3-H) ppm. C21H42OSi (338.6): calcd. C 74.48, H
12.50; found C 74.48, H 12.78.
1
(S)-16: n2D3 = 1.4576. [α]2D2 = +1.1 (c = 1.10, CHCl3). Its IR and H
NMR spectra were identical with those of ( )-16. C18H36OSi
(296.6): calcd. C 72.90, H 12.24; found C 73.15, H 12.52.
(R)-17: n2D5 = 1.4486. [α]2D5 = +65.4 (c = 1.05, CHCl ). IR (film): ν
˜
3
= 2180 cm–1 (w, CϵC), 1750 (s, C=O) cm–1. 1H NMR (90 MHz,
CDCl3): δ = 0.17 (m, 9 H, Si–Me), 0.80 (m, 3 H, 15-H3), 1.27–1.45
(m, 22 H, 4–14-H2), 2.08 (s, 3 H, OAc), 5.37 (t, J = 6.7 Hz, 1 H,
3-H) ppm. C20H38O2Si (338.6): calcd. C 70.94, H 11.31; found C
70.99, H 11.56.
tert-Butyl (4S,1ЈR,4ЈR)-4-(1Ј,4Ј-Dihydroxy-2Ј-hexadecynyl)-2,2-di-
methyl-3-oxazolidinecarboxylate [(4S,1ЈR,4ЈR)-22]: To
a stirred
solution of (R)-18 (1.00 g, 4.45 mmol) in dry THF (40 mL) was
added n-butyllithium (1.56 m in hexane; 6.28 mL, 9.8 mmol) drop-
wise at –78 °C under Ar. After the reaction mixture had been
stirred at –78 °C for 1.5 h, a solution of Garner’s aldehyde [(S)-21,
1.53 g, 6.68 mmol] in dry THF (40 mL) was added dropwise at
–78 °C. The mixture was then stirred overnight while being warmed
to 0 °C, before being quenched with NH4Cl aq. and extracted with
diethyl ether. The extract was successively washed with water and
brine, dried with MgSO4, and concentrated under reduced pressure
to give (4S,1ЈR,4ЈR)-22 (2.52 g, crude) as a yellow oil. Because of its
instability, this compound was used for the next reaction without
purification.
(R)-1-Pentadecyn-3-ol [(R)-18]: To a stirred solution of (R)-17
(13.7 g, 40.4 mmol) in MeOH (175 mL) was added K2CO3 (16.4 g,
119 mmol) at room temperature. The mixture was stirred for 4 h at
room temperature, before the solvent was removed under reduced
pressure. The concentrate was diluted with water and extracted
with diethyl ether. The resulting extract was successively washed
with water, satd. NaHCO3 aq. and brine, dried with MgSO4, and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel (300 g, hexane/ethyl acetate, 30:1) to give (R)-
18 (8.33 g, 92%) as a colorless solid, m.p. 40.5–42.0 °C. [α]2D4
=
+2.49 (c = 1.10, CHCl3). ref.[6]: m.p. 40–41 °C, [α]2D5 = +2.60 (c =
1.0, CHCl3); ref.[7]: m.p. not reported, [α]D = +1.3 (c = 1.05,
tert-Butyl (4S,1ЈR,4ЈS)-4-(1Ј,4Ј-Dihydroxy-2Ј-hexadecynyl)-2,2-di-
methyl-3-oxazolidinecarboxylate [(4S,1ЈR,4ЈS)-22]: In the same
manner as that just described, (S)-18 (1.00 g, 4.45 mmol) and Gar-
ner’s aldehyde [(S)-21, 1.53 g, 6.68 mmol] were converted into
2.01 g of (4S,1ЈR,4ЈS)-22 (2.78 g) as a yellow oil. Because of its
instability, this compound was used for the next reaction without
purification.
CHCl ). IR (film): ν = 3335 cm–1 (m, O–H), 3280 (m, HCϵC–),
˜
3
2115 (w, HCϵC–) cm–1. 1H NMR (90 MHz, CDCl3): δ = 0.78–
0.95 (m, 3 H, 15-H3), 1.08–1.81 (m, 23 H, 4–14-H2, OH), 2.46 (d,
J = 2.2 Hz, 1 H, 1-H), 4.25–4.40 (dt, J = 2.2, 6.4 Hz, 1 H, 3-H)
ppm. C15H28O (224.4): calcd. C 80.29, H 12.58; found C 80.06, H
12.85.
(2S,3R,4E,6R)-2-Amino-1,3,6-tris(tert-butyldimethylsilyloxy)-4-
octadecene [(2S,3R,4E,6R)-23]: A solution of crude (4S,1ЈR,4ЈR)-
22 (2.62 g) in dry THF (40 mL) was added dropwise to a blue solu-
tion of lithium (1.00 g, 148 mmol) in ethylamine (30 g) while stir-
ring at –78 °C for 1 h. The mixture was then stirred overnight while
being warmed to room temperature, before being quenched with
NH4Cl (ca. 16 g, 295 mmol). After removing ethylamine by evapo-
ration, the mixture was diluted with saturated aq. NH4Cl and ex-
tracted with CHCl3. The extract was dried with Na2SO4, and con-
(S)-1-Pentadecyn-3-ol [(S)-18]: In the same manner as that just de-
scribed, (S)-16 (13.2 g, 44.4 mmol) was converted into 9.46 g (95%)
of (S)-18 as a colorless solid, m.p. 42.0–43.0 °C. [α]2D5 = –2.30 (c =
1.00, CHCl3). ref.[6]: m.p. 40–41 °C. [α]2D5 = –2.59 (c = 1.00, CHCl3);
1
ref.[7]: m.p. 36–36.5 °C. [α]D = –1.8 (c = 1.6, CHCl3) Its IR and H
NMR spectra were identical with those of (R)-18. C15H28O (224.4):
calcd. C 80.29, H 12.58; found C 80.11, H 12.86.
Determination of the Enantiomeric Purity of (R)-18 and (S)-18: (S)-
2-Methoxy-2-phenyl-2-(trifluoromethyl)acetyl chloride (MTPACl; centrated under reduced pressure to give the reduction product
25 mg, 0.1 mmol) was added to a solution of (R)-18 (5 mg,
0.02 mmol) in dry pyridine (2 mL), and the mixture was stirred at
0 °C for 12 h. The reaction was quenched by adding water, and the
mixture was diluted with diethyl ether. The extract was successively
washed with saturated aq. CuSO4, saturated aq. NaHCO3, water
and brine, dried with Na2SO4, and concentrated under reduced
pressure. The residue was analyzed by HPLC (Pegasil-Senshu col-
umn, 25 cm×4.6 mm; eluent n-hexane/ethyl acetate, 60:1; flow rate,
1 mL /min). (R)-MTPA ester of (R)-18: tR = 9.78 min (99.5%); (R)-
MTPA ester of (S)-18: tR = 10.96 min (0.5%). The enantiomeric
purity of (R)-18 was therefore 99% ee. In the same manner as that
just described, (S)-18 was converted into its (R)-MTPA derivative,
(2.2 g) as a brown waxy solid. This reduction product was used for
the next reaction without purification. To a stirred solution of the
reduction product (2.2 g) in dry CH2Cl2 (30 mL) were added 2.6-
lutidine (2,6-dimethylpyridine; 5.8 mL, 50 mmol) and TBSOTf
(13.5 mL, 60 mmol) at 0 °C. After having been stirred at room tem-
perature for 1 h, the resulting solution was quenched with MeOH.
It was then poured into water and extracted with diethyl ether. The
extract was successively washed with water, saturated aq. NaHCO3
and brine, dried with MgSO4, and concentrated under reduced
pressure. The residue was chromatographed on silica gel (40 g, hex-
ane/ethyl acetate, 50:1) to give (2S,3R,4E,6R)-23 (420 mg, 3 steps,
15%) as a pale yellow oil, n2D4 = 1.4572. [α]2D4 = +0.8 (c = 1.0,
4796
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 4789–4800

Ceramides in Human Skin
FULL PAPER
CHCl ). IR (film): ν = 3395 cm–1 (w, N–H), 1255 (m, Si–Me) cm–1. 1 H, 4-H), 5.66 (dd, J = 6.1, 15.6 Hz, 1 H, 5-H), 7.32–7.44 (m, 6
˜
3
1H NMR (400 MHz, CDCl3): δ = 0.01, 0.02, 0.045, 0.051 (each s, H, aromatic), 7.65–7.68 (m, 4 H, aromatic) ppm. HRFABMS:
18 H, Si–Me), 0.86–0.91 (m, 30 H, tBu, 18-H3), 1.22–1.31 (m, 24
H, 7–17-H2, N–H2), 2.78 (dt, J = 5.6, 6.6 Hz, 1 H, 2-H), 3.53 (dd,
J = 6.6, 9.6 Hz, 1 H, 1-Ha), 3.64 (dd, J = 4.8, 9.6 Hz, 1 H, 1-Hb),
4.04–4.16 (m, 2 H, 3-H, 6-H), 5.53 (dd, J = 6.8, 15.6 Hz, 1 H, 4-
H), 5.65 (dd, J = 6.7, 15.6 Hz, 1 H, 5-H) ppm. C36H79NO3Si3
(658.3): calcd. C 65.68, H 12.10, N 2.13; found C 65.54, H 12.22,
N 2.15.
m/z [M + H]+: calcd. for C82H155NO5Si4Na, 1369.0880; found,
1369.0880.
(2S,3R,4E,6R)-1,3,6-Trihydroxy-2-(30-hydroxytriacontanoylamino)-
4-octadecene [(2S,3R,4E,6R)-1]: To a stirred solution of
(2S,3R,4E,6R)-24 (256 mg, 0.19 mmol) in dry THF (8 mL) was
added TBAF in THF (1.01 m, 1.52 mL, 0.152 mmol) at 0 °C under
Ar, and stirring was continued overnighat at room temperature.
The reaction mixture was concentrated under reduced pressure.
The residue was chromatographed on silica gel (50 g, CHCl3/
MeOH, 50:1) and the obtained 1 was recrystallized from MeOH to
give (2S,3R,4E,6R)-1 (90 mg, 62%) as a colorless solid, m.p. 113.5–
(2S,3R,4E,6S)-2-Amino-1,3,6-tris(tert-butyldimethylsilyloxy)-4-
octadecene [(2S,3R,4E,6S)-23]: In the same manner as that just de-
scribed, (4S,1ЈR,4ЈS)-22 (2.01 g) was converted into 350 mg (3
steps, 12%) of (2S,3R,4E,6S)-23 as a pale yellow oil, n2D5 = 1.4616.
117.0 °C. IR (KBr): ν = 3470 cm–1 (w, N–H), 3310 (m, O–H), 1620
[α]2D5 = –4.2 (c = 1.0, CHCl ). IR (film): ν = 3385 cm–1 (w, N–H),
˜
˜
3
(m, C=O), 1120 (w, C–O), 1060 (m, C–O) cm–1 1H NMR
.
1670 (w, C=C), 1255 (m, Si–Me) cm–1
.
1H NMR (500 MHz,
(400 MHz, CDCl3/CD3OD, 1:1): δ = 0.51 (t, J = 6.6 Hz, 3 H, 18-
H3), 0.80–1.22 (m, 81 H, 7–17-H2, 3Ј–29Ј-H2, N–H, OH), 1.84 (t,
J = 7.9 Hz, 2 H, 2Ј-H2), 3.17 (t, J = 6.7 Hz, 2 H, 30Ј-H2), 3.27 (dd,
J = 4.5, 11.3 Hz, 1 H, 1-Ha), 3.37 (dd, J = 5.3, 11.3 Hz, 1 H, 1-
Hb), 3.51 (dt-like, J = 5.1, 5.3 Hz, 1 H, 2-H), 3.67 (dt-like, J = 5.7,
6.1 Hz, 1 H, 6-H), 3.83 (t-like, J = 5.8 Hz, 1 H, 3-H), 5.29 (dd, J
= 5.8, 15.6 Hz, 1 H, 4-H), 5.37 (dd, J = 5.7, 15.6 Hz, 1 H, 5-H)
ppm. HRFABMS: m/z [M + H]+: calcd. for C48H96NO5, 766.7210;
found, 766.7269.
CDCl3): δ = 0.016–0.15 (m, 18 H, Si–Me), 0.87–0.90 (m, 30 H, tBu,
18-H3), 1.22–1.31 (m, 24 H, 7–17-H2, N–H2), 2.78 (q-like, J =
5.5 Hz, 1 H, 2-H), 3.53 (dd, J = 7.0, 9.8 Hz, 1 H, 1-Ha), 3.63 (dd,
J = 5.2, 9.8 Hz, 1 H, 1-Hb), 4.02–4.20 (m, 2 H, 3-H, 6-H), 5.54
(dd, J = 6.7, 15.4 Hz, 1 H, 4-H), 5.64 (dd, J = 5.8, 15.4 Hz, 1 H,
5-H) ppm. C36H79NO3Si3 (658.3): calcd. C 65.68, H 12.10, N 2.13;
found C 65.88, H 12.25, N 2.15.
(2S,3R,4E,6R)-1,3,6-Tris(tert-butyldimethylsilyloxy)-2-[30-(tert-
butyldiphenylsilyloxy)triacontanoylamino]-4-octadecene
[(2S,3R,4E,6R)-24]: To a stirred solution of 8 (415 mg, 0.59 mmol)
and (2S,3R,4E,6R)-23 (200 mg, 0.29 mmol) in dry CH2Cl2 (10 mL)
were added EDC [3-(3-dimethylaminopropyl)-1-ethylcarbodiimide
hydrochloride; 113 mg, 0.61 mmol] and HOBt (1-hydroxybenzotri-
azole; 163 mg, 1.26 mmol) at room temperature. The reaction mix-
ture was stirred for 12 h at room temperature and then quenched
with water. It was extracted with ethyl acetate, and the extract was
successively washed with water and brine, dried with MgSO4, and
concentrated under reduced pressure. The residue was chromato-
graphed (60 g, hexane/ethyl acetate, 60:1) on silica gel to give
(2S,3R,4E,6R)-24 (282 mg, 72%) as a colorless oil, n2D4 = 1.4877.
[α]2D4 = –0.51 (c = 1.00, CHCl ). IR (film): ν = 3445 cm–1 (w, N–
(2S,3R,4E,6S)-1,3,6-Trihydroxy-2-(30-hydroxytriacontanoylamino)-
4-octadecene [(2S,3R,4E,6S)-1]: In the same manner as that just
described, (2S,3R,4E,6S)-24 (174 mg, 0.12 mmol) was converted
into 53 mg (58 %) of (2S,3R,4E,6S)-1 as a colorless solid, m.p.
115.0–118.0 °C. IR (KBr): ν = 3335 cm–1 (br. m, N–H, O–H), 3310
˜
(m, O–H), 1660 (s, NCO), 1550 (m, NCO), 1075 (w, C–O) cm–1.
HRFABMS: m/z [M + H]+: calcd. for C48H96NO5, 766.7210;
found, 766.7269.
(2S,3R,4E,6R)-1,3,6-Triacetoxy-2-(30-acetoxytriacontanoylamino)-
4-octadecene [(2S,3R,4E,6R)-1Ј]: To a stirred solution of
(2S,3R,4E,6R)-1 (21 mg, 0.028 mmol) in dry pyridine (2 mL) was
added Ac2O (3 mL). The reaction mixture was stirred overnight at
room temperature. It was then poured into water and extracted
with diethyl ether. The extract was successively washed with water,
saturated aq. CuSO4, saturated aq. NaHCO3 and brine, dried with
MgSO4, and concentrated under reduced pressure. The residue was
chromatographed on silica gel (2 g, hexane/ethyl acetate, 5:1) to
give 22 mg (84%) of (2S,3R,4E,6R)-1Ј as a colorless powder, m.p.
˜
3
H), 1685 (m, C=O), 1255 (m, Si–Me), 1110 (s, C–O) cm–1 1H
.
NMR (500 MHz, CDCl3): δ = 0.01, 0.02, 0.04, 0.05 (each s, 18 H,
Si–Me), 0.86–0.91 (m, 27 H, tBu), 1.01–1.08 (m, 12 H, tBu, 18-
H3), 1.22–1.28 (m, 76 H, 7–17-H2, 3Ј–29Ј-H2), 1.99–2.15 (m, 2 H,
2Ј-H2), 3.54 (dd, J = 5.8, 10.4 Hz, 1 H, 1-Ha), 3.64 (t, J = 6.4 Hz,
2 H, 30Ј-H), 3.78 (dd, J = 5.5, 10.4 Hz, 1 H, 1-Hb), 3.97 (m, 1 H,
2-H), 4.12 (dt, J = 5.7, 6.4 Hz, 1 H, 6-H), 4.42 (t-like, J = 4.9 Hz,
1 H, 3-H), 5.46 (d, J = 8.6 Hz, 1 H, N–H), 5.60 (dd, J = 5.5,
15.3 Hz, 1 H, 4-H), 5.71 (dd, J = 5.7, 15.3 Hz, 1 H, 5-H), 7.34–
7.45 (m, 6 H, aromatic), 7.64–7.68 (m, 4 H, aromatic) ppm.
HRFABMS: m/z [M + H]+: calcd. for C82H155NO5Si4Na,
1369.0880; found, 1369.0883.
76.0–78.0 °C. [α]2D2 = +2.3 (c = 0.90, CHCl ). IR (KBr): ν =
˜
3
3310 cm–1 (br.w, N–H), 1740 (s, C=O), 1650 (m, NCO), 1540 (m,
NCO) cm–1. 1H NMR (500 MHz, CDCl3/D2O): δ = 0.86 (t, J =
6.9 Hz, 3 H, 18-H3), 1.21–1.40 (m, 72 H, 8–17-H2, 3Ј–28Ј-H2),
1.54–1.60 (m, 4 H, 7-, 29Ј-H2), 2.02, 2.03, 2.04, 2.05 (each s, 12 H,
OAc), 2.12–2.17 (m, 2 H, 2Ј-H2), 3.98 (dd, J = 4.6, 11.6 Hz, 1 H,
1-Ha), 4.02 (t, J = 6.7 Hz, 2 H, 30Ј-H2), 4.21 (dd, J = 6.4, 11.6 Hz,
1 H, 1-Hb), 4.46 (dd, J = 4.6, 6.4 Hz, 1 H, 2-H), 5.15 (dt, J = 6.4,
6.5 Hz, 1 H, 6-H), 5.32 (t-like, J = 5.8 Hz, 1 H, 3-H), 5.60 (dd, J
= 6.4, 15.6 Hz, 1 H, 4-H), 5.69 (dd, J = 6.5, 15.6 Hz, 1 H, 5-H)
ppm. 13C NMR (126 MHz, CDCl3): δ = 14.1, 21.0, 22.8, 25.9, 28.5,
29.21, 29.24, 29.31, 29.33, 29.36, 29.48, 29.50, 29.52, 29.55, 29.62,
29.65, 29.69, 31.89, 34.1, 36.8, 50.3, 62.4, 64.6, 72.7, 73.6, 126.5,
133.7, 169.8, 170.4, 170.8, 171.2, 172.9 ppm. HRFABMS: m/z [M
+ H]+: calcd. for C56H104NO9, 934.7711; found, 934.7709.
(2S,3R,4E,6S)-1,3,6-Tris(tert-butyldimethylsilyloxy)-2-(30-tert-
butyldiphenylsilyloxytriacontanoylamino)-4-octadecene
[(2S,3R,4E,6S)-24]: In the same manner as that just described,
(2S,3R,4E,6S)-23 (49 mg, 0.07 mmol) and 8 (62 mg, 0.09 mmol)
were converted into 66 mg (70%) of (2S,3R,4E,6S)-24 as a colorless
oil, n2D3 = 1.4869. [α]2D3 = –3.4 (c, 1.10, CHCl ). IR (film): ν =
˜
3
3445 cm–1 (w, N–H), 1685 (s, NCO), 1550 (m, NCO), 1255 (m, Si–
1
Me), 1110 (s, C–O) cm–1. H NMR (500 MHz, CDCl3): δ = 0.01–
0.05 (m, 18 H, Si–Me), 0.85–0.90 (m, 27 H, tBu), 1.01–1.06 (m, 12
H, tBu, 18-H3), 1.07–1.26 (m, 76 H, 7–17-H2, 3Ј–29Ј-H2), 2.07– (2S,3R,4E,6S)-1,3,6-Triacetoxy-2-(30-acetoxytriacontanoylamino)-
2.15 (m, 2 H, 2Ј-H2), 3.54 (dd, J = 5.8, 10.4 Hz, 1 H, 1-Ha), 3.65
(t, J = 6.4 Hz, 2 H, 30Ј-H), 3.81 (dd, J = 5.2, 10.4 Hz, 1 H, 1-Hb),
4-octadecene [(2S,3R,4E,6S)-1Ј]: In the same manner as that just
described, (2S,3R,4E,6S)-1 (21 mg, 0.028 mmol) was converted into
3.92–3.99 (m, 1 H, 2-H), 4.06–4.12 (m, 1 H, 6-H), 4.33–4.38 (m, 1 22 mg (84%) of (2S,3R,4E,6S)-1Ј as colorless powder, m.p. 71.5–
H, 3-H), 5.49 (d, J = 8.2 Hz, 1 H, N–H), 5.56 (dd, J = 6.1, 15.6 Hz,
73.0 °C. [α]2D2 = –11.6 (c = 0.8, CHCl ). IR (KBr): ν = 3305 cm–1
˜
3
Eur. J. Org. Chem. 2005, 4789–4800
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4797

Y. Masuda, K. Mori
FULL PAPER
(br.w, N–H), 1735 (s, C=O), 1650 (m, NCO), 1545 (m, NCO) cm–1. 32.0–34.5. [α]2D5 = –0.93 (c = 1.20, CHCl ). IR (nujol): ν =
˜
3
1H NMR (500 MHz, CDCl3/D2O): δ = 0.86 (t, J = 6.9 Hz, 3 H, 3445 cm–1 (w, N–H), 1685 (m, NCO), 1255 (s, Si–Me) cm–1. 1H
18-H3), 1.21–1.40 (m, 72 H, 8–17-H2, 3Ј–28Ј-H2), 1.53–1.60 (m, 4
H, 7-, 29Ј-H2), 2.02, 2.03, 2.04, 2.05 (each s, 12 H, OAc), 2.10–2.14
(m, 2 H, 2Ј-H2), 3.98–4.04 (m, 3 H, 1-Ha, 30Ј-H2), 4.23 (dd, J =
6.3, 11.4 Hz, 1 H, 1-Hb), 4.32–4.48 (m, 1 H, 2-H), 5.12 (dt, J = 6.2,
6.4 Hz, 1 H, 6-H), 5.33 (t-like, J = 5.3 Hz, 1 H, 3-H), 5.61 (dd, J
= 5.7, 15.6 Hz, 1 H, 4-H), 5.66 (dd, J = 5.9, 15.6 Hz, 1 H, 5-H)
ppm. 13C NMR (126 MHz, CDCl3): δ = 14.1, 20.7, 21.0, 21.2, 22.6,
25.0, 25.6, 25.8, 28.5, 29.2, 29.3, 29.37, 29.47, 29.50, 29.53, 29.55,
29.57, 29.63, 29.65, 29.69, 31.9, 34.1, 36.7, 50.2, 62.4, 64.6, 72.4,
NMR (500 MHz, CDCl3): δ = 0.012, 0.013, 0.035, 0.042, 0.049
(each s, 18 H, Si–Me), 0.86–0.90 (m, 33 H, tBu, 18-, 24Ј-H3), 1.10–
1.37 (m, 64 H, 7–17-H2, 3Ј–23Ј-H2), 2.09–2.14 (m, 2 H, 2Ј-H), 3.54
(dd, J = 6.0, 10.0 Hz, 1 H, 1-Ha), 3.78 (dd, J = 5.5, 10.5 Hz, 1 H,
1-Hb), 3.97 (ddt, J = 5.0, 6.0, 8.5 Hz, 1 H, 2-H), 4.12 (dt, J = 5.5,
6.0 Hz, 1 H, 6-H), 4.42 (t-like, J = 5.0 Hz, 1 H, 3-H), 5.47 (d, J =
8.5 Hz, 1 H, N–H) 5.60 (ddd, J = 1.5, 5.5, 15.5 Hz, 1 H, 4-H), 5.70
(ddd, J = 1.0, 5.5, 15.5 Hz, 1 H, 5-H) ppm. C60H125NO4Si3
(1008.9): calcd. C 71.43, H 12.49, N 1.39; found C 71.14, H 12.76,
74.0, 126.4, 133.5, 169.7, 170.5, 170.9, 171.2, 172.9 ppm. N 1.43.
HRFABMS: m/z [M + H]+: calcd. for C56H104NO9, 934.7711;
found, 934.7714.
To a stirred solution of protected ceramide (216 mg, 0.21 mmol) in
dry THF (5 mL) was added TBAF in THF (1.01 m, 6.3 mL,
6.3 mmol) at 0 °C under Ar, and stirring was continued overnight
at room temperature. The reaction mixture was concentrated under
reduced pressure. The residue was chromatographed on silica gel
(20 g, CHCl3/MeOH, 50:1) and recrystallized from MeOH to give
(2S,3R,4E,6R)-2 (103 mg, 72%) as a colorless solid, m.p. 113.5–
tert-Butyl (4S,1ЈR,4ЈR)-4-(4Ј-tert-Butyldimethylsilyloxy-1Ј-hydroxy-
2Ј-hexadecynyl)-2,2-dimethyl-3-oxazolidinecarboxylate
[(4S,1ЈR,4ЈR)-25]: To a stirred solution of (R)-20 (8.00 g,
23.6 mmol) in dry THF (200 mL) was added n-butyllithium (1.58 m
in hexane; 15.7 mL, 25.0 mmol) dropwise at –78 °C under Ar. After
the reaction mixture had been stirred at –78 °C for 1.5 h, a solution
of Garner’s aldehyde [(S)-21, 6.5 g, 23.6 mmol] in dry THF
(200 mL) and HMPA (20 mL) was added dropwise at –78 °C. The
mixture was then stirred overnight while being warmed to 0 °C,
before being quenched with NH4Cl aq. and extracted with diethyl
ether. The extract was successively washed with water and brine,
dried with MgSO4, and concentrated under reduced pressure. The
residue was chromatographed on silica gel (500 g, hexane/ethyl ace-
tate, 20:1) to give 10.9 g (81%) of (4S,1ЈR,4ЈR)-25 as a colorless
115.0 °C. IR (KBr): ν = 3470 cm–1 (br. m, N–H), 3310 (m, O–H),
˜
1620 (s, NCO), 1545 (m, NCO) cm–1. 1H NMR (500 MHz, CDCl3,
50 °C): δ = 0.89 (t, J = 7.0 Hz, 6 H, 18-, 24Ј-H3), 1.24–1.38 (m, 61
H, 8–17-H2, 4Ј–23Ј-H2), 1.51–1.69 (m, 4 H, 7-, 3Ј-H2), 2.23 (t, J =
7.5 Hz, 2 H, 2Ј-H), 2.34–2.41 (m, 1 H, OH), 2.75 (d, J = 5.5 Hz, 1
H, OH), 3.70–3.77 (m, 1 H, 1-Ha), 3.90–3.99 (m, 1 H, 1-Hb, 2-H),
4.12–4.19 (m, 1 H, 6-H), 4.39 (dt, J = 4.5, 5.0 Hz, 1 H, 3-H), 5.79
(dd, J = 5.5, 15.5 Hz, 1 H, 4-H) 5.85 (dd, J = 6.0, 15.5 Hz, 1 H, 5-
H), 6.18 (d, J = 6.5 Hz, 1 H, N–H) ppm. HRFABMS: m/z [M +
H]+: calcd. for C42H83NO4Na, 688.6946; found, 688.6226.
oil, n2D3 = 1.4641. [α]2D3 = –4.9 (c = 1.0, CHCl ). IR (film): ν =
˜
3
3445 cm–1 (w, N–H), 1705 (w, C=C), 1255 (m, Si–Me) cm–1. 1H
NMR (500 MHz, CDCl3): δ = 0.08, 0.11 (each s, 6 H, Si–Me),
0.85–0.90 (m, 12 H, tBu, 16Ј-H3), 1.24–1.31 (m, 15 H, tBu, 2–Me,
2–Me), 1.35–1.66 (m, 23 H, 5Ј–15Ј-H2, OH), 3.90–4.18 (m, 3 H 1Ј-
H, 3-H2), 4.35 (t, J = 6.5 Hz, 1 H, 4Ј-H), 4.58–4.76 (m, 1 H, 4-H)
ppm. C32H61NO5Si (567.9): calcd. C 67.68, H 10.83, N 2.47; found
C 67.38, H 10.97, N 2.44.
(2S,3R,4E,6R)-1,3,6-Trihydroxy-4N-[30Ј-(linoleoyloxy)triacon-
tanoyl]octadecene [(2S,3R,4E,6R)-3]: A solution of 9 (267 mg,
0.36 mmol) and (2S,3R,4E,6R)-23 (250 mg, 0.36 mmol) in pyridine
(10 mL) was stirred at 45 °C for 21 h. The mixture was diluted with
benzene (50 mL) and concentrated under reduced pressure. This
operation was repeated three times to remove pyridine. The residue
was chromatographed on silica gel (30 g, hexane/ethyl acetate,
100:1) to give the TBS-protected ceramide (377 mg 76%) as a col-
orless oil, n2D4 = 1.4712. [α]2D4 = –0.82 (c = 1.05, CHCl3). IR (film):
(2S,3R,4E,6R)-2-Amino-1,3,6-tris(tert-butyldimethylsilyloxy)-4-oc-
tadecene [(2S,3R,4E,6R)-23]: To a stirred solution of (4S,1ЈR,4ЈR)-
25 (500 mg, 0.88 mmol) in dry THF (20 mL) was added TBAF in
THF (1.01 m, 1.76 mL, 1.74 mmol) at 0 °C under Ar, and stirring
was continued overnighat at room temperature and then the reac-
tion was quenched with water. It was extracted with ethyl acetate.
The extract was successively washed with water and brine, dried
with MgSO4, and concentrated under reduced pressure. Because of
its instability, this product 22 was used for the next reaction without
purification.
ν = 3445 cm–1 (w, N–H), 1740 (m, C=O), 1685 (m, NCO), 1255 (s,
˜
1
Si–Me) cm–1. H NMR (500 MHz, CDCl3): δ = 0.01, 0.012, 0.03,
0.04, 0.047 (each s, 18 H, Si–Me), 0.86–0.91 (m, 33 H, tBu, 18-,
18ЈЈ-H3), 1.20–1.40 (m, 80 H, 8–14-H2, 4Ј–28Ј-H2, 3ЈЈ–6ЈЈ-H2, 16ЈЈ–
17ЈЈ-H2), 1.58–1.68 (m, 12 H, 7-, 3Ј-, 29Ј-, 3ЈЈ-, 7ЈЈ-, 15ЈЈ-H2), 2.01–
2.08 (m, 4 H, 8ЈЈ-, 14ЈЈ-H2), 2.10–2.15 (m, 2 H, 2ЈЈ-H2), 2.28 (t, J
= 7.5 Hz, 2 H, 2Ј-H2), 2.77 (t, J = 6.7 Hz, 2 H, 11ЈЈ-H2), 3.54 (dd,
J = 6.0, 10.0 Hz, 1 H, 1-Ha), 3.77 (dd, J = 5.5, 10.0 Hz, 1 H, 1-
Hb), 3.97 (ddt-like, J = 5.0, 5.5, 8.0 Hz, 1 H, 2-H), 4.05 (t, J =
6.8 Hz, 2 H, 30Ј-H2), 4.11 (dt, J = 6.0, 12.0 Hz,1 H, 6-H), 4.41 (t-
like, J = 5.0 Hz, 1 H, 3-H), 5.28–5.41 (m, 4 H, 9ЈЈ-, 10ЈЈ-, 12ЈЈ-,
13ЈЈ-H), 5.47 (d, J = 8.0 Hz, 1 H, N–H), 5.60 (ddd, J = 1.0, 5.5,
15.5 Hz, 1 H, 4-H), 5.70 (ddd, J = 1.0, 6.0, 15.5 Hz, 1 H, 5-H)
ppm.
In the same manner as that just described, the crude product of
(4S,1ЈR,4ЈR)-22 was converted into 1.10 g (3 steps, 37 %) of
(2S,3R,4E,6R)-23 as a pale yellow oil. Its physical data were iden-
tical with those of (2S,3R,4E,6R)-23 which was derived from (R)-
18.
(2S,3R,4E,6R)-1,3,6-Trihydroxy-2-tetracosanoylamino-4-octadecene
[(2S,3R,4E,6R)-2]: To a stirred solution of tetracosanoic acid (10)
(118 mg, 0.32 mmol) and (2S,3R,4E,6R)-23 (200 mg, 0.29 mmol) in
dry CH2Cl2 (15 mL) was added EDC (110 mg, 0.58 mmol) and
HOBt (160 mg, 1.18 mmol) at room temperature. The reaction mix-
ture was stirred for 12 h at room temperature and then quenched
with water. It was extracted with ethyl acetate, and the extract was
successively washed with water and brine, dried with MgSO4, and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel (20 g, hexane/ethyl acetate, 50:1) to give the
TBS-protected ceramide (280 mg, 96%) as a colorless solid, m.p.
To a stirred mixture of the TBS-protected ceramide (337 mg,
0.25 mmol) in dry THF (5 mL) was added TBAF in THF (1.0 m,
1.1 mL, 1.1 mmol) at 0 °C under Ar, and stirring was continued
overnight at room temperature. The mixture was then diluted with
chloroform (10 mL) and concentrated under reduced pressure. This
operation was repeated three times to remove THF. The residue
was chromatographed on silica gel (20 g, CHCl3/MeOH, 100:1) to
give (2S,3R,4E,6R)-3 (182 mg, 73 %) as colorless powder, m.p.
111.5–112.5 °C. IR (KBr): ν = 3470 cm–1 (w, N–H), 3310 (m, O–
˜
H), 1740 (m, C=O), 1620 (m, NCO) 1545 (w, NCO) cm–1. 1H NMR
4798
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 4789–4800

Ceramides in Human Skin
FULL PAPER
(500 MHz, CDCl3): δ = 0.88, 0.89 (each t, each J = 7.0 Hz, 6 H,
less oil, n2D4 = 1.4669. [α]2D4 = +5.0 (c = 1.00, CHCl ). IR (film): ν
˜
3
18-, 18ЈЈ-H3), 1.21–1.39 (m, 83 H, 8–14-H2, 4Ј–28Ј-H2, 3ЈЈ–6ЈЈ-H2, = 3395 cm–1 (br. m, N–H, O–H), 1655 (m, NCO), 1525 (m, NCO),
16ЈЈ–17ЈЈ-H2, OH), 1.49–1.67 (m, 12 H, 7-, 3Ј-, 29Ј-, 3ЈЈ-, 7ЈЈ-, 15ЈЈ- 1255 (w, Si–Me) cm–1 1H NMR (500 MHz, CDCl3): δ = 0.02,
.
H2), 2.02–2.07 (m, 4 H, 8Ј-, 14ЈЈ-H2), 2.23 (t, J = 7.5 Hz, 2 H, 2Ј-
H2), 2.29 (t, J = 7.5 Hz, 2 H, 2ЈЈ-H2), 2.77 (t-like, J = 7.0 Hz, 1 H,
0.039, 0.042, 0.052 (each s, 18 H, Si–Me), 0.85–0.90 (m, 33 H, tBu,
18-H3, 24Ј-H3), 1.13–1.60 (m, 65 H, 7–17-H2, 3Ј–23Ј-H2, O–H),
11ЈЈ-H2), 3.67–3.75 (m, 1 H, 2-H), 3.90–3.98 (m, 2 H, 1-H2), 4.05 3.57 (dd, J = 5.5, 10.0 Hz, 1 H, 1-Ha), 3.77 (dd, J = 6.0, 10.0 Hz,
(t, J = 6.5 Hz, 2 H, 30Ј-H2), 4.15 (dt, J = 5.5, 6.0 Hz, 1 H, 6-H), 1 H, 1-Hb), 3.98–4.07 (m, 2 H, 2-, 2Ј-H), 4.12 (dt, J = 5.5, 6.0 Hz,
4.36–4.41 (m, 1 H, 3-H), 5.28–5.42 (m, 4 H, 9ЈЈ-, 10ЈЈ-, 12ЈЈ-, 13ЈЈ- 1 H, 6-H), 4.43 (t-like, J = 5.5 Hz, 1 H, 3-H), 5.61 (ddd, J = 1.0,
H), 5.77 (ddd, J = 1.0, 5.5, 15.5 Hz, 1 H, 4-H), 5.84 (ddd, J = 1.0,
6.0, 15.5 Hz, 1 H, 5-H), 6.29 (d, J = 7.0 Hz, 1 H, N–H) ppm.
5.5, 15.5 Hz, 1 H, 4-H), 5.72 (ddd, J = 1.5, 5.5, 15.5 Hz, 1 H, 5-
H), 6.36 (d, J = 8.5 Hz, 1 H, N–H) ppm. C60H125NO5Si3 (1024.9):
calcd. C 70.31, H 12.29, N 1.37; found C 70.33, H 12.54, N 1.35.
(2S,3R,4E,6R,2ЈR)-2-(2Ј-Acetoxytetracosanoylamino)-1,3,6-
tris(tert-butyldimethylsilyloxy)-4-octadecene [(2S,3R,4E,6R,2ЈR)-
26]: To a stirred solution of (R)-13 (171 mg, 0.4 mmol) and
(2S,3R,4E,6R)-23 (250 mg, 0.36 mmol) in dry CH2Cl2 (15 mL) was
added EDC (138 mg, 0.72 mmol) and HOBt (199 mg, 1.48 mmol)
at room temperature. The reaction mixture was stirred for 12 h at
room temperature and then quenched with water. It was extracted
with ethyl acetate, and the extract was successively washed with
water and brine, dried with MgSO4, and concentrated under re-
duced pressure. The residue was chromatographed on silica gel (6 g,
hexane/ethyl acetate, 100:1) to give (2S,3R,4E,6R,2ЈR)-26 (280 mg,
73%) as a colorless oil, n2D4 = 1.4622. [α]2D4 = +8.9 (c = 1.00, CHCl3).
(2S,3R,4E,6R,2ЈS)-1,3,6-Tris(tert-butyldimethylsilyloxy)-2-(2Ј-hy-
droxytetracosanoylamino)-4-octadecene [(2S,3R,4E,6R,2ЈS)-27]: In
the same manner as that just described, (2S,3R,4E,6R,2ЈS)-26
(271 mg, 0.25 mmol) was converted into 216 mg (83 %) of
(2S,3R,4E,6R,2ЈS)-27 as a colorless oil, n2D4 = = 1.4661. [α]2D4
=
–9.7 (c = 1.05, CHCl ). IR (film): ν = 3395 cm–1 (br. m, N–H, O–
˜
3
H), 1650 (m, NCO), 1525 (m, NCO), 1255 (w, Si–Me) cm–1. 1H
NMR (500 MHz, CDCl3): δ = 0.02, 0.03, 0.04, 0.05 (each s, 18 H,
Si–Me), 0.84–0.91 (m, 33 H, tBu, 18-H3, 24Ј-H3), 1.14–1.63 (m, 65
H, 7–17-H2, 3Ј–23Ј-H2, O–H), 3.56 (dd, J = 5.5, 10.5 Hz, 1 H, 1-
Ha), 3.78 (dd, J = 5.0, 10.5 Hz, 1 H, 1-Hb), 3.97–4.00 (m, 2 H, 2-,
2Ј-H), 4.11 (dt, J = 5.5, 6.0 Hz, 1 H, 6-H), 4.43 (t-like, J = 5.5 Hz,
1 H, 3-H), 5.60 (ddd, J = 1.0, 5.5, 15.5 Hz, 1 H, 4-H), 5.71 (ddd,
J = 1.0, 5.5, 15.5 Hz, 1 H, 5-H), 6.43 (d, J = 9.0 Hz, 1 H, N–H)
ppm. C60H125NO5Si3 (1024.9): calcd. C 70.31, H 12.29, N 1.37;
found C 70.31, H 12.44, N 1.36.
IR (film): ν = 3445 cm–1 (w, N–H), 1755 (m, C=O), 1690 (m,
˜
1
NCO), 1255 (m, Si–Me) cm–1. H NMR (500 MHz, CDCl3): δ =
0.018, 0.022, 0.026, 0.034, 0.046, 0.060 (each s, 18 H, Si–Me), 0.85–
0.91 (m, 33 H, tBu, 18-H3, 24Ј-H3), 1.13–1.51 (m, 64 H, 7–17-H2,
3Ј–23Ј-H2), 2.10 (s, 3 H, OAc), 3.54 (dd, J = 6.0, 10.5 Hz, 1 H, 1-
Ha), 3.74 (dd, J = 6.0, 10.5 Hz, 1 H, 1-Hb), 4.04 (ddt, J = 4.5, 6.0,
8.5 Hz, 1 H, 2-H), 4.13 (dt, J = 5.5, 6.0 Hz, 1 H, 6-H), 4.46 (t-like,
J = 5.0 Hz, 1 H, 3-H), 5.21 (dt, J = 2.5, 4.5 Hz, 1 H, 2Ј-H), 5.62
(ddd, J = 1.0, 5.5, 15.5 Hz, 1 H, 4-H), 5.74 (ddd, J = 1.5, 5.0,
15.5 Hz, 1 H, 5-H), 6.16 (d, J = 8.5 Hz, 1 H, N–H) ppm.
C62H127NO6Si3 (1066.93): calcd. C 69.79, H 12.00, N 1.31; found
C 69.78, H 12.16, N 1.25.
(2S,3R,4E,6R,2ЈR)-1,3,6-Trihydroxy-2-(2Ј-hydroxytetracosanoyl-
amino)-4-octadecene [(2S,3R,4E,6R,2ЈR)-4]: To a stirred solution of
(2S,3R,4E,6R,2ЈR)-27 (181 mg, 0.18 mmol) in dry THF (5 mL) was
added TBAF in THF (1.01 m, 10 mL, 10.1 mmol) at 0 °C under
Ar, and stirring was continued overnighat at room temperature.
The reaction mixture was concentrated under reduced pressure.
The residue was chromatographed on silica gel (15 g, CHCl3/
MeOH, 50:1) and the obtained 4 was recrystallized from MeOH to
give (2S,3R,4E,6R,2ЈR)-4 (90 mg, 75%) as a colorless solid, m.p.
(2S,3R,4E,6R,2ЈS)-2-(2Ј-Acetoxytetracosanoylamino)-1,3,6-
tris(tert-butyldimethylsilyloxy)-4-octadecene [(2S,3R,4E,6R,2ЈS)-
26]: In the same manner as that just described, (S)-13 (137 mg,
0.32 mmol) and (2S,3R,4E,6R)-23 (200 mg, 0.29 mmol) were con-
verted into 291 mg (94%) of (2S,3R,4E,6R,2ЈS)-26 as a colorless
115.5–117.0 °C. IR (KBr): ν = 3265 cm–1 (br. m, N–H), 3200 (m,
˜
O–H), 1650 (m, C=O), 1560 (m, NCO) cm–1. 1H NMR (500 MHz,
CDCl3, 50 °C): δ = 0.89 (t, J = 7.0 Hz, 6 H, 18-H3, 24Ј-H3), 1.20–
1.38 (m, 60 H, 8–17-H2, 4Ј–23Ј-H2), 1.51–1.69 (m, 4 H, 7-, 3Ј-H2),
1.82–1.89 (m, 1 H, OH), 2.50 (dd, J = 2.5, 5.0 Hz, 1 H, 1-Ha),
2.51–2.54 (m, 1 H, OH), 2.63 (dd, J = 2.0, 5.0 Hz, 1 H, 1-Hb),
3.75–3.80 (m, 1 H, OH), 3.87–3.92 (m, 1 H, 2-H), 3.99 (dt, J = 4.5,
11.5 Hz, 1 H, 2Ј-H), 4.10–4.19 (m, 2 H, 6-H, OH), 4.42 (q, J =
5.0 Hz, 1 H, 3-H), 5.77 (dd, J = 5.5, 15.5 Hz, 1 H, 4-H), 5.84 (dd,
J = 5.5, 15.5 Hz, 1 H, 5-H), 7.05 (d, J = 5.5 Hz, 1 H, N–H) ppm.
13C NMR (126 MHz, CDCl3/CD3OD, 1:1): δ = 13.2, 22.1, 24.8,
25.0, 28.75, 28.77, 29.0, 29.06, 29.09, 29.13, 31.4, 54.4, 60.7, 71.2,
71.60, 71.66, 128.8, 135.3, 175.7 ppm. C42H83NO5 (682.1): calcd. C
73.95, H 12.26, N 2.05; found C 73.83, H 12.43, N 2.04.
oil, n2D2 = 1.4631. [α]2D4 = –4.8 (c = 1.00, CHCl ). IR (film): ν =
˜
3
3445 cm–1 (w, N–H), 1755 (m, C=O), 1690 (m, NCO), 1255 (m, Si–
Me) cm–1. 1H NMR (500 MHz, CDCl3): δ = –0.02, –0.016, –0.014,
0.018, 0.03, 0.05 (each s, 18 H, Si–Me), 0.85–0.90 (m, 33 H, tBu,
18-H3, 24Ј-H3), 1.10–1.52 (m, 64 H, 7–17-H2, 3Ј–23Ј-H2), 2.09 (s,
3 H, OAc), 3.52 (dd, J = 5.5, 10.5 Hz, 1 H, 1-Ha), 3.80 (dd, J =
6.0, 10.5 Hz, 1 H, 1-Hb), 3.95 (ddt, J = 5.0, 6.0, 8.0 Hz, 1 H, 2-H),
4.10 (dt, J = 5.5, 6.0 Hz, 1 H, 6-H), 4.44 (t-like, J = 5.0 Hz, 1 H,
3-H), 5.21 (dt, J = 2.0, 5.0 Hz, 1 H, 2Ј-H), 5.59 (ddd, J = 1.0, 5.5,
15.5 Hz, 1 H, 4-H), 5.69 (ddd, J = 1.5, 5.0, 15.5 Hz, 1 H, 5-H), 6.24
(d, J = 8.0 Hz, 1 H, N–H) ppm. C62H127NO6Si3 (1066.9): calcd. C
69.79, H 12.00, N 1.31; found C 69.83, H 12.25, N 1.30.
(2S,3R,4E,6R,2ЈS)-1,3,6-Trihydroxy-2-(2Ј-hydroxytetracosanoyl-
amino)-4-octadecene [(2S,3R,4E,6R,2ЈS)-4]: In the same manner as
that just described, (2S,3R,4E,6R,2ЈS)-27 (216 mg, 0.21 mmol) was
converted into 103 mg (72%) of (2S,3R,4E,6R,2ЈS)-4 as a colorless
(2S,3R,4E,6R,2ЈR)-1,3,6-tris(tert-Butyldimethylsilyloxy)-2-(2Ј-hy-
droxytetracosanoylamino)-4-octadecene [(2S,3R,4E,6R,2ЈR)-27]: To
a stirred solution of (2S,3R,4E,6R,2ЈR)-26 (280 mg, 0.26 mmol) in
MeOH (5 mL) was added K2CO3 (72 mg, 0.52 mmol) at room tem-
perature. The mixture was stirred for 2 h at room temperature, be-
fore the solvent was removed under reduced pressure. The concen-
trate was diluted with water and extracted with diethyl ether. The
resulting extract was successively washed with water and brine,
dried with MgSO4, and concentrated under reduced pressure. The
solid, m.p. 106.5–109.5 °C. IR (KBr): ν = 3365 cm–1 (br. m, N–H,
˜
O–H), 1655 (s, NCO), 1550 (m, NCO) cm–1. 13C NMR (126 MHz,
CDCl3:CD3OD, 1:1): δ = 13.2, 22.2, 24.8, 25.1, 28.9, 29.0, 29.12,
29.18, 29.20, 31.5, 34.0, 36.7, 54.3, 60.9, 71.4, 71.60, 71.72, 129.0,
135.4, 175.9 ppm. C42H83NO5 (682.1): calcd. C 73.95, H 12.26, N
2.05; found C 73.76, H 12.36, N 2.02.
residue was chromatographed on silica gel (6 g, hexane/ethyl ace- (2S,3R,4E,6R,2ЈR)-1,3,6-Triacetoxy-2-(2Ј-acetoxytetracosanoyl-
tate, 70:1) to give (2S,3R,4E,6R,2ЈR)-27 (209 mg, 78%) as a color- amino)-4-octadecene [(2S,3R,4E,6R,2ЈR)-4Ј]: To a stirred solution
Eur. J. Org. Chem. 2005, 4789–4800
www.eurjoc.org
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4799

Y. Masuda, K. Mori
of (2S,3R,4E,6R,2ЈR)-4 (20 mg, 0.03 mmol) in dry pyridine (2 mL) Our thanks are due to Dr. S. Hamanaka (Hamanaka Dermatologi-
FULL PAPER
was added Ac2O (3 mL). The reaction mixture was stirred over-
night at room temperature. It was then poured into water and ex-
tracted with diethyl ether. The extract was successively washed with
water, saturated aq. CuSO4, saturated aq. NaHCO3 and brine,
dried with MgSO4, and concentrated under reduced pressure. The
residue was chromatographed on silica gel (4 g, hexane/ethyl ace-
tate, 5:1) to give (2S,3R,4E,6R,2ЈR)-4Ј (22 mg, 92%) as colorless
powder, m.p. 91.0–93.0 °C. [α]2D5 = +12.1 (c = 0.6, CHCl3). IR
cal Clinic, Saitama) for discussion. We thank Dr. Y. Hirose (Amano
Enzyme, Inc., Gifu) for his generous gifts of lipase PS. We are
grateful to Dr. K. Sakai (Seikagaku Corporation, Tokyo) for his
interest in this work.
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S. Hamanaka, M. Suzuki, A. Suzuki, T. Yamakawa, Proc. Ja-
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S. Brodesser, P. Sawatzki, T. Kolter, Eur. J. Org. Chem. 2003,
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(KBr): ν = 3355 cm–1 (m, N–H), 1740 (s, OAc), 1660 (s, NCO),
˜
1530 (m, NCO), 1235 (s, C–OAc) cm–1
.
1H NMR (500 MHz,
CDCl3): δ = 0.88 (t, J = 7.0 Hz, 6 H, 18-, 24Ј-H3), 1.21–1.32 (m, 6
H, 8–17-H2, 4Ј–23Ј-H2), 1.76–1.85 (m, 4 H, 7-, 3Ј-H), 2.02, 2.04,
2.06, 2.16 (each s, 12 H, OAc), 3.94 (dd, J = 4.3, 11.6 Hz, 1 H, 1-
Ha), 4.31 (dd, J = 7.8, 11.6 Hz, 1 H, 1-Hb), 4.39–4.49 (m, 1 H, 2-
H), 5.08 (dd, J = 4.5, 7.5 Hz, 1 H, 2Ј-H), 5.17 (dt, J = 6.5, 6.5 Hz,
1 H, 6-H), 5.36 (t-like, J = 5.5 Hz, 1 H, 3-H), 5.61 (ddd, J = 1.0,
6.4, 15.7 Hz, 1 H, 4-H), 5.70 (ddd, J = 1.0, 6.3, 15.7 Hz, 1 H, 5-
H), 6.38 (d, J = 9.0 Hz, 1 H, N–H) ppm. 13C NMR (126 MHz,
CDCl3): δ = 14.1, 20.7, 20.9, 21.0, 21.2, 22.7, 24.8, 25.1, 29.25,
29.34, 29.47, 29.49, 29.57, 29.59, 29.64, 29.66, 29.70, 31.8, 31.9,
34.1, 50.9, 61.7, 72.6, 73.6, 74.0, 126.3, 133.6, 169.7, 169.9, 170.1,
170.4, 171.4 ppm. HRFABMS: m/z [M + H]+ : calcd. for
C50H91NO9Na, 872.6592; found, 872.6592.
K. J. Robson, M. E. Stewart, S. Michelsen, N. D. Lazo, D. T.
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(2S,3R,4E,6R,2ЈS)-1,3,6-Triacetoxy-2-(2Ј-acetoxytetracosanoyl-
amino)-4-octadecene [(2S,3R,4E,6S,2ЈS)-4Ј]: In the same manner as
that just described, (2S,3R,4E,6R,2ЈS)-4 (20 mg, 0.03 mmol) was
converted into 20 mg (84%) of (2S,3R,4E,6R,2ЈS)-4Ј as colorless
powder, m.p. 80.0–81.5 °C. [α]2D5 = –4.8 (c = 0.7, CHCl3). IR (KBr):
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ν = 3340 cm–1 (m, N–H), 1735 (s, OAc), 1650 (s, NCO), 1540 (s,
˜
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NCO), 1235 (s, C–OAc) cm–1. 1H NMR (500 MHz, CDCl3): δ =
0.86 (t, J = 6.5 Hz, 6 H, 18-, 24Ј-H3), 1.18–1.32 (m, 60 H, 8–17-
H2, 4Ј–23Ј-H2), 1.71–1.82 (m, 4 H, 7-, 3Ј-H), 2.043, 2.047, 2.059,
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4.18 (dd, J = 7.0, 11.5 Hz, 1 H, 1-Hb), 4.40–4.48 (m, 1 H, 2-H),
5.08 (dd, J = 5.0, 7.5 Hz, 1 H, 2Ј-H), 5.15 (dt, J = 6.5, 6.5 Hz, 1
H, 6-H), 5.31 (t-like, J = 5.5 Hz, 1 H, 3-H), 5.58 (ddd, J = 0.5, 6.4,
15.5 Hz, 1 H, 4-H), 5.69 (ddd, J = 1.0, 6.5, 15.5 Hz, 1 H, 5-H),
6.37 (d, J = 9.0 Hz, 1 H, N–H) ppm. 13C NMR (126 MHz, CDCl3):
δ = 14.1, 20.7, 20.8, 20.9, 21.2, 22.7, 24.7, 25.1, 29.27, 29.30, 29.34,
29.44, 29.47, 29.56, 29.63, 29.65, 29.69, 31.7, 31.9, 34.1, 50.5, 62.0,
72.9, 73.6, 74.0, 126.2, 133.6, 169.8, 169.9, 170.0, 170.4, 170.9 ppm.
HRFABMS: m/z [M + H]+: calcd. for C50H91NO9Na, 872.6592;
found, 872.6589.
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Acknowledgments
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Dermatol. 2003, 120, 581–588.
We thank Prof. M. Taniguchi (Director, RIKEN Research Center
for Allergy and Immunology, Yokohama) for his encouragemant.
Received: May 19, 2005
Published Online: September 23, 2005
4800
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2005, 4789–4800