Sex pheromones of the hair crab Erimacrus isenbeckii. II. Synthesis of ceramides

Article abstract of DOI:10.1021/np010177m

To confirm their structures and to assess the pheromonal activity, novel ceramides, possible sex pheromones of the hair crab Erimacrus isenbeckii, were synthesized from D-galactose. The synthetic ceramides were identical with the natural ceramides.

Full text of DOI:10.1021/np010177m

1210
J . Nat. Prod. 2001, 64, 1210-1215
Sex P h er om on es of th e Ha ir Cr a b Er im a cr u s isen beckii. II. Syn th esis of
Cer a m id es
Naoki Asai, Nobuhiro Fusetani,* and Shigeki Matsunaga
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, J apan
Received April 13, 2001
To confirm their structures and to assess the pheromonal activity, novel ceramides, possible sex
pheromones of the hair crab Erimacrus isenbeckii, were synthesized from D-galactose. The synthetic
ceramides were identical with the natural ceramides.
The hair crab Erimacrus isenbeckii, a commercially
important species, exhibits a series of typical mating
behaviors: precopulatory guarding (male grasps female),
molting of female, copulation, and postcopulatory guard.
The involvement of a pheromone released from pre- and
postmolt females in these behaviors was shown by “sponge
assay”. A sexually competent male exhibits the typical
mating behaviors toward a bath sponge containing water
conditioned with pre- or postmolt females. Bioassay-guided
isolation furnished a mixture of novel ceramides as possible
sex pheromones.1 It is well known that either pheromone
structures or the combination of components are crucial
for pheromonal activity in insect pheromones.2 To confirm
the structures proposed for the sex pheromones and to
assess pheromonal activity of this ceramide mixture, we
attempted to synthesize all ceramides. This paper deals
with total synthesis of the ceramides.
metrically isomeric alcohols 8a . Mesylation of 8a followed
by hydrogenation gave rise to a tetraol monomesylate 10a
in 73% yield from 6. The mesylate 10a was converted to
an azide 11a with sodium azide in 82% yield. Finally,
catalytic hydrogenation of the azide afforded the C15
phytosphingosine 12a in 32% overall yield.
Similarly, C16 phytosphingosine was synthesized. The
aldehyde 7 and C11 phosphonium salt prepared from
bromododecane were coupled to give alcohols 8b. A four-
step reaction sequence from 8b afforded the C16 phytosph-
ingosine 12b in 23% overall yield from 7.
Syn th esis of C23 F a tty Acid 21a . The isopropyl ter-
minus was initially introduced by treatment of 9-bromo-
1-nonanal (14), which was obtained by PCC oxidation of
9-bromo-1-nonanol (13), with the ylide of isobutyltriph-
enylphosphonium bromide, which furnished a mixture of
geometric isomers of branched bromoalkenes 15. The
mixture was heated with triphenylphosphine to give phos-
phonium salts 16. Without purification, 16 was again
subjected to a Wittig reaction with 8-bromo-1-octanal. The
resulting diene 17a showed complex 1H NMR signals
probably due to olefin migration, which could consequently
be converted to a single bromoalkane 18a by hydrogena-
tion. Then, conventional acetamidomalonic ester synthesis
was applied to 18a . The resulting mixture was hydrolyzed
and decarboxylated to afford racemic amino acid 19a .
Deamination of 19a was effected with NaNO2 in an acidic
solution (H2SO4/H2O/1,4-dioxane) to give the 2-hydroxy
fatty acid 20a . This was acetylated to afford 21a with Ac2O
and pyridine for the following condensation step (5.7%
overall yield from 13).
Syn th esis of C24 (21b) a n d C25 (21c) F a tty Acid s. In
a similar manner used for the preparation of the C23 fatty
acid 21a , C24 21b and C25 21c fatty acids were synthesized
using 9-bromononanal or 10-bromodecanal in the second
Wittig reaction. Acetates of the C24 and C25 fatty acids were
synthesized in 4.9% and 14.5% overall yield, respectively.
Syn th esis of th e Ha ir Cr a b Cer a m id e Mixtu r e (1a ).
Prior to the condensation step, each of the sphingosines
and fatty acids was mixed according to the natural abun-
dance [12a :12b (4:5); 21a :21b:21c (5:5:3)], which had been
roughly estimated from both HPLC and FABMS data of
the natural ceramide mixture. Coupling of these compo-
nents by using WSCI (water-soluble carbodiimide)/HOBt
(1-hydroxybenzotriazole) and subsequent deacetylation
with K2CO3 gave a diastereomeric mixture of ceramides
1a and 1b. Silica gel TLC (CHCl3/MeOH, 95:5) clearly
showed two well-separated spots, of which the slower-
moving spot (1a ) corresponded to the natural 2′R ceram-
Resu lts a n d Discu ssion
There are many reports of syntheses of ceramides
(cerebrosides);3-7 syntheses of ceramides consisting of
2-hydroxy fatty acids and phytosphingosines have also been
established. Usually, (2S,3S,4R)-phytosphingosines were
synthesized from natural chiral pools such as D-galac-
tose,9,10 D-xylose,11 L-ascorbic acid,12 glyceraldehyde,13 and
L-serine.14 The present work employed a protocol of Ak-
iyama and Natori15 starting from D-galactose, because of
its advantage in large-scale synthesis. On the other hand,
racemic synthesis of the fatty acid portion starting from
9-bromononanol was envisioned.16 After the individual
synthesis of two sphingosines (C15, C16) and three fatty
acids (C23, C24, C25), a combination of these components via
mixed condensation was expected to afford a ceramide
mixture. Acylation of the phytosphingosines with the
racemic fatty acids was expected to give a diastereomeric
mixture from which the desired 2′R isomer could be
separated. Our synthetic strategy for ceramides is depicted
in Scheme 1.
Syn th eses of th e C15 P h ytosp h in gosin e (12a ) a n d
C
16 P h ytosp h in gosin e (12b). Starting from â-D-galactose
pentaacetate (2), 3,4,6-tri-O-benzyl-D-galactopyranose (6)
was prepared in four steps in 54% yield from 2.17 Oxidation
of 6 with sodium metaperiodate provided an aldehyde 7,18
which was directly subjected to Wittig reaction without
purification; 7 was treated with a C10 phosphonium salt
prepared from bromodecane to afford a mixture of geo-
* To whom correspondence should be addressed. Tel: +813-5841-5299.
Fax: +813-5841-8166. E-mail: anobu@mail.ecc.u-tokyo.ac.jp.
10.1021/np010177m CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/11/2001

Sex Pheromones of the Hair Crab Erimacrus
J ournal of Natural Products, 2001, Vol. 64, No. 9 1211
Sch em e 1. Synthetic Plan for the Ceramide Mixture
Sch em e 2a
a
Reagents and conditions: (i) HBr/AcOH; (ii) MeOH/TEA; (iii) BnCl/KOH; (iv) H2SO4; (v) NaIO4; (vi) n-C10H21PPh3Br or n-C11H23PPh3Br/n-BuLi; (vii)
MsCl/pyridine; (viii) H2/Pd-C; (ix) NaN3; (x) H2/Pd-C.
Sch em e 3a
a
Reagents and conditions: (i) PCC; (ii) i-BuPPh3Br/n-BuLi; (iii) PPh3; (iv) 8-bromooctanal or 9-bromononanal or 10-bromodecanal/n-BuLi; (v) H2/Pd-C;
(vi) (EtO2C)2CHNHAc/EtONa, then HCl; (vii) NaNO2; (viii) Ac2O/pyridine.
ides. This was then separated by silica gel column chro-
matography with CHCl3/MeOH solvent systems.
ior of male crabs could be observed for the synthetic
ceramide mixture, because no sexually competent males
were available.
The synthetic ceramide mixture 1a was identical with
1
the natural product in the H and 13C NMR spectra.19 In
Exp er im en ta l Section
addition, HPLC profiles of 1a were almost superimposable
on those of the natural ceramide mixture, except for the
absence of minor components. However, no mating behav-
Gen er a l Exp er im en ta l P r oced u r es. NMR spectra were
1
recorded on either a Bruker AC-300 (300 MHz for H, 75 MHz

1212 J ournal of Natural Products, 2001, Vol. 64, No. 9
Asai et al.
Sch em e 4a
a
Reagents and conditions: (i) WSCI/HOBt; (ii) K2CO3; (iii) Si gel column separation.
for 13C), a J EOL J MN-R600 (600 MHz for 1H, 150 MHz for
The precipitated salts were removed by filtration, and the
filtrate was evaporated to dryness. The resulting residue was
partitioned between CHCl3 and water; the organic layer was
washed with brine and concentrated in vacuo to afford 15.5 g
of 6 (34.4 mmol, 92%): 1H NMR of the R-anomer (300 MHz,
CDCl3) δ 7.36-7.20 (15H, m), 5.27 (1H, d, J ) 4.0 Hz), 4.84
(1H, d, J ) 11.6 Hz), 4.69 (1H, d, J ) 11.9 Hz), 4.62 (1H, d, J
) 11.6 Hz), 4.52 (1H, d, J ) 11.6 Hz), 4.46 (1H, d, J ) 11.9
Hz), 4.38 (1H, d, J ) 11.9 Hz), 4.10 (1H, dd, J ) 6.1, 6.1 Hz),
4.10 (1H, dd, J ) 10.0, 4.0 Hz), 3.88 (1H, m), 3.68 (1H, dd, J
) 9.8, 2.8 Hz), 3.52 (1H, dd, J ) 9.2, 6.7 Hz), 3.42 (1H, dd, J
) 9.5, 6.1 Hz).
(2S,3S,4R)-2,3,5-Tr iben zyloxy-4-h yd r oxyp en ta n a l (7).
Sodium metaperiodate (7.2 g, 33.6 mmol) was added to a
solution of diol 6 (7.6 g, 16.9 mmol) in EtOH/H2O (4:1, 100
mL), and the mixture stirred for 17 h at room temperature.
The reaction mixture was concentrated and partitioned be-
tween ether and water. The organic layer was washed with
brine and dried (Na2SO4), and the solvent evaporated to give
an oil 7 (7.3 g), which was used in the next step without further
purification.
1
13C), or a J EOL J MN-R500 (500 MHz for H, 125 MHz for 13C)
NMR spectrometer at 303 K. 1H chemical shifts were refer-
enced to residual solvent peaks: δ 7.24 for CHCl3 in CDCl3; δ
3.30 for CD2HOD in CD3OD; δ 3.30 for CD2HOD in CDCl3/
CD3OD (1:1). 13C chemical shifts were referenced to 13C signals
of CDCl3 (δ 77.0) or CD3OD (δ 49.0). FAB mass spectra were
measured on a J EOL J MX-SX102/SX102 tandem mass spec-
trometer. Glycerol or m-nitrobenzyl alcohol (NBA) was used
as a matrix. Air- and moisture-sensitive reactions were carried
out under an atmosphere of argon. When necessary, solvents
were distilled from dehydrating agents (THF from sodium and
benzophenone; EtOH from sodium and diethylphthalate; CH2-
Cl2, toluene, MeOH, and 1,2-dichloroethane from CaH2).
Routine monitoring of reactions and preparative TLC were
performed using precoated Si gel TLC plates (Merck 60 F254).
Spots were visualized with either H2SO4, anisaldehyde-H2SO4,
or ninhydrin reagent. Column chromatography was performed
on Merck Si gel 60 (60-230 mesh).
3,4,6-Tr i-O-a cetyl-1,2-O-(1-m eth oxyeth ylid en e)-r-D-ga -
la ctop yr a n ose (4). To penta-O-acetyl-â-D-galactose (2) (25.0
g, 64.1 mmol) was added 30% HBr/AcOH (50 mL), and the
mixture was stirred for 3 h at room temperature. The reaction
mixture was azeotropically evaporated with toluene under
reduced pressure to afford a bromide 3. Without purification,
it was dissolved in 1,2-dichloroethane (200 mL) and triethy-
lamine (18.0 mL, 129 mmol), MeOH (2.74 mL, 67.6 mmol), and
n-tetrabutylammonium bromide (10.3 g, 31.9 mmol) were
added. The mixture was stirred for 16 h at 45 °C, and the
precipitated salt was removed by filtration. The filtrate was
washed with brine and concentrated in vacuo. The residue was
chromatographed over Si gel. Elution with n-hexane/EtOAc
(9:1, 8:2, and 7:3) gave 22.1 g of 4 (61.0 mmol, 95% from 2):
1H NMR (300 MHz, CDCl3) δ 5.78 (1H, d, J ) 4.8 Hz), 5.40
(1H, dd, J ) 2.9, 2.7 Hz), 5.03 (1H, dd, J ) 6.8, 3.3 Hz), 4.29
(2H, m), 4.10 (2H, m), 3.27 (3H, s), 2.08 (3H, s), 2.04 (3H, s),
2.03 (3H, s), 1.64 (3H, s).
3,4,6-Tr i-O-ben zyl-1,2-O-(1-m eth oxyeth ylid en e)-r-D-ga -
la ct op yr a n ose (5). To a stirred solution of 4 (22.0 g, 60.8
mmol) in toluene (60 mL) were added powdered KOH (35.0 g,
620 mmol) and benzyl chloride (48.0 mL, 417 mmol). The
mixture was gradually heated over 1 h and refluxed for 1.5 h.
It was then diluted with toluene and washed with water and
brine. The toluene solution was concentrated in vacuo and
chromatographed over Si gel. Elution with n-hexane/EtOAc
(8:2) gave 19.1 g of 5 (37.5 mmol, 62%): 1H NMR (300 MHz,
CDCl3) δ 7.40-7.20 (15H, m), 5.73 (1H, d, J ) 4.5 Hz), 4.90
(1H, d, J ) 11.5 Hz), 4.79 (1H, d, J ) 12.2 Hz), 4.66 (1H, d, J
) 12.2 Hz), 4.59 (1H, d, J ) 11.9 Hz), 4.49 (1H, d, J ) 11.9
Hz), 4.42 (1H, d, J ) 11.9 Hz), 4.03-3.98 (2H, m), 3.63-3.58
(3H, m), 3.25 (3H, s), 1.56 (3H, s).
3,4,6-Tr i-O-ben zyl-D-ga la ctop yr a n ose (6). To a stirred
solution of 5 (19.0 g, 37.5 mmol) in 1,4-dioxane (280 mL) was
added 1 M H2SO4 (80 mL), and the mixture was refluxed (110
°C) for 2 h. The reaction mixture was cooled to room temper-
ature, then solid NaHCO3 was carefully added until the
mixture was neutralized; stirring was continued for 30 min.
(2R,3R,4R)-1,3,4-Tr iben zyloxy-5-p en ta d ecen e-2-ol (8a ).
A suspension of decyltriphenylphosphonium bromide (18.0 g,
37.2 mmol) in THF (80 mL) was stirred at 0 °C, while 16.0
mL of n-BuLi (1.6 M solution in n-hexane) was added dropwise.
The resulting solution was stirred for 30 min, and then a
solution of the aldehyde 7 (4.6 g, 10.7 mmol) in THF (20 mL)
was added. The mixture was warmed to room temperature and
stirred for 1 h. The reaction was quenched by adding water
and extracted with ether. The ether layer was dried with Na2-
SO4 and concentrated in vacuo. SiO2 chromatography with
n-hexane/EtOAc (8:2) gave the alkene 8a (4.41 g, 8.1 mmol,
76%) as a pale yellow oil: 1H NMR of the 5Z-isomer (300 MHz,
CDCl3) δ 7.35-7.20 (15H, m), 5.72 (1H, m), 5.45 (1H, dd, J )
11.0, 10.9 Hz), 4.68 (1H, d, J ) 11.3 Hz), 4.60 (1H, d, J ) 11.7
Hz), 4.50-4.45 (3H, m), 4.40 (1H, dd, J ) 9.5, 5.2 Hz), 4.33
(1H, d, J ) 11.8 Hz), 4.07 (1H, td, J ) 5.9, 2.9 Hz), 3.56 (1H,
dd, J ) 5.6, 2.9 Hz), 3.51 (2H, d, J ) 5.9 Hz), 2.81 (1H, brs),
1.93 (2H, m), 1.30-1.20 (14H, m), 0.88 (3H, t, J ) 7.0 Hz).
(2R,3R,4R)-1,3,4-Tr iben zyloxy-5-h exa d ecen e-2-ol (8b).
A suspension of undecyltriphenylphosphonium bromide (16.5
g, 33.2 mmol) in THF (70 mL) was stirred at 0 °C, while 18.0
mL of n-BuLi (1.6 M solution in n-hexane) was added dropwise.
The resulting solution was stirred for 10 min, and then a
solution of the aldehyde 7 (4.6 g, 10.7 mmol) in THF (15 mL)
was added. The mixture was warmed to room temperature and
stirred for 30 min. The reaction was quenched by adding water
and then extracted with ether. The ether layer was dried with
Na2SO4 and concentrated in vacuo. SiO2 chromatography with
n-hexane/EtOAc (8:2) gave the alkene 8b (4.15 g, 7.43 mmol,
69%) as a pale yellow oil: 1H NMR of the 5Z-isomer (300 MHz,
CDCl3) δ 7.35-7.20 (15H, m), 5.71 (1H, dt, J ) 11.0, 7.0 Hz),
5.44 (1H, dd, J ) 11.0, 11.0 Hz), 4.66 (1H, d, J ) 11.3 Hz),
4.61 (1H, d, J ) 9.5 Hz), 4.50-4.45 (3H, m), 4.42 (1H, dd, J )
10.3, 5.0 Hz), 4.33 (1H, d, J ) 11.8 Hz), 4.06 (1H, td, J ) 5.9,
2.9 Hz), 3.54 (1H, dd, J ) 5.5, 2.9 Hz), 3.50 (2H, d, J ) 5.9

Sex Pheromones of the Hair Crab Erimacrus
J ournal of Natural Products, 2001, Vol. 64, No. 9 1213
Hz), 1.94 (1H, ddt, J ) 13.5, 7.0, 7.0 Hz), 1.87 (1H, ddt, J )
13.5, 7.0, 7.0 Hz), 1.30-1.20 (16H, m), 0.86 (3H, t, J ) 7.0
Hz).
eluting with CHCl3/MeOH (95:5) gave 191 mg (0.61 mmol,
74%) of the azide 11b: 1H NMR (300 MHz, CDCl3/CD3OD, 1:1)
δ 3.90 (1H, dd, J ) 11.9 and 3.4 Hz), 3.75 (2H, m), 3.55 (2H,
m), 1.65-1.47 (4H, m), 1.40-1.20 (18H, m), 0.84 (3H, t, J )
6.9 Hz).
(2R,3R,4R)-1,3,4-Tr iben zyloxy-2-m eth a n esu lfon yloxy-
5-p en ta d ecen e (9a ). A solution of 8a (4.0 g, 7.17 mmol) in
pyridine (10.0 mL) was ice-cooled, and then MsCl (1.20 mL,
15.5 mmol) was added. The mixture was stirred for 7 h at room
temperature, then toluene was added, and the solution was
concentrated in vacuo. The resulting residue was dissolved in
ether, and the solution was washed successively with water
and brine. The organic layer was dried over Na2SO4 and
evaporated to dryness, and the residue was chromatographed
over Si gel. Elution with n-hexane/EtOAc (9:1) gave the
O-mesylate 9a (4.37 g, 6.87 mmol, 96%): 1H NMR of the 5Z-
isomer (500 MHz, CDCl3) δ 7.35-7.20 (15H, m), 5.77 (1H, dt,
J ) 11.2, 7.3 Hz), 5.46 (1H, t, J ) 11.2 Hz), 5.03 (1H, ddd, J
) 7.1, 4.0, 4.0 Hz), 4.73 (1H, d, J ) 11.0 Hz), 4.51-4.35 (6H,
m), 3.74 (1H, dd, J ) 11.0, 3.7 Hz), 2.90 (3H, s), 2.04 (1H, ddt,
J ) 14.5, 7.3, 7.3 Hz), 1.94 (1H, ddt, J ) 14.5, 7.3, 7.3 Hz),
1.35-1.20 (16H, m), 0.84 (3H, t, J ) 7.0 Hz).
(2S,3S,4R)-2-Am in o-1,3,4-p en ta d eca n etr iol (12a ). To a
solution of the azide 11a (500 mg, 1.66 mmol) in EtOH (20
mL) was added 10% palladium on carbon (500 mg). The
reaction flask was flushed with hydrogen gas and the solution
vigorously stirred for 20 h (room temperature, 1 atm). The
mixture was then filtered through a pad of Celite (with CHCl3/
MeOH rinse), the solvent concentrated, and the residue
chromatographed over Si gel under basic conditions. The
column was eluted with CHCl3/MeOH/H2O/25% NH4OH (8:2:
0.15:0.05), and the eluate was concentrated, diluted with
water, and lyophilized to afford the C15 phytosphingosine 12a
(433 mg, 1.57 mmol, 95%): 1H NMR (300 MHz, CDCl3) δ 3.72
(1H, dd, J ) 11.1, 4.3 Hz), 3.58 (1H, dd, J ) 11.1, 6.2 Hz),
3.51 (1H, m), 3.32 (1H, dd, J ) 7.7, 5.5 Hz), 2.94 (1H, dd, J )
10.1, 5.5 Hz), 1.70 (2H, m), 1.53 (2H, m), 1.40-1.20 (16H, m),
0.85 (3H, t, J ) 6.9 Hz).
(2R,3R,4R)-2-Meth a n esu lfon yloxy-1,3,4-p en ta d eca n e-
tr iol (10a ). To an ice-cooled solution of 20a (4.0 g, 7.35 mmol)
in pyridine (15.0 mL) was added MsCl (1.15 mL, 14.9 mmol).
The mixture was stirred for 15 h at room temperature, then
toluene was added, and the solution was concentrated in vacuo.
The resulting residue was dissolved in ether, and the solution
was washed successively with water and brine. The organic
layer was dried over Na2SO4 and evaporated to dryness, and
the residue was chromatographed over Si gel. Elution with
n-hexane/EtOAc (9:1, 8:2) afforded the O-mesylate 9a (4.66 g,
quant.). Then the O-mesylate 9a (2.26 g, 3.63 mmol) was
dissolved in EtOH (30 mL), and 2.30 g of 10% palladium on
carbon was added. Hydrogen gas was introduced to the
reaction flask and the solution vigorously stirred for 20 h (room
temperature, 1 atm). The reaction mixture was filtered
through a pad of Celite (with CHCl3 rinse), and the filtrate
was concentrated to give 1.24 g (3.50 mmol, 96%) of 10a as a
white powder: 1H NMR (300 MHz, CDCl3/CD3OD, 1:1) δ 4.89
(1H, ddd, J ) 6.5, 5.3, 2.1 Hz), 3.90-3.75 (2H, m), 3.53-3.48
(1H, m), 3.46 (1H, dd, J ) 8.5, 2.1 Hz), 3.14 (3H, s), 1.74 (2H,
m), 1.52 (2H, m), 1.40-1.20 (16H, m), 0.84 (3H, t, J ) 6.7 Hz).
(2S,3S,4R)-2-Am in o-1,3,4-h exa d eca n etr iol (12b). To a
solution of the azide 11b (100 mg, 0.32 mmol) in EtOH (11
mL) was added 10% palladium on carbon (110 mg). The
reaction flask was flushed with hydrogen gas and the solution
vigorously stirred for 20 h (room temperature, 1 atm). The
mixture was then filtered through a pad of Celite (with CHCl3/
MeOH rinse), the solvent concentrated, and the residue
chromatographed over Si gel. Elution with CHCl3/MeOH/H2O
(8:2:0.2, 7:3:0.5) gave the C16 phytosphingosine12b (51.0 mg,
0.18 mmol, 56%): 1H NMR (300 MHz, CDCl3/CD3OD, 1:1) δ
3.87 (1H, dd, J ) 11.8, 4.0 Hz), 3.75 (1H, dd, J ) 11.8, 7.5
Hz), 3.62 (1H, m), 3.52 (1H, dd, J ) 8.9, 4.4 Hz), 3.45 (1H, dd,
J ) 7.6, 3.7 Hz), 1.73 (2H, m), 1.49 (2H, m), 1.40-1.20 (18H,
m), 0.83 (3H, t, J ) 7.0 Hz).
9-Br om on on a n a l (14). To a stirred solution of 9-bro-
mononanol (13) (4.9 g, 22.0 mmol) in CH2Cl2 (30 mL) was
added pyridinium chlorochromate (10.0 g, 46.4 mmol). Stirring
was continued for 2 h at room temperature, and then ether
(10 mL) and Na2SO4 were added to the solution. The suspen-
sion was filtered and rinsed with ether, and the solvent
concentrated in vacuo. The residue was chromatographed over
Si gel. Elution with n-hexane/EtOAc (96:4, 9:1) gave the
aldehyde 14 (4.3 g, 19.5 mmol, 89%): 1H NMR (300 MHz,
CDCl3) δ 9.74 (1H, t, J ) 1.8 Hz), 3.38 (2H, t, J ) 7.0 Hz),
2.40 (2H, brt, J ) 7.0 Hz), 1.82 (2H, quint, J ) 7.0 Hz), 1.60
(2H, quint, J ) 7.3 Hz), 1.40 (2H, quint, J ) 7.0 Hz), 1.35-
1.25 (6H, m).
12-Br om o-2-m eth yl-3-d od ecen e (15). To an ice-cooled
suspension of isobutyltriphenylphosphonium bromide (10.0 g,
25.0 mmol) in THF (50 mL) was added 14.0 mL of n-BuLi (1.6
M solution in n-hexane). The resulting solution was stirred
for 30 min at 0 °C, and then a solution of 8-bromooctanal (4.1
g, 18.5 mmol) in THF (30 mL) was added. The mixture was
warmed to room temperature and stirring was continued for
2 h. The reaction was quenched by addition of water, and the
mixture extracted with ether. The combined ethereal fraction
was washed with brine, dried over MgSO4, and concentrated.
Chromatography on Si gel with n-hexane gave the alkene 15
as a colorless oil (3.8 g, 14.4 mmol, 78%): 1H NMR of the 3Z-
isomer (300 MHz, CDCl3) δ 5.20 (1H, dt, J ) 11.0, 6.9 Hz),
5.16 (1H, ddt, J ) 10.7, 10.7, 1.2 Hz), 3.38 (2H, t, J ) 6.7 Hz),
2.56 (1H, m), 2.00 (2H, dt, J ) 7.0, 7.0 Hz), 1.83 (2H, quint, J
) 6.9 Hz), 1.42 (2H, m), 1.40-1.20 (8H, m), 0.92 (6H, d, J )
6.7 Hz).
11-Met h yl-9-d od ecen ylt r ip h en ylp h osp h on iu m Br o-
m id e (16). The mixture of the bromide 15 (3.6 g, 16.2 mmol)
and triphenylphosphine (4.3 g, 16.4 mmol) was heated at 120
°C for 2 days to give 16. In the same manner, this reaction
was repeated twice for each of the next Wittig reactions. The
resulting viscous materials were used in the next step without
further purification.
(2R,3R,4R)-2-Met h a n esu lfon yloxy-1,3,4-h exa d eca n e-
tr iol (10b). To a solution of 9b (1.35 g, 2.12 mmol) in EtOH
(30 mL) was added 10% palladium on carbon (1.36 g). The
mixture was stirred for 22 h under a hydrogen atmosphere at
room temperature (1 atm). The Pd/C was then removed by
filtration through
a pad of Celite, and the filtrate was
concentrated to give the triol 10b as a white powder (1.24 g,
3.50 mmol, 89%): 1H NMR (300 MHz, CDCl3/CD3OD, 1:1) δ
4.90 (1H, ddd, J ) 6.6, 5.3, 2.0 Hz), 3.86 (1H, dd, J ) 12.2, 6.5
Hz), 3.80 (1H, dd, J ) 12.1, 4.9 Hz), 3.52 (1H, ddd, J ) 8.4,
8.4, 2.3 Hz), 3.46 (1H, dd, J ) 8.4, 1.9 Hz), 3.15 (3H, s), 1.75
(2H, m), 1.52 (2H, m), 1.40-1.20 (18H, m), 0.84 (3H, t, J )
6.7 Hz).
(2S,3S,4R)-2-Azid o-1,3,4-p en t a d eca n et r iol (11a ). So-
dium azide (600 mg, 9.2 mmol) was added to a solution of the
mesylate 10a (609 mg, 1.72 mmol) in DMF (20 mL) at room
temperature. The mixture was slowly heated to 100 °C over
30 min and then stirred for 3 h. The mixture was diluted with
EtOAc and washed with water and brine. The organic layer
was dried (Na2SO4) and concentrated in vacuo, and remaining
DMF was removed by lyophilization. The resulting residue was
chromatographed over Si gel with CHCl3/MeOH (97:3, 96:4)
to yield 425 mg (1.41 mmol, 82%) of the azide 11a : 1H NMR
(600 MHz, CDCl3/CD3OD, 1:1) δ 3.90 (1H, dd, J ) 12.0, 3.6
Hz), 3.74 (2H, m), 3.54 (2H, m), 1.62 (2H, m), 1.56 (2H, m),
1.40-1.20 (16H, m), 0.85 (3H, t, J ) 7.0 Hz).
(2S,3S,4R)-2-Azid o-1,3,4-h exa d eca n etr iol (11b). Sodium
azide (200 mg, 3.08 mmol) was added to a solution of the
mesylate 10b (300 mg, 0.82 mmol) in DMF (12 mL) at room
temperature. It was slowly heated to 100 °C over 30 min and
then stirred for 3 h. The reaction mixture was partitioned
between EtOAc and H2O. The organic layer was dried (Na2-
SO4) and concentrated in vacuo. Chromatography (SiO2)
1-Br om o-19-m eth yleicosa n e (18a ). To a suspension of the
phosphonium salt 16 (prepared from 3.6 g of 15) in THF (20

1214 J ournal of Natural Products, 2001, Vol. 64, No. 9
Asai et al.
mL) was added 8.5 mL of n-BuLi (1.6 M solution in n-hexane)
at 0 °C. The resulting solution was stirred for 30 min at 0 °C,
and then a solution of 8-bromoctanal (3.4 g, 16.4 mmol) in THF
(15 mL) was added. The mixture was warmed to room
temperature, and stirring was continued for 1 h. The reaction
was quenched by the addition of water, and the mixture
extracted with ether. The combined ethereal fraction was
washed with brine, dried over MgSO4, and concentrated.
Chromatography over Si gel with n-hexane afforded the E,Z-
mixture of the 1-bromo-19-methyl-8,17-eicosadiene 17a as a
(1H, dd, J ) 7.6, 4.5 Hz), 1.80-1.50 (2H, m), 1.50-1.20 (37H,
m), 1.12 (2H, m), 0.82 (6H, d, J ) 6.5 Hz).
2-Acetoxy-21-m eth yld ocosa n oic a cid (21a ). Acetic an-
hydride (4.0 mL) was added to a solution of the alcohol 20a
(117 mg, 0.32 mmol) in pyridine (4.0 mL). The mixture was
stirred for 13 h at room temperature. It was then diluted with
ice water and extracted with ether. The ethereal extract was
washed with brine, dried (MgSO4), and concentrated. The
residue was chromatographed over Si gel. Elution with n-
hexane/EtOAc/AcOH (5:5:0.2) gave the acetate 21a as a white
solid (102 mg, 0.25 mmol, 77%): 1H NMR (600 MHz, CDCl3)
δ 4.94 (1H, dd, J ) 7.3, 5.0 Hz), 2.07 (3H, s), 1.79 (2H, m),
1.44 (2H, m), 1.36 (2H m), 1.40-1.20 (30H, m), 1.07 (2H, m),
0.79 (6H, d, J ) 6.5 Hz).
2-Acetoxy-22-m eth yltr icosa n oic a cid (21b). In the same
manner as described above for the preparation of 21a , the
alcohol 20b (109 mg, 0.28 mmol) was converted to 105 mg (0.25
mmol, 88%) of 21b: 1H NMR (600 MHz, CDCl3) δ 4.99 (1H, t,
J ) 6.3 Hz), 2.12 (3H, s), 1.84 (2H, m), 1.49 (2H, m), 1.41 (2H
m), 1.40-1.20 (30H, m), 1.13 (2H, m), 0.84 (6H, d, J ) 6.9
Hz).
1
colorless oil (2.7 g, 7.3 mmol, 45%). The H NMR spectrum of
17a exhibited additional signals for 1-bromo-19-methyl-8,16-
eicosadiene and 1-bromo-19-methyl-8,18-eicosandiene probably
produced through migration of the double bond, which were
not separated, but were used as such in the next reaction. The
mixture of 17a (2.7 g, 7.3 mmol) and 10% palladium on carbon
(2.7 g) in 30 mL of EtOH was stirred under a hydrogen
atmosphere for 20 h (room temperature, 1 atm). The reaction
mixture was filtered through a pad of Celite (with CHCl3
rinse), and the filtrate was evaporated to dryness. Chroma-
tography over Si gel, eluting with n-hexane, yielded the
bromoalkane 18a (2.0 g, 5.4 mmol, 74%) as a colorless oil: 1H
NMR (300 MHz, CDCl3) δ 3.39 (2H, t, J ) 6.8 Hz), 1.84 (2H,
quint, J ) 7.3 Hz), 1.50 (1H, m), 1.41 (2H, m), 1.35-1.20 (28H,
m), 1.20-1.15 (2H, m), 0.85 (6H, d, J ) 6.7 Hz).
1-Br om o-20-m eth ylh en eicosa n e (18b). Following the
same procedure as described above for the preparation of 18a ,
9-bromononanal (2.0 g, 9.0 mmol) gave 18b (1.1 g, 2.8 mmol,
31%): 1H NMR (300 MHz, CDCl3) δ 3.39 (2H, t, J ) 7.1 Hz),
1.83 (2H, quint, J ) 6.7 Hz), 1.49 (1H, m), 1.40 (2H, m), 1.35-
1.20 (30H, m), 1.13 (2H, m), 0.84 (6H, d, J ) 6.8 Hz).
2-Acetoxy-23-m eth yltetr a cosa n oic a cid (21c). In the
same manner as described above for the preparation of 21a ,
the alcohol 20c (180 mg, 0.45 mmol) was converted to 168 mg
(0.38 mmol, 84%) of 21c: 1H NMR (600 MHz, CDCl3) δ 4.98
(1H, dd, J ) 7.3, 6.4 Hz), 2.12 (3H, s), 1.84 (2H, m), 1.49 (2H,
m), 1.40 (2H, m), 1.40-1.20 (30H, m), 1.13 (2H, m), 0.83 (6H,
d, J ) 6.5 Hz).
Syn th esis of th e Cer a m id e Mixtu r e (1a , 1b). The
mixture of phytosphingosines 12a (25.0 mg, 0.091 mmol) and
12b (33.0 mg, 0.114 mmol), 2-acetoxy fatty acids 21a (30.0 mg,
0.073 mmol), 21b (30.0 mg, 0.070 mmol), and 21c (20.0 mg,
0.045 mmol), and HOBt (1-hydroxybendotriazole) (40.0 mg,
0.30 mmol) was diluted with benzene, evaporated, and lyoph-
ilized to remove water. The residue was dissolved in CH2Cl2
(5.0 mL) and added to WSCI [1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride] (60.0 mg, 0.31 mmol). The
mixture was stirred for 20 h at room temeperature, then
diluted with CHCl3, washed with water, dried (NaSO4), and
concentrated. To the residue were added MeOH (9.0 mL), THF
(3.0 mL), and K2CO3 (90 mg), and the mixture was stirred for
2.5 h at room temperature. The reaction mixture was concen-
trated in vacuo and chromatographed over Si gel. Elution with
CHCl3/MeOH (97:3, 96:4) furnished 2′S (1 b, 45.0 mg) and 2′R
(1 a , 36.0 mg) ceramide mixtures. 1a : 1H NMR (600 MHz,
CDCl3/CD3OD, 1:1) δ 4.08 (1H, ddd, J ) 4.8, 4.6, 3.8 Hz), 4.00
(1H, dd, J ) 8.1, 3.8 Hz), 3.76 (1H, dd, J ) 11.2, 4.6 Hz), 3.70
(1H, dd, J ) 11.3, 4.8 Hz), 3.51 (2H, m), 1.74 (1H, m), 1.64
(1H, m), 1.60-1.15 (brm), 1.12 (2H, m), 0.84 (3H, t, J ) 6.9
Hz), 0.82 (6H, d, J ) 6.9 Hz); 13C NMR (150 MHz, CDCl3/CD3-
OD, 1:1) δ 176.4s, 75.9d, 72.9d, 72.4d, 61.6t, 52.1d, 39.6t, 35.1t,
33.2t, 32.5t, 30.4t, 30.2 (18-21C), 29.9t, 28.5t, 27.9t, 26.4t,
25.6d, 23.2q (2C), 22.9t, 14.3q; HRFABMS for C15/C23 m/z (M
+ H)+ 628.5873 (calcd for C38H78NO5 ∆ -0.7 mmu); C16/C23
m/z (M + H)+ 642.6085 (calcd for C39H80NO5 ∆ +4.8 mmu);
1-Br om o-21-m eth yld ocosa n e (18c). Following the same
procedure as described above for the preparation of 18a , 10-
bromodecanal (3.7 g, 15.7 mmol) afforded 18c (4.0 g, 10.0
mmol, 64%): 1H NMR (300 MHz, CDCl3) δ 3.40 (2H, t, J )
7.0 Hz), 1.83 (2H, quint, J ) 6.8 Hz), 1.50 (1H, m), 1.40 (2H,
m), 1.35-1.20 (32H, m), 1.15 (2H, m), 0.85 (6H, d, J ) 6.8
Hz).
2-Hyd r oxy-21-m eth yld ocosa n oic a cid (20a ). To a sus-
pension of diethyl acetamidomalonate (5.0 g, 23.0 mmol) in
EtOH (10.0 mL) was added 9.0 mL of sodium ethoxide (2.4 M
solution in EtOH, prepared from Na and EtOH). The mixture
was stirred for 30 min at room temperature. Then, 7.0 mL
(6.0 mmol) of the resulting solution was added to a solution of
the bromide 18a (935 mg, 2.49 mmol) in EtOH (5.0 mL), and
the mixture was stirred and heated under reflux for 10 h. The
reaction mixture was concentrated, diluted with water, and
extracted with ether. The ether extract was concentrated in
vacuo. The residue was dissolved in concentrated HCl (7.5 mL),
water (2.5 mL), and 1,4-dioxane (5.0 mL), and the resulting
mixture was stirred and heated under reflux for 12 h. The
reaction mixture was lyophilized to give 500 mg (1.36 mmol)
of amino acid 19a as a white powder. Part of the resulting
amino acid (91.0 mg, 0.26 mmol) was mixed with 2 N H2SO4
(5.0 mL) and 1,4-dioxane (5.0 mL). A solution of sodium nitrite
(75.0 mg, 1.09 mmol) in water (1.0 mL) was added to the
suspension, which was stirred for 1 h at 80 °C. The resulting
solution was diluted with ether, washed with water, dried
(MgSO4), and concentrated. Chromatography on Si gel with
n-hexane/EtOAc/AcOH (5:5:0.2) afforded the 2-hydroxy acid
20a (56.0 mg, 0.15 mmol, 32% from 18a ): 1H NMR (300 MHz,
CDCl3/CD3OD, 1:1) δ 4.09 (1H, dd, J ) 7.6, 4.5 Hz), 1.80-
1.50 (2H, m), 1.50-1.20 (33H, m), 1.12 (2H, m), 0.82 (6H, d, J
) 6.7 Hz).
C
15/C25 m/z (M + H)+ 656.6244 (calcd for C40H82NO5 ∆ +5.1
mmu); C16/C25 m/z (M + H)+ 670.6400 (calcd for C41H84NO5 ∆
+5.0 mmu). 1b: 1H NMR (600 MHz, CDCl3/CD3OD, 1:1) δ 4.09
(1H, ddd, J ) 5.0, 4.6, 3.8 Hz), 4.00 (1H, dd, J ) 8.1, 3.8 Hz),
3.76 (1H, dd, J ) 11.2, 4.6 Hz), 3.70 (1H, dd, J ) 11.2, 5.0
Hz), 3.52 (2H, m), 1.76 (1H, m), 1.64 (1H, m), 1.60-1.15 (brm),
1.12 (2H, m), 0.84 (3H, t, J ) 6.9 Hz), 0.82 (6H, d, J ) 6.6
Hz).
2-Hyd r oxy-22-m eth yltr icosa n oic a cid (20b). Following
the same procedure as described above for the preparation of
20a , the bromoalkane 18b was converted to the 2-hydroxy acid
20b (18%): 1H NMR (300 MHz, CDCl3/CD3OD, 1:1) δ 4.09 (1H,
dd, J ) 7.6, 4.5 Hz), 1.80-1.50 (2H, m), 1.50-1.20 (35H, m),
1.12 (2H, m), 0.82 (6H, d, J ) 6.5 Hz).
Ack n ow led gm en t. We thank Professor Paul J . Scheuer
of the University of Hawaii for reading the manuscript. This
study was partly supported by a research fund from the Nissan
Science Foundation. N.A. is grateful for a Research Fellowship
of the J apan Society for the Promotion of Science for Young
Scientists.
2-Hyd r oxy-23-m eth yltetr a cosa n oic a cid (20c). Follow-
ing the same procedure as described above for the preparation
of 20a , the bromoalkane 18c was converted to the 2-hydroxy
acid 20c (27%); 1H NMR (300 MHz, CDCl3/CD3OD, 1:1) δ 4.09
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra and HPLC
profiles of the natural and synthetic ceramide mixtures. This material
is available free of charge via the Internet at http://pubs.acs.org.

Sex Pheromones of the Hair Crab Erimacrus
J ournal of Natural Products, 2001, Vol. 64, No. 9 1215
(12) Nakashima, H.; Hirata, N.; Iwamura, T.; Yamagiwa, Y.; Kamikawa,
T. J . Chem. Soc., Perkin. Trans. 1 1994, 2849-2857.
(13) Mulzer, J .; Brand, C. Tetrahedron 1986, 42, 5961-5968.
(14) Sugiyama, S.; Honda, M.; Komori, T. Liebigs Ann. Chem. 1990, 1069-
1078.
(15) Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994,
50, 2771-2784.
Refer en ces a n d Notes
(1) Asai, N.; Fusetani, N.; Matsunaga, S.; Sasaki, J . Tetrahedron 2000,
56, 9895-9899.
(2) Roelofs, W. L. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 44-49, and
references therein.
(3) Reist, E. J .; Christie, P. H. J . Org. Chem. 1970, 35, 4127-4130.
(4) Tkaczuk, P.; Thornton, E. R. J . Org. Chem. 1981, 46, 4393-4403.
(5) Obayashi, M.; Schlosser, M. Chem. Lett. 1985, 1715-1718.
(6) Schmidt R. R.; Zimmermann, P. Tetrahedron Lett. 1986, 27, 481-
484.
(7) J ulina, R.; Herzig, T.; Bernet, B.; Vasella, A. Helv. Chim. Acta 1986,
69, 368-373.
(8) Mori, K.; Kinsho, T. Liebigs Ann. Chem. 1988, 807-814.
(9) Akimoto, K.; Natori, T.; Morita, M. Tetrahedron Lett. 1993, 34, 5593-
5596.
(16) Sugiyama, S.; Honda, M.; Komori, T. Liebigs Ann. Chem. 1990, 1063-
1068.
(17) Mori, K.; Funaki, Y. Tetrahedron 1985, 41, 2369-2377.
(18) Koike, K.; Numata, M.; Sugimoto, M.; Nakahara, Y.; Ogawa, T.
Carbohydr. Res. 1986, 158, 113-123.
(19) 13C NMR data for the natural ceramide mixture (150 MHz, CDCl3/
CD3OD, 1:1) δ 176.5s, 75.7d, 72.7d, 72.5d, 61.5t, 52.0d, 34.8t, 33.0t,
30.0t, 29.5d, 24.0t, 22.7q(2C), 14.2q.
(10) Gigg, J .; Gigg, R. J . Chem. Soc. (C) 1966, 1876-1879.
(11) Schmidt, R. R.; Maier, T. Carbohydr. Res. 1988, 174, 169-179.
NP010177M