Syntheses of sphingomyelin methylene, aza, and sulfur analogues by the versatile olefin cross-metathesis method

Article abstract of DOI:10.1016/j.tet.2008.10.018

The syntheses of optically homogeneous sphingomyelin analogues, which possess CH₂, NH, and S instead of the phosphate oxygen connecting the phosphocholine head group to the sphingosine backbone, were successfully achieved by employing the olefi

Full text of DOI:10.1016/j.tet.2008.10.018

Tetrahedron 64 (2008) 11647–11660
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Syntheses of sphingomyelin methylene, aza, and sulfur analogues
by the versatile olefin cross-metathesis method
Tetsuya Yamamoto, Hiroko Hasegawa, Sekimi Ishii, Satoshi Kaji, Tatsuro Masuyama,
*
Syuji Harada, Shigeo Katsumura
Department of Chemistry and Open Research Center on Organic Tool Molecules, School of Science and Technology,
Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2008
Received in revised form 8 October 2008
Accepted 8 October 2008
Available online 15 October 2008
The syntheses of optically homogeneous sphingomyelin analogues, which possess CH2, NH, and S instead
of the phosphate oxygen connecting the phosphocholine head group to the sphingosine backbone, were
successfully achieved by employing the olefin cross-metathesis protocol between 1-pentadecene and the
amino alcohol parts possessing the suitable building block or functional group for construction of the
phosphocholine moiety. In addition, fluorescence-labeled sphingomyelin methylene analogue was also
synthesized.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
groups to examine their physical properties in the microdomain.
Since the replacement of the phosphate oxygen is likely to affect
Sphingolipids are regarded as lipid secondary messengers in
mammalian cells and cell membranes, and are now accepted to
play an important role in signal transduction and molecular rec-
ognition processes in cell membranes.1 Among them, sphingo-
myelin (SM) is known as a major component that forms a raft
domain, which is the lipid microdomain consisting of SM and
cholesterol as the major components.2 Generally, this particular
domain is considered to play a key role in certain processes, such as
membrane trafficking and signal transduction.2c,3 However, the
distinct role of SM in constructing the raft domain is still not clearly
understood. It has been suggested that there exists an intra-
molecular hydrogen bond between the hydroxy group in the
sphingosine backbone and the phosphate oxygen, which is the one
connecting the phosphocholine head group to the sphingosine
backbone.4a,b Meanwhile, the amide hydrogen has also been sug-
gested to act as a donor of the intermolecular hydrogen bond.4b,c
These two kinds of hydrogen bonds have been suggested to affect
the phase transition temperature of bilayer membranes consisting
of SM and cholesterol. The size of the phosphocholine head group
in sphingolipids, which is connected by phosphate oxygen, has also
been found important in the interactions with cholesterol.5
the properties of the SM analogues, we intended to examine how
these modifications affect the SM intermolecular property in ad-
dition to the SM/cholesterol interaction in monolayers and multi-
lamellar vesicles. Meanwhile, we previously synthesized the
corresponding saturated short chain analogues, which were origi-
nally designed as sphingomyelinase inhibitors.6 However, both the
double bond and the same number of the carbon chain to that of
natural SM are required for understanding the property of the raft
domain. The olefin cross-metathesis protocol is very attractive for
providing these molecules,7 and a number of papers on the func-
tionalized sphingolipid synthesis were reported.8 We also de-
veloped the olefin cross-metathesis protocol as the efficient
synthesis of SM.9 Furthermore, very recently, we have established
a stereocontrolled versatile method for the synthesis of various
kinds of natural sphingolipids, such as sphingosine, ceramide,
sphingosine 1-phosphate, SM, and functionalized sphingosine de-
rivatives by two types of combinations of the olefin cross-metath-
esis reaction. One was between the same olefin part and
appropriate amino alcohols, which were prepared starting from
L-serine, and the other was between the fluorescence and photo-
affinity labeled olefin parts and the same amino alcohol part as
We then planned to synthesize three types of SM analogues, in
which the phosphate oxygen was replaced with CH2, NH, and S
shown in Figure 2.10
In this paper, we describe the highly efficient syntheses of the
SM methylene 1, aza 2, and sulfur 3 analogues in addition to the
synthesis of the fluorescence-labeled SM methylene analogue 4
using the versatile olefin cross-metathesis protocol as a key step
(Fig. 1).
* Corresponding author. Tel.: þ81 79 565 8314; fax: þ81 79 565 9077.
E-mail address: katsumura@kwansei.ac.jp (S. Katsumura).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.10.018

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T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
amino alcohol derivative 7 in 78% yield. The olefin cross-metathesis
O
O
intermolecular
hydrogen bond
reaction between 7 and 1-pentadecene required a rather longer
reaction time to complete the reaction than the results of Figure 2,
and the coupling product 8 was obtained in 68% yield after 7 h. In
this case, the cis-isomer was not admitted. The Cbz protecting
group at the secondary hydroxy group seemed to decrease the re-
activity compared with that of the free hydroxy derivative in the
olefin cross-metathesis reaction. The treatment of 8 with TFA fol-
lowed by an acylation with palmitoyl chloride and K2CO3 produced
9, which was transformed into the desired phosphonic acid de-
rivative 10 by the TMSBr treatment in good yield.12 The final part of
the synthesis of 1 was the introduction of a choline group. The
treatment of 10 with 3.0 equiv of N-Boc aminoethanol and 5.0 equiv
of trichloroacetonitrile in pyridine at 65 ꢀC successfully produced
11 in 89% yield.6d,13 Meanwhile, the use of a large amount of the
reagents afforded the undesired product, which possessed two
choline moieties. The treatment of 11 with TFA to remove the Boc
group gave the undesired product, which might be the benzyloxy-
carbonyl (Cbz) ester of the phosphonic acid resulting from the
rearrangement of the Cbz group at the secondary hydroxy group.
On the other hand, the Boc group was successfully removed after
methylation of the phosphonic acid moiety. Thus, the successive
treatment of 11 with Dowex 50W in chloroform to thoroughly
acidify followed by a diazomethane treatment quantitatively pro-
duced the corresponding methyl ester, which was treated with
bromotrimethylsilane and then methanol to produce the desired 12
resulting from the selective removal of both the Boc and the methyl
groups rather than the 2-aminoethyl group.14 Methylation of the
primary amine in 12 with MeI and K2CO3 produced the desired
compound 13 in 54% yield from 10. Finally, the Cbz group of 13 was
removed by hydrolysis with 2 N KOH in methanol at room tem-
perature. The desired 1 was successfully obtained in 76% yield by
neutralization of the reaction mixture with 2 N HCl, concentration
without extraction, and then purification by silica gel.
H
C15H31
C13H27
N
H
sphingomyelin (SM)
O
O
P
NMe3
O
O
intramolecular
hydrogen bond
O
1: X = CH2
C15H31
C13H27
NH
SM analogues 2: X = NH
3: X = S
O
X
P
NMe3
O
O
OH
O
O2N
C15H31
NH
(CH2)9
4: X = CH2
O
P
X
N
H
NMe3
N
O
O
O
N
OH
Figure 1. Sphingomyelin (SM) and SM analogues.
2. Results and discussion
2.1. Synthesis of sphingomyelin methylene analogue 1
We first planned to synthesize 1 in order to investigate the effect
of the intramolecular hydrogen bond in the SM molecule in con-
structing the raft domain. The synthesis of 1 is shown in Scheme 1.
Dimethyl phosphonate 6, a key synthetic intermediate of the SM
methylene analogue 1, was prepared by the method used for our
previous synthesis of the fluorescence-labeled shingosine 1-phos-
phate methylene analogue.10b Thus, the intermediary 5, which
Thus, we synthesized the SM methylene analogue 1; however,
very careful reaction conditions and purification procedures were
required. In order to synthesize 1 more efficiently, we then tried to
improve the construction method of the phosphonocholine moiety
as shown in Scheme 2. The selective removal of one methyl group of
the dimethyl phosphonate in 14, which was prepared from 5 by the
similar synthetic procedure to that of 6, was successful by the
could be easily furnished from L-serine over five steps, was con-
verted to 6 through the oxazolidinone formation and the re-
placement of the primary hydroxy group into the dimethyl
phosphonylmethyl group over seven steps. The oxazolidinone ring
opening of 6 was performed with benzyl alcohol in the presence of
cesium carbonate11 in THF to obtain the desired Cbz-protected
O
Boc
Boc
HN
NH2
NH
Boc
O
N
ref. 10a
5 steps
a
ref. 10b
7 steps
OMe
OMe
HO
OH
OTBS
P
P
OMe
OMe
O
P
O
OCbz
OH
O
5
L-serine
6
7
O
O
Boc
HN
C15H31
C13H27
NH
C15H31
C13H27
NH
C13H27
b
OMe
OMe
c
d
e
OH
OH
OMe
P
P
O
OCbz
O
O
OMe
OCbz
10
OCbz
9
8
O
O
O
C15H31
C13H27
NH
C15H31
C13H27
NH
C15H31
C13H27
NH
f,g
h
O
O
O
P
NHBoc
P
NH3
P
NMe3
O
OH
O
O
O
O
OCbz
OCbz
12
OR
13: R = Cbz
1: R = H
11
i
Scheme 1. (a) BnOH, Cs2CO3, THF, rt, 1 h, 78%; (b) 1-pentadecene, second generation Grubbs catalyst, CH2Cl2, reflux, 7 h, 68%; (c) TFA, CH2Cl2, 0 ꢀC, 3 h, then C15H31COCl, K2CO3 aq,
CHCl3, 0 ꢀC, 15 min, 76%; (d) TMSBr, CH2Cl2, rt, 2 h, then MeOH, rt, 1 h, 86%; (e) HOC2H4NHBoc, CCl3CN, pyridine, 65 ꢀC, 2 h, 89%; (f) Dowex 50W, CHCl3, rt, 5 min, then CH2N2, CHCl3,
Et2O, 0 ꢀC, 99%; (g) TMSBr, CH2Cl2, 0 ꢀC, 1 h, then MeOH, rt, 1 h; (h) MeI, K2CO3, CHCl3, rt, 2 days, 61% for two steps; (i) 2 N KOH, MeOH, rt, 2 h, 76%.

T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
11649
O
R1
R1
Grubbs cat. 2nd
(0.03 equiv)
NH
NH
C15H31
C13H27
NH
Z
OR2
Z
OR2
O
O
CH2Cl2, reflux, 2 h
55-76%
P
NMe3
(4.0 equiv)
olefins
O
O
OH
aminoalcohols
OH
OH
sphingomyelin (SM)
olefins
aminoalcohols
and
O
CnH2n+1
(n = 7, 13)
C15H31 NH
O
OR2
R2 = H,
P
Br
O2N
N
sphingosine
ceramide
O
OMe
OH
(CH2)9
N
H
N
O
H, TBS
fluorescence-labeled olefin
sphingosine 1-phosphate
Boc
NH
OMe
P
N N
F3C
OR2
R2 =
labeled analogues
O
OMe
OH
(CH2)9
O
O
OMe
P
Br
O
photoaffinity-labeled olefin
Figure 2. The established synthetic method of sphingolipids.
treatment with excess aqueous trimethylamine in methanol to af-
ford 15 in 87% yield. Treatment of 15 with 5.0 equiv of 1,2-dibro-
moethane in the presence of potassium carbonate resulted in the
introduction of the 2-bromoethyl group into 15 to afford 16 in 61%
yield.15,16 After the PMB group of 16 was exchanged with a Boc
group, the resulting 17 was treated with methanol in the presence
of cesium carbonate to give the amino alcohol 18 in 51% yield. The
intermediary corresponding methylcarbonate of the secondary
hydroxy group, which was detected by mass spectra, was gradually
changed to 18. In the olefin cross-metathesis between 18 and 1-
pentadecene, the coupling smoothly proceeded again to produce 19
in 60% yield along with a very small amount of the corresponding
cis-isomer. The treatment of the obtained 19 with TFA followed by
the reaction with palmitoyl chloride produced 20 in 56% yield. The
desired methylene analogue 1 was obtained in 73% yield by the
treatment of 20 with trimethylamine in methanol for 2 days. Thus,
the synthesis of 1 was efficiently achieved by modifying the in-
troduction step of the phosphonocholine moiety.
2.2. Synthesis of fluorescence-labeled analogue 4
As further demonstration, the synthetic route of 1 was applied
for the synthesis of the fluorescence-labeled SM methylene ana-
logue 4 as shown in Scheme 3. This analogue might be regarded as
a second generation fluorescence-labeled one of SM, because the
phosphate oxygen connecting the phosphocholine part with the
backbone skeleton was replaced by a CH2 group and hence com-
pound 4 was not hydrolyzed at this position.6a,d Thus, the same
amino alcohol 18 was coupled to the olefin part 21, which possessed
a fluorescent NBD (nitrobenzo-2-oxa-1,3-diazole) group at the
terminus of the backbone skeleton, under the same reaction con-
ditions used for the methylene analogue synthesis. As a result, the
desired coupling product 22 was obtained in 78% yield as a mixture
of 15:1 of trans- and cis-isomer. The treatment of the resulting 22
with TFA followed by acylation, and then a trimethylamine treat-
ment easily produced the desired fluorescence-labeled methylene
analogue 4 in a manner similar to that for the methylene analogue
synthesis. Thus, we demonstrated the versatility of the olefin cross-
metathesis strategy for the synthesis of various sphingolipids.
O
O
PMB
PMB
b
a
O
N
O
N
OMe
OMe
OH
P
P
O
O
OMe
15
14
O
O
Boc
O
N
PMB
c,d
e
O
N
O
P
Br
O
P
Br
Br
O
OMe
O
OMe
16
17
Boc
Boc
NH
NH
f
C13H27
O
O
P
Br
P
2.3. Synthesis of sphingomyelin aza analogue 2
O
OMe
O
OMe
OH
OH
18
19
We next planned to synthesize the aza analogue 2, which has an
NH group instead of the phosphate oxygen, from the same in-
termediate 5 for the SM synthesis (Scheme 4).
O
NH
C15H31
C13H27
After several trials to introduce a nitrogen function at the ter-
minal position of 5, we found that the Mitsunobu reaction condi-
tions gave the best results (Scheme 4). Thus, compound 5 was
transformed into the N-Boc-oxazolidinone derivative 24 in three
steps in 73% yield, which was then reacted with N-Boc-protected
nosylamide in the presence of DIAD and triphenylphosphine17 to
successfully produce the nitrogen-substituted derivative 25, al-
though a similar Mitsunobu-type substitution reaction of the cor-
responding linear compound was not successful. The isolation of 25
g
h
O
P
1
Br
O
OMe
OH
20
Scheme 2. (a) NMe3 aq, MeOH, rt, 1 day; (b) 1,2-dibromoethane, K2CO3, DMF, 80 ꢀC,
4 h, 61%; (c) CAN, CH3CN, H2O, 0 ꢀC, 15 min, 80%; (d) Boc2O, NEt3, DMAP, CH2Cl2, 0 ꢀC,
15 min, 92%; (e) MeOH, Cs2CO3, THF, rt, 20 min, 51%; (f) 1-pentadecene, second gen-
eration Grubbs catalyst, CH2Cl2, reflux, 2 h, 60%; (g) TFA, CH2Cl2, 0 ꢀC, 3 h, then
C15H31COCl, K2CO3 aq, 0 ꢀC, 15 min, 56%; (h) NMe3, MeOH, rt, 2 days, 73%.

11650
T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
Boc
NH
O
P
Br
O
OMe
OH
O2N
N
Boc
NH
18
a
(CH2)9
O
N
H
P
Br
O
OMe
N
O2N
N
OH
22
O
(CH2)9
N
H
N
O
21
O
O2N
C15H31
(CH2)9
NH
b
O
N
H
P
Br
N
O
O
OMe
N
OH
23
O
O2N
N
C15H31
(CH2)9
NH
c
O
N
P
NMe3
H
O
O
N
OH
O
4
Scheme 3. (a) Second generation Grubbs catalyst, CH2Cl2, reflux, 2 h, 78%; (b) TFA, CH2Cl2, rt, 75 min, then C15H31COCl, K2CO3 aq, 0 ꢀC, 82%; (c) NMe3 aq, MeOH, rt, 2 days, 61%.
from the residue, however, was very difficult. The resulting mixture
was semi-purified by column chromatography, and then treated
O
O
with cesium carbonate in methanol. The 3-hydroxy-1,2-diamine
derivative 26 could be isolated by careful column chromatography
(2–3% of methanol in chloroform) in 87% yield for two steps.
The Boc group at the nosylamide of 25 transferred to the allylic
oxygen resulting from the solvolysis of the oxazolidinone ring. Next
was the key olefin cross-metathesis reaction. Compound 26 and
4.0 equiv of 1-pentadecene in the presence of 0.03 equiv of the
second generation Grubbs catalyst in CH2Cl2 for 7 h under reflux
produced the desired coupling product 27 in 72% yield. In this case,
the catalyst was not deactivated until the reaction was completed,
although the secondary hydroxy group was protected by a Boc
group. The desired trans-isomer was exclusively obtained in usual
cases of this reaction, but the cis-isomer was sometimes admitted
less than 10% portion in the repeated same reactions. The activity of
the used catalyst may be responsible. The treatment of 27 with TFA
and then acylation produced the N-acyl derivative 28 in 82% yield
for two steps. The hydroxy group of 28 was again protected with
a TES group followed by removal of the nosyl group with an
odorless dodecylmercaptane treatment18 to afford the 3-alkoxy-
1,2-diamine derivative 29 in 89% yield from 28. The next step was
the construction of the phosphocholine moiety. We applied 2-
bromoethyl dimethyl phosphite (30),19 which was previously used
for the rapid synthesis of the natural SM.9 Thus, compound 29 was
stirred with 30 in the presence of carbon tetrabromide and catalytic
amount of 2,6-lutidine to produce the desired phosphoramidate 31
along with a small amount of the residue produced from the re-
agent 30 in nearly 80% yield. The isolation of 31 was not successful
even after very careful column chromatography. When the
obtained compound 31 was reacted with trimethylamine in
a sealed tube followed by Dowex 50W treatment, unfortunately the
desired choline compound 2 was obtained in very low yield and the
reaction was not reproducible under the reaction conditions ap-
plied for the syntheses of natural SM and methylene analogue 1. In
order to obtain 31 as a pure form, we attempted the purification of
alcohol 32, which was easily derived from 31. The treatment of 31
with Dowex 50W in methanol gave the corresponding alcohol 32,
which was isolated by careful column chromatography. The
Boc
N
Boc
a-c
d
O
N
O
N
5
Boc
Ns
OH
24
25
Boc
Boc
NH
NH
H
N
H
N
e
f
C13H27
Ns
Ns
OBoc
26
OBoc
27
O
O
C15H31
NH
C15H31
C13H27
NH
g
h,i
H
N
C13H27
NH2
Ns
OH
28
OTES
29
O
MeO
O
P
Br
C15H31
NH
30
H
N
OMe
C13H27
O
P
Br
j
O
OMe
OR
31: R= TES
32: R=H
l
k
O
C15H31
C13H27
NH
H
N
m,n
O
P
NMe3
O
O
OH
2
Scheme 4. (a) NaH, THF, 50 ꢀC, 1 h, 93%; (b) Boc2O, DMAP, NEt3, CH2Cl2, 0 ꢀC, 1 h; (c)
2 N HCl, MeOH, rt, 16 h, 78% for two steps; (d) BocNHNs, DIAD, PPh3, toluene, rt, 16 h;
(e) Cs2CO3, MeOH, rt, 1 h, 87% for two steps; (f) 1-pentadecene, second generation
Grubbs catalyst, CH2Cl2, reflux, 7 h, 72%; (g) TFA, CH2Cl2, 0 ꢀC, 3.5 h, then C15H31COCl,
K2CO3, 0 ꢀC, 30 min, 84%; (h) TESCl, imidazole, DMF, 0 ꢀC, 5 min; (i) CH3(CH2)11SH,
NaH, DMF, 0 ꢀC, 1 h, 89% for two steps; (j) CBr4, 2,6-lutidine, 4 Å MS, CH2Cl2, 0 ꢀC, 1.5 h;
(k) Dowex 50W, MeOH, rt, 5 min, 54% for two steps; (l) TESCl, imidazole, DMF, 0 ꢀC,
5 min, 95%; (m) NMe3, toluene, 60 ꢀC, h, 2 days; (n) Dowex 50W, MeOH, rt, 5 min, 30%
for two steps.

T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
11651
hydroxy group of the isolated 32 was again protected with a TES
group to avoid the rearrangement of the phosphate moiety from
the amino group to the secondary hydroxy group of 32 under basic
condition such as the trimethylamine treatment. Thus, the pure 31
was obtained. The desired SM aza analogue 2 was finally synthe-
sized in 30% yield by the trimethylamine treatment of 31 at 60 ꢀC in
a sealed tube for 2 days followed by the Dowex 50W treatment.
This reaction condition was fortunately reproducible. Thus, we
achieved the synthesis of the relatively unstable aza analogue 2 of
SM.
SM, by Slotte’s group at åbo Academi University.20 The results
showed that the S-linkage increased and the NH- and CH2-linkage
decreased the stability of the SM-analogue bilayer membranes as
compared to SM. The obtained data also showed that all com-
pounds formed SM/cholesterol-rich domains, and the S-SM/cho-
lesterol and SM/cholesterol domains displayed
a
similar
thermostability, whereas the NH-SM/cholesterol and CH2-SM/
cholesterol domains were less thermostable. It was suggested from
the obtained data that the properties of the bond linking the
phosphocholine head group to the 1-hydroxy group on the
ceramide molecule is important for the stability of the SM/SM and
SM/cholesterol interactions.
2.4. Synthesis of sphingomyelin sulfur analogue 3
3. Experimental
3.1. General
Finally, we synthesized SM sulfur analogue 3 starting from L-
cystein as shown in Scheme 5. Thiol 34 corresponding to the in-
termediary amino alcohol 5 in the syntheses of methylene and aza
analogues was derived from the commercially available N-Boc-S-
All commercially available reagents were used without further
purification. All solvents were used after distillation. Tetrahydro-
furan, diethyl ether, and toluene were refluxed over and distilled
from sodium. Dichloromethane was refluxed over and distilled
from P2O5. Dimethylformamide (DMF) was distilled from CaH2.
Preparative separation was usually performed by column chroma-
tography on silica gel. 1H and 13C NMR spectra were recorded on
Bn-L-cystein 33 over seven steps according to the previously
reported procedure including monoalkylation of the corresponding
Weinreb amide with a vinyl Grignard reagent, stereoselective re-
duction of the produced ketone, and selective protection of the
resulting secondary hydroxy group.10b The phosphorylation of 34
smoothly proceeded to give thiophosphate 35 in 61% yield with
phosphite 30 under the similar conditions to those of the SM and
aza analogue 2 synthesis. The removal of the TBS group in 35 with
2 N HCl in MeOH afforded allyl alcohol 36 in 68% yield. The olefin
cross-metathesis reaction between 36 and 1-pentadecene pro-
duced 37 in 74% yield as a mixture of approximately 15:1 of trans-
and cis-isomer. The treatment of the obtained 37 with TFA followed
by the reaction with palmitoyl chloride produced 38 in 92% yield.
The synthesis of sulfur analogue 3 was achieved by the reaction of
38 with trimethylamine in methanol for 1 day in 50% yield.
JEOL
a-400 or a JMM-ECX 400KA spectrometer and chemical shifts
were represented as
d
values relative to the internal standard TMS.
IR spectra were recorded on a JASCO FT/IR-5300 and FT/IR-8100A
Fourier Transform Infrared Spectrometer. High-resolution mass
spectra (HRMS) were obtained on a JEOL JMS-T100LC spectrometer
for electrospray ionization. Optical rotations were determined us-
ing a JASCO DIP-370 polarimeter with a 100 mm cell.
3.2. Synthesis of SM methylene analogue 1
MeO
O
Boc
Boc
P
Br
3.2.1. (4S)-(tert-Butyldimethylsilyloxymethyl)-(5R)-
vinyloxazolidin-2-one (5a)
NH
NH
30
ref. 10b
7 steps
OMe
a
HO
SBn
SH
To a solution of 5 (7.304 g, 22.03 mmol) in THF (110 ml) was
added sodium hydride (793 mg, 33.0 mmol) at 0 ꢀC. After the re-
action mixture was stirred at 50 ꢀC for 1 h, saturated aqueous NH4Cl
solution was added and the resulting mixture was extracted with
ethyl acetate. The organic layers were combined, washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo to give
the crude products. Column chromatography on silica gel (from 13%
O
OTBS
34
33
Boc
R
NH
NH
c
S
O
C13H27
S
O
P
Br
P
Br
O
OMe
O
OMe
OR
OH
37: R = Boc
38: R = C15H31CO
35: R = TBS
36: R = H
to 33% ethyl acetate in hexane) gave 5a (5.264 g, 93%) as a colorless
b
d
20.5
solid. [
a
]
ꢁ51.1 (c 1.04, CHCl3); IR (KBr disk, cmꢁ1) 3252, 2932,
D
1775, 1391, 1258, 1103; 1H NMR (CDCl3, 400 MHz),
d 5.90 (ddd,
O
J¼17.2, 10.6, 6.8 Hz, 1H), 5.49 (ddd, J¼17.2, 2.1, 1.0 Hz, 1H), 5.37 (ddd,
J¼10.6, 2.0, 1.2 Hz, 1H), 5.09 (m, 1H), 3.89 (ddd, J¼8.1, 7.5, 4.3 Hz,
1H), 3.59 (dd, J¼10.3, 4.3 Hz, 1H), 3.54 (dd, J¼10.3, 7.4 Hz, 1H), 0.89
(s, 9H), 0.061 (s, 3H), 0.057 (s, 3H); 13C NMR (CDCl3, 100 MHz)
C15H31
C13H27
NH
e
S
O
P
NMe3
O
O
OH
d
158.7, 130.3, 120.2, 78.8, 62.4, 56.9, 25.7, 18.1, ꢁ5.5; ESI-HRMS m/z
3
calcd for C12H23NO3SiNa (MþNa)þ 280.1345, found 280.1349.
Scheme 5. (a) CBr4, 2,6-lutidine, 4 Å MS, CH2Cl2, 0 ꢀC, 2 h, 61%; (b) 2 N HCl, MeOH, rt,
1 h, 68%; (c) 1-pentadecene, second generation Grubbs catalyst, CH2Cl2, reflux, 2 h, 74%
(d) TFA, CH2Cl2, 0 ꢀC, 2.5 h, then C15H31COCl, K2CO3, 0 ꢀC, 15 min, 92%; (e) NMe3, MeOH,
rt, 1 day, 50%.
3.2.2. (4S)-(Hydroxymethyl)-3-(p-methoxybenzyl)-(5R)-
vinyloxazolidin-2-one (5b)
To a solution of 5a (7.795 g, 30.3 mmol) in THF (150 ml) was
added sodium hydride (1.820 g, 45.49 mmol) at 0 ꢀC. After the
reaction mixture was stirred at the same temperature for 30 min, p-
methoxybenzyl chloride (6.17 ml, 45.5 mmol) and tetrabutyl-
ammonium iodide (22.40 g, 60.52 mmol) were added at the same
temperature. After the reaction mixture was stirred at room tem-
perature for 16 h, saturated aqueous NH4Cl solution was added and
the resulting mixture was extracted with ethyl acetate. The organic
layers were combined, washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo to give the crude products. Col-
umn chromatography on silica gel (from 3.3% to 20% ethyl acetate in
Thus, we accomplished the syntheses of four analogues, SM
methylene 1, aza 2, and sulfur 3 analogues, and fluorescence-
labeled SM methylene one 4 by employing the olefin cross-
metathesis protocol.
2.5. The molecular properties and membrane behavior
The molecular properties and membrane behavior of the syn-
thesized methylene (CH2-SM) 1, aza (NH-SM) 2, and sulfur (S-SM) 3
analogues of SM were examined comparing with natural palmitoyl

11652
T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
hexane) gave the PMB protected oxazolidinone (10.73 g, 94%) as
1744, 1514, 1252, 1038; 1H NMR (CDCl3, 400 MHz)
d 7.23 (md,
20.5
a colorless solid. [
a]
ꢁ4.2 (c 0.62, CHCl3); IR (KBr disk, cmꢁ1
)
J¼8.7 Hz, 2H), 6.88 (md, J¼8.7 Hz, 2H), 5.90 (ddd, J¼17.2, 10.5,
6.8 Hz, 1H), 5.51 (ddd, J¼17.2, 1.2, 1.1 Hz, 1H), 5.44 (mdd, J¼10.5,
1.1 Hz, 1H), 4.88 (mdd, J¼7.8, 7.1 Hz, 1H), 4.80 (d, J¼15.1 Hz, 1H),
3.97 (d, J¼15.1 Hz, 1H), 3.80 (s, 3H), 3.71 (d, J¼11.0 Hz, 3H), 3.70 (d,
J¼10.7 Hz, 3H), 3.66 (td, J¼7.8, 3.2 Hz, 1H), 1.75–1.95 (m, 2H), 1.59–
D
2928, 1748, 1514, 1418, 1253, 839; 1H NMR (CDCl3, 400 MHz)
d 7.23
(md, J¼8.7 Hz, 2H), 6.87 (md, J¼8.7 Hz, 2H), 5.99 (ddd, J¼17.4, 10.6,
7.1 Hz, 1H), 5.42 (ddd, J¼17.4, 1.2, 1.1 Hz, 1H), 5.35 (ddd, J¼10.5, 1.1,
0.9 Hz, 1H), 4.90 (dddd, J¼8.2, 7.3, 0.9, 0.9 Hz, 1H), 4.86 (d,
J¼15.1 Hz, 1H), 3.99 (d, J¼15.1 Hz, 1H), 3.81 (s, 3H), 3.68 (m, 1H),
3.55–3.62 (m, 2H), 0.90 (s, 9H), 0.070 (s, 3H), 0.053 (s, 3H); 13C NMR
1.68 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d 159.3, 157.5, 130.2, 129.4,
127.5,121.0,114.2, 77.7, 56.6 (d, JC–P¼17.7 Hz), 52.45 (d, JC–P¼6.8 Hz),
52.40 (d, JC–P¼6.8 Hz), 45.6, 20.6, 19.9 (d, JC–P¼140 Hz). ESI-HRMS
m/z calcd for C17H24NO6PNa (MþNa)þ 392.1239, found 392.1248.
(CDCl3, 100 MHz) d 159.3, 158.1, 131.5, 129.4, 128.3, 120.3, 114.1, 77.6,
59.2, 58.5, 55.3, 45.9, 25.7, 18.0, ꢁ5.7; ESI-HRMS m/z calcd for
C20H31NO4SiNa (MþNa)þ 400.1920, found 400.1908.
To a solution of the compound obtained above (10.73 g,
28.43 mmol) in methanol (150 ml) was added 2 N HCl (57 ml) at
room temperature. After the reaction mixture was stirred at the
same temperature for 16 h, saturated aqueous NaHCO3 solution
was added and the solvent was removed in vacuo. The resulting
mixture was extracted with ethyl acetate. The organic layers were
combined, washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo to give the crude products. Column chro-
3.2.4. Dimethyl 2-[2-oxo-(5R)-vinyloxazolidin-(4S)-yl]-
ethylphosphonate (14a)
To a solution of 14 (2.733 g, 7.399 mmol) in acetonitrile (22 ml)
and water (7.5 ml) was added ceric ammonium nitrate (12.17 g,
22.19 mmol) at 0 ꢀC. After the reaction mixture was stirred at the
same temperature for 15 min, brine was added and the resulting
mixture was extracted with ethyl acetate for more than three
times. The organic layers were combined, washed with brine,
dried over MgSO4, filtered, and concentrated in vacuo to give the
crude products. Column chromatography on silica gel (from 0% to
matography on silica gel (from 33% to 66% ethyl acetate in hexane)
20.5
gave 5b (7.477 g, quant.) as a colorless solid. [
a
]
D
ꢁ17.8 (c 1.06,
CHCl3); IR (KBr disk, cmꢁ1) 3370, 2928, 1721, 1514, 1447, 1244, 1038;
9% methanol in chloroform) gave 14a (1.482 g, 80%) as a colorless
1H NMR (CDCl3, 400 MHz)
d
7.26 (md, J¼8.7 Hz, 2H), 6.88 (md,
oil. [
a]
–11.1 (c 1.12, CHCl3); IR (neat, cmꢁ1) 3245, 2957, 1759,
20.5
D
J¼8.7 Hz, 2H), 6.05 (ddd, J¼17.2, 10.5, 6.9 Hz, 1H), 5.50 (md,
J¼17.2 Hz, 1H), 5.40 (md, J¼10.6 Hz, 1H), 4.94 (mdd, J¼8.0, 7.1 Hz,
1H), 4.72 (d, J¼15.0 Hz, 1H), 4.21 (d, J¼15.0 Hz, 1H), 3.80 (s, 3H),
1393, 1236, 1035; 1H NMR (CDCl3, 400 MHz)
d 7.41 (br m, 1H),
5.90 (ddd, J¼17.2, 10.5, 6.9 Hz, 1H), 5.48 (mdd, J¼17.2, 1.2 Hz, 1H),
5.41 (mdd, J¼10.5, 0.9 Hz, 1H), 5.07 (dd, J¼7.8, 7.4 Hz, 1H), 3.94
(m, 1H), 3.75 (d, J¼10.8 Hz, 3H), 3.74 (d, J¼10.7 Hz, 3H), 1.65–2.13
3.62–3.74 (m, 3H); 13C NMR (CDCl3, 100 MHz)
d 159.4, 158.1, 131.4,
129.4, 128.3, 120.5, 114.3, 77.3, 59.2, 58.8, 55.3, 46.2; ESI-HRMS m/z
(m, 4H); 13C NMR (CDCl3, 100 MHz)
d 158.9, 130.4, 120.6, 80.2,
calcd for C14H17NO4Na (MþNa)þ 286.1055, found 286.1066.
55.8 (d, JC–P¼13.4 Hz), 52.7 (d, JC–P¼6.2 Hz), 52.7 (d, JC–P¼6.3 Hz),
24.4 (d, JC–P¼4.8 Hz), 21.2 (d, JC–P¼142.8 Hz); ESI-HRMS m/z calcd
for C9H16NO5PNa (MþNa)þ 272.0664, found 272.0674.
3.2.3. Dimethyl 2-[3-(p-methoxybenzyl)-2-oxo-(5R)-
vinyloxazolidin-(4S)-yl]ethylphosphonate (14)
To a solution of 5b (1.80 g, 6.84 mmol) in dichloromethane
(68 ml) were added 2,6-lutidine (0.95 ml, 8.17 mmol) and tri-
fluoromethanesulfonic anhydride (1.38 ml, 8.18 mmol) at ꢁ78 ꢀC.
After the reaction mixture was stirred at the same temperature for
20 min, it was poured into saturated aqueous NaHCO3 solution and
the resulting mixture was extracted with chloroform. The organic
layers were combined, washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo to give the crude products. Col-
umn chromatography on silica gel (from 20% to 33% ethyl acetate in
3.2.5. Dimethyl 2-[3-(tert-butyloxycarbonyl)-2-oxo-(5R)-
vinyloxazolidin-(4S)-yl]ethylphosphonate (6)
To a solution of 14a (320 mg, 1.28 mmol) in dichloromethane
(13 ml) were added triethylamine (0.36 ml, 2.57 mmol), 4-dime-
thylaminopyridine (78 mg, 0.64 mmol), and di-tert-butyldicar-
bonate (420 mg, 1.93 mmol) at 0 ꢀC. After the reaction mixture was
stirred at the same temperature for 20 min, saturated aqueous
NH4Cl solution and brine were added, and the resulting mixture
was extracted with chloroform. The organic layers were combined,
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo to give the crude products. Column chromatography on silica
hexane) gave the roughly purified corresponding triflate as a col-
21.5
orless solid. [
a]
þ2.1 (c 0.52, CHCl3); IR (KBr disk, cmꢁ1) 2938,
D
1748, 1514, 1416, 1248, 1179; 1H NMR (CDCl3, 400 MHz)
d
7.23 (md,
gel (from 0% to 1% ethyl methanol in chloroform) gave 6 (420 mg,
20.5
J¼8.7 Hz, 2H), 6.90 (md, J¼8.7 Hz, 2H), 5.85 (ddd, J¼17.1, 10.8,
6.4 Hz, 1H), 5.57 (ddd, J¼17.1, 1.2, 1.2 Hz, 1H), 5.50 (ddd, J¼10.8, 1.2,
1.2 Hz, 1H), 5.00 (dddd, J¼8.5, 6.4, 1.2, 1.1 Hz, 1H), 4.85 (d, J¼15.1 Hz,
1H), 4.48 (dd, J¼10.8, 4.8 Hz, 1H), 4.39 (dd, J¼10.8, 4.8 Hz, 1H), 4.10
(d, J¼15.1 Hz, 1H), 3.89 (dt, J¼8.2, 4.6 Hz, 1H), 3.81 (s, 3H); 13C NMR
94%) as a colorless oil. [
a]
þ18.2 (c 0.73, CHCl3); IR (KBr disk,
D
cmꢁ1) 2984, 1823, 1385, 1368, 1078, 1042; 1H NMR (CDCl3,
400 MHz)
d
5.90 (ddd, J¼17.2, 10.8, 6.6 Hz, 1H), 5.58 (md, J¼17.2 Hz,
1H), 5.51 (md, J¼10.8 Hz, 1H), 5.00 (m, 1H), 4.35 (td, J¼6.6, 4.8 Hz,
1H), 3.741 (d, J¼10.8 Hz, 3H), 3.737 (d, J¼10.8 Hz, 3H), 2.02–2.14
(m, 1H), 1.84–1.97 (m, 1H), 1.70–1.82 (m, 2H), 1.55 (s, 9H); 13C NMR
(CDCl3, 100 MHz)
d 159.7, 156.9, 129.6, 129.0, 126.9, 122.0, 118.4 (q,
JC–P¼320.1 Hz), 71.7, 55.8, 55.3, 46.5; ESI-HRMS m/z calcd for
(CDCl3,100 MHz)
d
151.3,149.2,129.0, 121.9, 84.3, 77.8, 58.1 (d, JC–P
¼
C15H16F3NO6SNa (MþNa)þ 418.0548, found 418.0539.
20.1 Hz), 52.5 (d, JC–P¼6.7 Hz), 52.46 (d, JC–P¼6.7 Hz), 27.9, 22.5 (d,
JC–P¼3.8 Hz), 20.6 (d, JC–P¼142.8 Hz); ESI-HRMS m/z calcd for
C14H24NO7PNa (MþNa)þ 372.1188, found 372.1176.
To
a solution of dimethyl methyl phosphonate (2.47 ml,
22.8 mmol) in THF (38 ml) was added 1.6 N n-butyllithium in
hexane (14.24 ml, 22.8 mmol) at ꢁ78 ꢀC, and the reaction mixture
was stirred at the same temperature for 30 min to prepare the
corresponding lithium anion, which was already added to a solu-
tion of the triflate obtained above in THF (30 ml) at –78 ꢀC until the
triflate was consumed. After the reaction mixture was stirred at the
same temperature for 10 min, saturated aqueous NH4Cl solution
was added and the resulting mixture was extracted with ethyl ac-
etate. The organic layers were combined, washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (from 0% to 4%
3.2.6. Dimethyl (4R)-(benzyloxycarbonyloxy)-(3S)-(tert-
butyloxycarbonylamino)hex-5-enylphosphonate (7)
To a solution of 6 (200 mg, 0.573 mmol) in THF (3 ml) were
added benzyl alcohol (0.30 ml, 2.86 mmol) and cesium carbonate
(933 mg, 2.86 mmol) at room temperature. After the reaction mix-
ture was stirred at the same temperature for 1 h, saturated aqueous
NH4Cl solution was added and the resulting mixture was extracted
with ethyl acetate. The organic layers were combined, washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo to give
the crude products. Column chromatography on silica gel (from 33%
to 83% ethyl acetate in hexane) gave 7 (205 mg, 78%) as a colorless
methanol in chloroform) gave 14 (1.75 g, 70%, two steps) as a col-
20.5
orless solid. [
a
]
þ25.0 (c 1.02, CHCl3); IR (KBr disk, cmꢁ1) 2955,
D

T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
11653
20.5
oil. [
a
]
D
þ0.755 (c 1.02, CHCl3); IR (neat, cmꢁ1) 3409, 2976, 1750,
solution was stirred for additional 1 h at room temperature. The
solvent was removed in vacuo to give the crude product. Column
1711, 1368, 1262, 1036, 820; 1H NMR (CDCl3, 400 MHz)
d 7.35–7.39
(m, 5H), 5.79 (ddd, J¼17.2, 10.8, 6.2 Hz, 1H), 5.34 (d, J¼17.4 Hz, 1H),
5.30 (d, J¼11.0 Hz, 1H), 5.16 (s, 2H), 5.11–5.15 (m, 1H), 4.68 (br d,
J¼9.2 Hz, 1H), 3.74–3.86 (m, 1H), 3.72 (d, J¼10.9 Hz, 6H), 1.56–1.96
chromatography on silica gel (from 0% to 9% methanol in chloro-
23.0
form) gave 10 (173 mg, 86%) as a colorless solid. [
a]
ꢁ8.8 (c 1.12,
D
CHCl3); IR (KBr disk, cmꢁ1) 3281, 2922, 2853,1748, 1647, 1545, 1466,
(m, 4H),1.43 (s, 9H); 13C NMR (CDCl3,100 MHz)
d
155.5,154.3,134.9,
1254, 970; 1H NMR (CD3OD, 400 MHz)
d
7.31–7.36 (m, 5H), 5.81 (dt,
132.1, 128.54, 128.47, 128.3, 128.2, 119.3, 80.2, 79.8, 69.8, 53.3 (d,
JC–P¼16.3 Hz), 52.41 (d, JC–P¼6.7 Hz), 52.40 (d, JC–P¼6.7 Hz), 28.3,
22.7 (d, JC–P¼2.9 Hz), 21.4 (d, JC–P¼142.8 Hz); ESI-HRMS m/z calcd
for C21H32NNaO8P (MþNa)þ 480.1749, found 480.1763.
J¼15.6, 6.6 Hz, 1H), 5.43 (dd, J¼15.3, 7.8 Hz, 1H), 5.13 (s, 2H), 5.04
(m, 1H), 4.09 (m, 1H), 2.18 (t, J¼7.3 Hz, 2H), 2.05 (dt, J¼6.9, 6.6 Hz,
2H), 1.53–1.79 (m, 6H), 1.28 (s, 46H), 0.88 (t, J¼6.9 Hz, 6H); 13C NMR
(CD3OD, 100 MHz)
d 176.6, 155.8, 138.6, 137.1, 129.6, 129.4, 129.1,
125.6, 81.5, 70.5, 58.3, 53.3 (d, JC–P¼18.2 Hz), 33.4, 33.1, 30.9, 30.70,
30.65, 30.60. 30.52, 30.40, 30.3, 30.0, 27.2, 25.0(d, JC–P¼139.9 Hz),
24.5 (d, JC–P¼1.9 Hz), 23.8, 14.5; ESI-HRMS m/z calcd for
C43H75NNaO7P (MþNa)þ 794.5096, found 794.5077.
3.2.7. Dimethyl (4R)-(benzyloxycarbonyloxy)-(3S)-(tert-
butyloxycarbonylamino)nonadec-(5E)-enylphosphonate (8)
To a solution of 7 (200 mg, 0.437 mmol) in dichloromethane
(2 ml) were added 1-pentadecene (368 mg, 1.750 mmol) and sec-
ond generation Grubbs catalyst (11 mg, 0.013 mmol) at room
temperature. After the reaction mixture was stirred for 7 h under
reflux, the solvent was removed in vacuo to give the crude prod-
3.2.10. 2-(tert-Butyloxycarbonyl)ethyl hydrogen (4R)-
(benzyloxycarbonyloxy)-(3S)-1-oxohexadecylaminononadec-
(5E)-enylphosphonate (11)
ucts. Column chromatography on silica gel (from 33% to 75% ethyl
To a solution of 10 (240 mg, 0.320 mmol) in pyridine (3 ml) were
added 2-(N-tert-butyloxycarbonylamino)ethanol (155 mg, 0.960 mmol)
and trichloroacetonitrile (0.16 ml, 1.60 mmol) at room tempera-
ture. After the reaction mixture was stirred for 2 h at 65 ꢀC, 2 N
HCl was added and the resulting mixture was extracted with ethyl
acetate. The organic layers were combined, washed with brine,
dried over MgSO4, filtered, and concentrated in vacuo to give the
crude products. Column chromatography on silica gel (from 0% to
20.5
acetate in hexane) gave 8 (190 mg, 68%) as a colorless oil. [a]
D
ꢁ8.5 (c 1.00, CHCl3); IR (neat, cmꢁ1) 3281, 2926, 2854, 1748, 1713,
1365, 1246, 1034, 822, 754; 1H NMR (CDCl3, 400 MHz)
d 7.33–7.38
(m, 5H), 5.81 (dt, J¼15.6, 6.9 Hz, 1H), 5.38 (dd, J¼15.3, 7.1 Hz, 1H),
5.15 (s, 2H), 5.06 (m, 1H), 4.61 (br d, J¼9.8 Hz, 1H), 3.73–3.77 (m,
1H), 3.74 (d, J¼10.8 Hz, 6H), 2.03 (dt, J¼7.3, 6.9 Hz, 2H), 1.58–1.90
(m, 4H), 1.42 (s, 9H), 1.25 (s, 22H), 0.88 (t, J¼6.8 Hz, 3H); 13C NMR
(CDCl3, 100 MHz)
d
155.6, 154.3, 137.6, 135.1, 128.55, 128.50, 128.3,
9% methanol in chloroform) gave 11 (255 mg, 89%) as a colorless
23.0
123.4, 80.6, 79.7, 69.7, 53.5 (d, JC–P¼18.2 Hz), 52.7 (m), 32.4, 31.9,
29.68, 29.59, 29.46, 29.36, 29.18, 28.79, 28.38, 28.31, 28.23, 23.0 (m),
22.7, 21.4 (d, JC–P¼142.8 Hz), 14.1; ESI-HRMS m/z calcd for
C34H58NNaO8P (MþNa)þ 662.3770, found 662.3798.
solid. [
a
]
ꢁ19.1 (c 0.97, CHCl3); IR (KBr disk, cmꢁ1) 3416, 2920,
D
2851, 1736, 1684, 1638, 1468, 1262, 1071; 1H NMR (CD3OD,
400 MHz)
d
7.30–7.35 (m, 5H), 5.80 (dt, J¼15.3, 6.6 Hz, 1H), 5.44
(dd, J¼15.6, 7.8 Hz, 1H), 5.12 (s, 2H), 5.00 (m 1H), 4.22 (m, 1H), 3.84
(m, 2H), 3.23 (m, 2H), 2.20 (t, J¼7.3 Hz, 2H), 2.04 (dt, J¼6.9, 6.6 Hz,
2H), 1.53–1.68 (m, 6H), 1.42 (s, 9H) 1.28 (s, 46H), 0.89 (t, J¼6.9 Hz,
3.2.8. Dimethyl (4R)-(benzyloxycarbonyloxy)-(3S)-(1-
oxohexadecylamino)nonadec-(5E)-enylphosphonate (9)
6H); 13C NMR (CD3OD, 100 MHz)
d 176.7, 158.4, 155.8, 138.7, 137.0,
To a solution of 8 (180 mg, 0.282 mmol) in dichloromethane
(3.0 ml) was added trifluoroacetic acid (0.56 ml) at 0 ꢀC. After the
mixture was stirred for 3 h at the same temperature, saturated
aqueous K2CO3 solution was added and the resulting mixture was
stirred for further 5 min at the same temperature. To the mixture
was added palmitoyl chloride (1.00 ml, 3.27 mmol) at 0 ꢀC. After
the reaction mixture was stirred for 15 min, saturated aqueous
NH4Cl solution was added and the resulting mixture was extracted
with chloroform. The organic layers were combined, washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo to give
the crude products. Column chromatography on silica gel (from 33%
129.5, 129.4, 129.1, 125.6, 81.8, 80.2, 71.3, 70.5, 63.85, 63.8, 42.5 (d,
JC–P¼15.7 Hz), 40.2, 37.3, 33.4, 33.1, 31.6, 30.9, 30.8, 30.74, 30.68,
30.63, 30.5, 30.3, 30.2, 30.1, 28.8, 27, 25.2, 24.5 (d, JC–P¼141.1 Hz),
23.8, 18.4, 14.5, 14.4; ESI-HRMS m/z calcd for C50H89N2NaO9P
(MþNa)þ 891.6238, found 891.6277.
3.2.11. (4R)-(Benzyloxycarbonyloxy)-(3S)-(1-oxohexadecylamino)-
nonadec-(5E)-enylphosphonocholine (13)
To a solution of 11 (170 mg, 0.190 mmol) in chloroform (2 ml)
was added a small amount of Dowex 50W-X4 at room temperature.
After the reaction mixture was stirred for 5 min at the same tem-
perature, it was filtered and a diazomethane diethyl ether solution
was added dropwise to the filtrate at 0 ꢀC until the reaction mixture
became yellow. A saturated aqueous NH4Cl solution was added to
the reaction mixture and the resulting mixture was extracted with
chloroform. The organic layers were combined, washed with brine,
dried over MgSO4, filtered, and concentrated in vacuo to give the
crude products. Column chromatography on silica gel (from 0% to
to 80% ethyl acetate in hexane) gave 9 (167 mg, 76%) as a colorless
23.0
solid. [
a
]
ꢁ6.9 (c 0.99, CHCl3); IR (KBr disk, cmꢁ1) 3306, 2919,
D
2851, 1742, 1649, 1260, 1032; 1H NMR (CDCl3, 400 MHz)
d 7.35–7.38
(m, 5H), 5.81 (dt, J¼15.1, 6.9 Hz, 1H), 5.67 (br d, J¼9.6 Hz, 1H), 5.38
(dd, J¼15.6, 7.1 Hz, 1H), 5.15 (s, 2H), 5.08 (dd, J¼4.4, 6.9 Hz, 1H), 4.18
(m, 1H), 3.72 (d, J¼10.8 Hz, 3H), 3.71 (d, J¼10.8 Hz, 3H), 2.14 (t,
J¼7.6 Hz, 2H), 2.03 (td, J¼6.9, 6.9 Hz, 2H), 1.69–1.98 (m, 6H), 1.25 (s,
46H), 0.88 (t, J¼6.6 Hz, 6H); 13C NMR (CDCl3, 100 MHz)
d
173.2,
2% methanol in chloroform) gave the corresponding methyl phos-
23.0
154.3, 137.4, 135.1, 128.57, 128.54, 128.30, 123.5, 80.3, 69.7, 52.4 (d,
JC–P¼6.7 Hz), 51.7 (d, JC–P¼16.3 Hz), 36.8, 32.3, 31.9, 29.70, 29.66,
29.61, 29.52, 29.47, 29.39, 29.36, 29.29, 29.20, 28.83, 25.7, 22.7, 22.5
(d, JC–P¼4.8 Hz), 21.3 (d, JC–P¼142.8 Hz), 14.1; ESI-HRMS m/z calcd
for C45H80NNaO7P (MþNa)þ 800.5552, found 800.5570.
phonate (170 mg, 99%) as a colorless solid. [
a]
ꢁ6.8 (c 1.01,
D
CHCl3); IR (KBr disk, cmꢁ1) 3360, 2919, 2851, 1738, 1688, 1649,1260,
1032; 1H NMR (CDCl3, 400 MHz)
d
7.33–7.38 (m, 5H), 5.81 (dt,
J¼15.3, 6.9 Hz, 1H), 5.73 (br d, J¼9.6 Hz, 1H), 5.39 (dd, J¼15.6, 7.1 Hz,
1H), 5.14 (s, 2H), 5.08 (m, 1H), 4.21 (m, 1H), 4.05 (m, 2H), 3.71 (dd,
J¼10.8, 3.0 Hz, 3H), 3.37 (m, 2H), 2.15 (t, J¼7.3 Hz, 2H), 2.03 (dt,
J¼6.9, 6.6 Hz, 2H), 1.72–1.82 (m, 2H), 1.57–1.61 (m, 4H), 1.44 (s, 9H),
1.25 (s, 46H), 0.88 (t, J¼6.9 Hz, 6H); 13C NMR (CDCl3, 100 MHz)
3.2.9. (4R)-(Benzyloxycarbonyloxy)-(3S)-(1-oxohexadecylamino)-
nonadec-(5E)-enylphosphonic acid (10)
To a solution of 9 (210 mg, 0.270 mmol) in dichloromethane
(2 ml) was added bromotrimethylsilane (0.36 ml, 2.70 mmol) at
room temperature. After the reaction mixture was stirred for 2 h at
the same temperature, the solvent was removed in vacuo. The
resulting crude silylester was dissolved in methanol (2 ml) and the
d 173.3, 155.8, 154.3, 137.4, 135.0, 128.56, 128.3, 123.5, 80.3, 79.5,
65.1, 65.0, 53.4, 52.5, 51.7, 51.5, 32.3, 31.9, 29.70, 29.66, 29.6, 29.52,
29.46, 29.4, 29.3, 29.2, 28.9, 28.4, 22.7, 22.6 (m), 21.7 (d, JC–P
¼
142.8 Hz), 14.1; ESI-HRMS m/z calcd for C51H91N2NaO9P (MþNa)þ
929.6371, found 929.6360.

11654
T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
To a solution of the methyl phosphonate thus obtained (44 mg,
121.1, 115.2, 79.9, 59.4 (d, JC–P¼18.2 Hz), 55.7, 51.6 (d, JC–P¼4.8 Hz),
46.2, 23.1 (d, JC–P¼2.9 Hz), 22.7 (d, JC–P¼138.9 Hz); ESI-HRMS m/z
calcd for C16H22NO6P (MꢁH)ꢁ 354.1106, found 354.1102.
0.049 mmol) in dichloromethane (1 ml) was added bromo-
trimethylsilane (0.065 ml, 0.49 mmol) at 0 ꢀC. After the reaction
mixture was stirred for 1 h at the same temperature, the solvent
was removed in vacuo. The resulting crude silylester was dissolved
in methanol and the solution was stirred for additional 1 h at room
temperature. The solvent was removed in vacuo to give the crude
products. Column chromatography on silica gel (from 0% to 9%
methanol in chloroform) gave the roughly purified corresponding
amine 12. To a solution of the obtained 12 in chloroform (1 ml)
were added methyl iodide (0.03 ml, 0.485 mmol) and potassium
carbonate (67 mg, 0.485 mmol) at room temperature. After the
reaction mixture was stirred for 48 h at the same temperature,
precipitates were removed by filtration and the filtrate was con-
centrated in vacuo to give the crude products. Column chroma-
tography on silica gel (from 9% methanol in chloroform to 4.3%
water and 26.6% methanol in chloroform) gave 13 (41 mg, 61% for
two steps) as colorless solid. IR (KBr disk, cmꢁ1) 3424, 2920, 2851,
1638, 1468, 1256, 1188, 1053, 968 cm; 1H NMR (CD3OD, 400 MHz)
3.3.2. 2-Bromoethyl methyl 2-[3-(p-methoxybenzyl)-2-oxo-(5R)-
vinyloxazolidin-(4S)-yl]ethylphosphonate (16)
To a solution of potassium carbonate (1.167 mg, 8.444 mmol)
and 1,2-dibromoethane (0.36 ml, 4.222 mmol) in acetonitrile
(4.2 ml) was added 15 (300 mg, 0.844 mmol) in acetonitrile
(4.2 ml) dropwise at 80 ꢀC. After the reaction mixture was stirred
for 4 h at the same temperature, saturated aqueous NH4Cl solution
was added and the resulting mixture was extracted with ethyl ac-
etate. The organic layers were combined, washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (from 0% to 2%
methanol in chloroform) gave 16 (238 mg, 61%) as a colorless oil.
22.0
[a
]
þ19.0 (c 0.90, CHCl3); IR (neat, cmꢁ1) 3459, 2928, 1748, 1512,
D
1418, 1250, 1038; 1H NMR (CDCl3, 400 MHz)
d
7.23 (d, J¼8.7 Hz, 2H),
6.87 (d, J¼8.4 Hz, 2H), 5.90 (ddd, J¼17.2, 10.5, 7.1 Hz, 1H), 5.51 (dd,
J¼17.2, 0.9 Hz, 1H), 5.44 (dd, J¼15.0, 0.9 Hz, 1H), 4.88 (dd, J¼7.5,
7.3 Hz, 1H), 4.80 (d, J¼15.1 Hz, 1H), 4.28 (m, 2H), 3.98 (dd, J¼15.1,
2.3 Hz, 1H), 3.80 (s, 3H), 3.719 (d, J¼11.0 Hz, 3/2H), 3.716 (d,
J¼10.8 Hz, 3/2H), 3.67 (dd, J¼7.8, 7.7 Hz, 1/2H), 3.66 (dd, J¼8.1,
7.8 Hz, 1/2H), 3.51 (dd, J¼6.0, 5.7 Hz, 1H), 3.50 (dd, J¼5.9, 5.7 Hz,
1H), 1.80–1.97 (m, 2H), 1.64–1.73 (m, 2H); 13C NMR (CDCl3,
d
7.31–7.36 (m, 5H), 5.81 (dt, J¼15.6, 6.9 Hz, 1H), 5.44 (dd, J¼15.6,
7.8 Hz,1H), 5.13 (s, 2H), 5.02 (m, 1H), 4.23 (m, 2H), 4.15 (m, 1H), 3.58
(m, 2H), 3.19 (s, 9H), 2.19 (t, J¼7.6 Hz, 2H), 2.05 (dt, J¼6.9, 6.9 Hz,
2H), 1.51–1.68 (m, 6H), 1.28 (s, 46H), 0.89 (t, J¼6.9 Hz, 6H); 13C NMR
(CD3OD, 100 MHz)
d 176.6, 155.8, 138.6, 137.1 129.6, 129.4, 129.2,
125.5, 81.8, 70.5, 67.8, 58.6 (d, JC–P¼4.8 Hz), 54.72, 54.68, 54.72, 37.3
(d, JC–P¼5.8 Hz), 33.4, 33.1, 30.8, 30.69, 30.66, 30.59, 30.52, 30.3,
30.1, 27.3, 25.1 (d, JC–P¼3.8 Hz), 24.6 (d, JC–P¼137.0 Hz), 14.5; ESI-
HRMS m/z calcd for C48H87N2NaO7P (MþNa)þ 857.6188, found
857.6149.
100 MHz)
d 159.4, 157.5, 130.2, 129.4, 127.5, 121.1, 114.2, 77.7, 65.10
(d, JC–P¼5.8 Hz, 1/2C), 65.05 (d, JC–P¼5.8 Hz, 1/2C), 56.62 (d,
J¼18.2 Hz, 1/2C), 56.58 (d, J¼18.2 Hz, 1/2C), 55.3, 52.5 (d, J¼4.8 Hz,
1/2C), 52.4 (d, JC–P¼5.8 Hz, 1/2C), 45.6, 30.4 (d, JC–P¼5.8 Hz, 1/2C),
30.3 (d, JC–P¼5.7 Hz, 1/2C), 20.6 (d, JC–P¼3.8 Hz), 20.5 (d, JC–P
¼
3.2.12. SM methylene analogue 1
143.7 Hz); ESI-HRMS m/z calcd for C18H25BrNO6PNa (MþNa)þ
To a solution of 13 (20 mg, 0.0239 mmol) in methanol (1 ml) was
added 2 N KOH (1 ml) at room temperature. After the reaction
mixture was stirred for 2 h at the same temperature, 2 N HCl was
added and the resulting mixture was concentrated in vacuo to give
the crude products. Column chromatography on silica gel (from 9%
484.0497, found 484.0501.
3.3.3. 2-Bromoethyl methyl 2-[3-(tert-butyloxycarbonyl)-2-oxo-
(5R)-vinyloxazolidin-(4S)-yl]ethylphosphonate (17)
To a solution of 16 (235 mg, 0.508 mmol) in acetonitrile (1.5 ml)
was added ceric ammonium nitrate (836 mg, 1.525 mmol) at 0 ꢀC.
After the reaction mixture was stirred at the same temperature for
15 min, brine was added and the resulting mixture was extracted
with ethyl acetate. The organic layers were combined, washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo to give
the crude products. Column chromatography on silica gel (from 0%
methanol in chloroform to 14% water and 43% methanol in chlo-
23.0
roform) gave 1 (13 mg, 76%) as a colorless form. [
a
]
ꢁ12.3 (c 0.13,
D
CHCl3); IR (KBr disk, cmꢁ1) 3441, 2920, 2851, 1638, 1468, 1192, 1053,
968; 1H NMR (CD3OD, 400 MHz)
d
5.57 (dt, J¼15.3, 6.6 Hz, 1H), 5.34
(dd, J¼15.3, 7.3 Hz, 1H), 4.15 (m, 2H), 3.77 (t, J¼7.0 Hz, 1H), 3.67 (m,
1H), 3.51 (m, 2H), 3.12 (s, 9H), 2.08 (t, J¼7.6 Hz, 2H), 1.93 (dt, J¼7.1,
6.9 Hz, 2H), 1.43–1.57 (m, 6H), 1.19 (s, 46H), 0.80 (t, J¼6.9 Hz, 6H);
to 9% methanol in chloroform) gave the PMB-deprotected oxazo-
22.0
13C NMR (CD3OD, 100 MHz)
d
176.3, 134.6, 131.3, 76.2, 67.9, 58.7 (d,
lidinone (185 mg, 80%) as a colorless oil. [
a
]
ꢁ21.9 (c 0.32,
D
JC–P¼5.8 Hz), 54.73, 54.70, 54.66, 37.4, 33.5, 33.1, 30.84, 30.77, 30.7,
30.6, 30.51, 30.47, 27.3, 25.7 (d, JC–P¼3.9 Hz), 24.7 (d, JC–P¼137.0 Hz),
23.8, 14.5; ESI-HRMS m/z calcd for C40H81N2NaO5P (MþNa)þ
723.8655, found 723.5781.
CHCl3); IR (neat, cmꢁ1) 3254, 2957, 1753, 1392, 1233, 1017, 957, 826,
770; 1H NMR (CDCl3, 400 MHz)
d
6.71 (br d, J¼8.3 Hz, 1H), 5.88
(ddd, J¼10.5, 7.4, 7.1 Hz, 1H), 5.49 (ddd, J¼17.2, 1.2, 1.1 Hz, 1H), 5.41
(ddd, J¼10.5, 1.1, 1.0 Hz, 1H), 5.07 (dd, J¼7.6, 7.6 Hz, 1H), 4.33 (m,
2H), 3.95 (dd, J¼8.5, 8.4 Hz, 1/2H), 3.94 (dd, J¼8.7, 8.4 Hz, 1/2H),
3.77 (d, J¼11.0 Hz, 3/2H), 3.76 (d, J¼10.9 Hz, 3/2H), 3.544 (dd, J¼6.0,
5.7 Hz, 1H), 3.537 (dd, J¼6.0, 5.7 Hz, 1H), 1.70–1.95 (m, 4H); 13C
3.3. Improved synthesis of SM methylene analogue 1
3.3.1. Methyl hydrogen 2-[3-(p-methoxybenzyl)-2-oxo-(5R)-
vinyloxazolidin-(4S)-yl]ethylphosphate (15)
To a solution of 14 (1.078 g, 2.919 mmol) in methanol (8.1 ml)
was added 30% trimethylamine aqueous solution (8.1 ml) at room
temperature. After the reaction mixture was stirred for 24 h at the
same temperature, the solvent was removed in vacuo to give the
NMR (CDCl3, 100 MHz)
d
158.8, 130.4, 120.7, 80.1, 65.32 (d, JC–P
¼
5.7 Hz, 1/2C), 65.26 (d, JC–P¼5.8 Hz, 1/2C), 55.7 (d, JC–P¼12.4 Hz),
52.65 (d, JC–P¼6.7 Hz, 1/2C), 52.62 (d, JC–P¼6.7 Hz, 1/2C), 30.4 (d,
JC–P¼6.7 Hz, 1/2C), 30.3 (d, JC–P¼7.7 Hz, 1/2C), 24.5 (d, JC–P¼4.8 Hz),
21.89 (d, JC–P¼143.6 Hz, 1/2C), 21.87 (d, JC–P¼143.6 Hz, 1/2C); ESI-
HRMS m/z calcd for C10H17BrNO5PNa (MþNa)þ 363.9925, found
363.9917.
crude products. Column chromatography on silica gel (from 0% to
21.5
9% methanol in chloroform) gave 15 (1.031 g, 87%). [
a]
þ17.0 (c
To a solution of the oxazolidinone compound thus obtained
(148 mg, 0.433 mmol) in dichloromethane (2 ml) were added
triethylamine (0.12 ml, 0.865 mmol), DMAP (26 mg, 0.216 mmol),
and Boc2O (113 mg, 0.519 mmol) at 0 ꢀC. After the reaction mixture
was stirred at the same temperature for 15 min, saturated aqueous
NH4Cl solution was added and the resulting mixture was extracted
with dichloromethane. The organic layers were combined, washed
with brine, dried over MgSO4, filtered, and concentrated in vacuo to
D
1.01, CHCl3); IR (KBr disk, cmꢁ1) 3409, 2951, 1744, 1514, 1441, 1246,
1181, 1069, 949, 826; 1H NMR (CD3OD, 400 MHz)
d
7.25 (d, J¼8.5 Hz,
2H), 6.90 (d, J¼8.7 Hz, 2H), 6.03 (ddd, J¼17.4, 10.5, 7.4 Hz, 1H), 5.49
(d, J¼17.2 Hz, 1H), 5.41 (d, J¼10.6 Hz, 1H), 4.99 (dd, J¼7.5, 7.5 Hz,
1H), 4.66 (d, J¼15.1 Hz, 1H), 4.11 (d, J¼15.1 Hz, 1H), 3.77 (s, 3H), 3.75
(m, 1H), 3.51 (s, J¼10.3 Hz, 3H), 1.90 (m, 1H), 1.78 (m, 1H), 1.45 (m,
2H); 13C NMR (CD3OD, 100 MHz)
d 160.8, 160.3, 132.4, 130.3, 129.3,

T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
11655
give the crude products. Column chromatography on silica gel
cooled to 0 ꢀC. Saturated aqueous K2CO3 solution was added and
the mixture was stirred for 10 min at the same temperature, and
then palmitoyl chloride (0.20 ml, 0.65 mmol) was added at the
same temperature. After the mixture was stirred for 15 min at the
same temperature, saturated aqueous NH4Cl solution was added
and the resulting mixture was extracted with chloroform. The or-
ganic layers were combined, washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo to give the crude products.
(from 0% to 2% methanol in chloroform) gave 17 (176 mg, 92%) as
22.0
a colorless oil. [
a
]
D
þ14.1 (c 0.33, CHCl3); IR (neat, cmꢁ1) 3459,
2975, 1813, 1721, 1368, 1252, 1074; 1H NMR (CDCl3, 400 MHz)
d
5.894 (ddd, J¼17.2, 10.5, 6.7 Hz, 1/2H), 5.891 (ddd, J¼17.2, 10.5,
6.4 Hz, 1/2H), 5.57 (dt, J¼17.2, 1.2 Hz, 1H), 5.50 (dt, J¼10.8, 1.2 Hz,
1H), 5.00 (m, 1H), 4.35 (m, 1H), 4.31 (m, 2H), 3.75 (d, J¼11.0 Hz, 3/
2H), 3.74 (d, J¼11.0 Hz, 3/2H), 2.01–2.14 (m, 1H), 1.88–1.99 (m, 1H),
1.76–1.86 (m, 1H), 1.54 (s, 9H); 13C NMR (CDCl3, 100 MHz)
d
151.2,
Column chromatography on silica gel (from 0% to 2% methanol in
25.5
149.2, 128.9, 122.0, 84.4, 77.8, 65.14 (d, JC–P¼5.8 Hz, 1/2C), 65.11 (d,
JC–P¼5.7 Hz, 1/2C), 58.00 (d, JC–P¼10.2 Hz, 1/2C), 57.97 (d, JC–P
9.1 Hz, 1/2C), 52.50 (d, JC–P¼6.7 Hz, 1/2C), 52.47 (d, JC–P¼6.7 Hz, 1/
chloroform) gave 20 (0.223 g, 56%) as a colorless solid. [
a
]
ꢁ7.95
D
¼
(c 0.4, CHCl3); IR (KBr disk, cmꢁ1) 3287, 2930,1649,1468,1215,1051,
721; 1H NMR (CDCl3, 400 MHz)
d
6.13 (dd, J¼8.5, 7.3 Hz, 1H), 5.74
2C), 30.2 (d, JC–P¼6.7 Hz), 27.9, 22.5 (d, JC–P¼3.8 Hz), 21.2 (d, JC–P
¼
(dt, J¼14.9, 6.8 Hz, 1H), 5.44 (dd, J¼14.6, 6.4 Hz, 1H), 4.26–4.38 (m,
2H), 4.16–4.13 (m, 1H), 3.95–4.05 (m, 1H), 3.75 (d, J¼11.0 Hz, 3/2H),
3.74 (d, J¼11.0 Hz, 3/2H), 3.53 (td, J¼6.0, 1.8 Hz, 2H), 2.20 (t,
J¼7.8 Hz, 2H), 2.04 (dt, J¼7.1, 7.1 Hz, 2H), 1.74–1.92 (m, 6H), 1.56–
1.67 (m, 2H), 1.18–1.30 (m, 48H), 0.88 (t, J¼7.1 Hz, 6H); 13C NMR
142.8 Hz); ESI-HRMS m/z calcd for C15H25BrNO7PNa (MþNa)þ
464.0450, found 464.0436.
3.3.4. 2-Bromoethyl methyl (3S)-(tert-butyloxycarbonylamino)-
(4R)-hydroxyhex-5-enylphosphate (18)
(CDCl3, 100 MHz)
d
174.3, 134.4, 128.1, 75.1, 65.1 (d, JC–P¼5.7 Hz),
To a solution of 17 (90 mg, 0.204 mmol) in methanol (1 ml) was
added potassium carbonate (42 mg, 0.244 mmol) at room tem-
perature. After the reaction mixture was stirred for 20 min at the
same temperature, saturated aqueous NH4Cl solution was added
and the resulting mixture was extracted with ethyl acetate. The
organic layers were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (from 0% to 2%
54.4 (d, JC–P¼15.3 Hz), 52.5 (d, JC–P¼6.7 Hz), 36.8, 35.9, 32.4, 32.3,
31.9, 30.2 (d, JC–P¼3.8 Hz), 29.7, 29.7, 29.5, 29.5, 29.4, 25.8, 25.5,
22.7, 14.1; ESI-HRMS m/z calcd for C38H75BrNO5PNa (MþNa)þ
758.4464, found 758.4448.
3.3.7. SM methylene analogue 1
To a solution of 20 (0.258 g, 0.350 mmol) in methanol (1.8 ml)
was added trimethylamine (1.1 ml) at room temperature. After the
reaction mixture was stirred for 48 h at the same temperature, the
solvent was removed in vacuo to give the crude products. Column
chromatography on silica gel (from 9% methanol in chloroform to
14% water and 43% methanol in chloroform) gave 1 (0.179 g, 73%) as
a colorless foam.
methanol in chloroform) gave 18 (43 mg, 51%) as a colorless oil.
21.5
[
a]
ꢁ2.9 (c 0.44, CHCl3); IR (neat, cmꢁ1) 3356, 2976, 1528, 1453,
D
1368, 1244, 1171, 1047; 1H NMR (CDCl3, 400 MHz)
d 5.86 (ddd,
J¼17.2, 10.5, 5.5 Hz, 1H), 5.35 (dt, J¼17.4, 1.4 Hz,1H), 5.25 (dt, J¼10.5,
1.4 Hz, 1H), 4.88 (br d, J¼2.9 Hz, 1H), 4.31 (m, 1H), 4.23 (md,
J¼2.7 Hz, 1H), 3.75 (d, J¼11.0 Hz, 3H), 3.69 (m, 1H), 3.53 (t, J¼5.9 Hz,
2H), 2.66 (s, 1H), 1.77–2.00 (m, 3H), 1.58–1.67 (m, 1H); 13C NMR
3.4. Synthesis of fluorescence-labeled SM methylene
analogue 4
(CDCl3, 100 MHz)
d
156.4, 136.9, 116.8, 79.8, 75.1, 65.0 (d, JC–P
¼
5.8 Hz), 55.3 (d, JC–P¼17.2 Hz), 52.4 (d, JC–P¼5.7 Hz), 30.22 (d, JC–P
¼
6.7 Hz, 1/2C), 30.20 (d, JC–P¼6.7 Hz, 1/2C), 28.3, 22.4 (m), 22.02 (d,
JC–P¼141.9 Hz, 1/2C), 22.00 (d, JC–P¼141.9 Hz, 1/2C); ESI-HRMS m/z
calcd for C14H27BrNO6PNa (MþNa)þ 438.0657, found 438.0653.
3.4.1. 2-Bromoethyl methyl 15-(7-nitrobenzo-2-oxa-1,3-diazol-4-
ylamino)-(3S)-(tert-butyloxycarbonylamino)-(4R)-hydroxypent-
(5E)-enylphosphonate (22)
To a solution of 18 (40 mg, 0.096 mmol) in dichloromethane
(2 ml) were added 21 (128 mg, 0.384 mmol) and second generation
Grubbs catalyst (2.5 mg, 0.029 mmol) at room temperature. After
the reaction mixture was stirred for 2 h at 40 ꢀC, the solvent was
removed in vacuo to give the crude products. Column chromatog-
3.3.5. 2-Bromoethyl methyl (4R)-hydroxy-(3S)-(tert-
butyloxycarbonylamino)nonadec-(5E)-enylphosphonate (19)
To a solution of 18 (0.250 g, 0.601 mmol) in dichloromethane
(3.0 ml) were added 1-pentadecene (0.65 ml, 2.40 mmol) and sec-
ond generation Grubbs catalyst (15 mg, 0.018 mmol) at room
temperature. After the reaction mixture was stirred for 2 h under
reflux, the solvent was removed in vacuo to give the crude prod-
raphy on silica gel (from 0% to 2% methanol in chloroform) gave 22
25.0
(54 mg, 78%) as a brownish foam. [
a
]
D
ꢁ12.2 (c 0.30, CHCl3); IR
(KBr disk) 3322, 2928, 2855, 1692, 1588, 1300, 1252, 1047 cmꢁ1; 1H
NMR (CDCl3, 400 MHz)
ucts. Column chromatography on silica gel (from 0% to 2% methanol
d
8.49 (d, J¼8.5 Hz, 1H), 6.51 (s, 1H), 6.17 (d,
25.5
in chloroform) gave 19 (0.216 mg, 60%) as a brownish foam. [
a]
J¼8.7 Hz, 1H), 5.73 (td, J¼6.5, 16.0 Hz, 1H), 5.45 (dd, J¼6.6, 15.6 Hz,
1H), 4.81 (br d, J¼6.9 Hz, 1H), 4.32 (m, 2H), 4.16 (m, 1H), 3.75 (d,
J¼10.9 Hz, 3H), 3.64 (m, 1H), 3.53 (t, J¼6.0 Hz, 2H), 3.49 (td, J¼6.0,
7.3 Hz, 2H), 2.35 (s, 1H), 2.04 (dt, J¼7.1, 7.1 Hz, 2H), 1.81 (tt, J¼7.3,
7.6 Hz, 2H), 1.77–1.96 (m, 3H), 1.58–1.69 (m, 1H), 1.44 (s, 9H), 1.24–
D
ꢁ0.31 (c 2.9, CHCl3); IR (neat, cmꢁ1) 3387, 2926, 1716, 1508, 1242,
1174, 1045; 1H NMR (CDCl3, 400 MHz)
d
5.73 (dt, J¼15.1, 7.3 Hz, 1H),
5.45 (dd, J¼15.6, 6.6 Hz, 1H), 5.01 (br d, J¼8.9 Hz, 1H), 4.25–4.37 (m,
2H), 4.14 (br m, 1H), 3.75 (d, J¼11.0 Hz, 3H), 3.57–3.65 (m, 2H), 3.53
(t, J¼6.2 Hz, 2H), 2.98 (br m, 1H), 2.03 (td, J¼6.9, 6.9 Hz, 2H), 1.60–
1.98 (m, 6H), 1.44 (s, 9H), 1.26–1.38 (m, 22H), 0.88 (t, J¼7.1 Hz, 3H);
1.38 (m,12H); 13C NMR (CDCl3, 100 MHz)
143.9, 136.5, 134.2, 128.4, 98.5, 79.8, 75.2, 65.1 (d, JC–P¼6.8 Hz), 55.6
d 157.3, 156.4, 144.3,144.0,
13C NMR (CDCl3, 100 MHz)
d
156.3, 134.0, 128.4, 79.5, 75.0, 65.0 (d,
(d, JC–P¼16.3 Hz), 52.4 (d, JC–P¼6.7 Hz), 44.0, 32.2, 30.3 (d, JC–P¼
JC–P¼5.7 Hz), 55.6 (d, JC–P¼15.3 Hz), 52.3 (d, J¼6.7 Hz), 32.3, 31.8,
30.2 (d, JC–P¼6.7 Hz), 29.6, 29.6, 29.6, 29.5, 29.3, 29.2, 29.1, 28.3,
28.2, 22.6, 22.5 (d, JC–P¼2.9 Hz), 22.0 (d, JC–P¼141.8 Hz, 1/2C), 21.9
(d, JC–P¼141.8 Hz, 1/2C), 14.0; ESI-HRMS m/z calcd for
C29H47BrN5O9PNa (MþNa)þ 620.2692, found 620.2697.
5.7 Hz), 29.23, 29.18, 29.1, 29.9 (d, JC–P¼2.9 Hz), 28.4, 28.3, 26.9,
22.6, 22.0 (d, JC–P¼141.8 Hz); ESI-HRMS m/z calcd for
C29H47BrN5O9PNa (MþNa)þ 742.2208, found 742.2192.
3.4.2. 2-Bromoethyl methyl 15-(7-nitrobenzo-2-oxa-1,3-diazol-4-
ylamino)-(4R)-hydroxy-(3S)-(1-oxohexadecylamino)pentadec-
(5E)-enylphosphonate (23)
3.3.6. 2-Bromoethyl methyl (4R)-hydroxy-(3S)-(1-
oxohexadecylamino)nonadec-(5E)-enylphosphonate (20)
To a solution of 19 (0.324 g, 0.541 mmol) in dichloromethane
(5.4 ml) was added trifluoroacetic acid (1.1 ml) at 0 ꢀC. After the
reaction mixture was stirred for 3 h at the room temperature, it was
To a solution of 22 (33 mg, 0.046 mmol) in dichloromethane
(0.5 ml) was added trifluoroacetic acid (0.09 ml) at 0 ꢀC. After the
reaction mixture was stirred for 2 h at the same temperature, it was
warmed to room temperature and then stirred for another 1 h at

11656
T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
the same temperature. Saturated aqueous NaHCO3 solution was
added at 0 ꢀC and the mixture was stirred for 5 min at the same
temperature, and then palmitoyl chloride (0.017 ml, 0.055 mmol)
was added at the same temperature. After the reaction mixture was
stirred for 15 min, saturated aqueous NH4Cl solution was added and
the resulting mixture was extracted with dichloromethane. The
organic layers were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (from 0% to 2%
1H), 3.99 (dd, J¼11.0, 3.4 Hz,1H), 3.71 (dd, J¼11.0,1.1 Hz,1H),1.55 (s,
9H), 0.88 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H); 13C NMR (CDCl3,
100 MHz)
d 151.6, 149.4, 130.8, 121.9, 83.6, 77.9, 59.5, 59.2, 28.0,
25.6, 17.9, ꢁ5.7; ESI-HRMS m/z calcd for C17H51NO5SiNa (MþNa)þ
380.1869, found 380.1850.
To a solution of the obtained oxazolidinone derivative (3.000 g,
8.391 mmol) in methanol (52 ml) was added 1 N HCl (25.2 ml) at
room temperature. After the reaction mixture was stirred for 16 h
at the same temperature, saturated aqueous NaHCO3 solution was
added and the resulting mixture was extracted with ethyl acetate.
The organic layers were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (from 33% to 83%
methanol in chloroform) gave 23 (32 mg, 82%) as a brownish foam.
23.0
[
a]
ꢁ8.7 (c 0.38, CHCl3); IR (neat, cmꢁ1) 3285, 2924, 2853, 1588,
D
1300, 1046; 1H NMR (CDCl3, 400 MHz)
d
8.50 (d, J¼8.4 Hz, 1H), 6.63
(m, 1H), 6.17 (d, J¼8.7 Hz, 1H), 6.08 (m, 1H), 5.72 (dt, J¼15.3, 6.7 Hz,
1H), 5.45 (dd, J¼15.6, 6.6 Hz, 1H), 4.32 (m, 2H), 4.16 (m, 1H), 4.01
(mdd, J¼9.4, 9.3 Hz, 1H), 3.748 (d, J¼11.0 Hz, 3/2H), 3.746 (d,
J¼11.0 Hz, 3/2H), 3.53 (t, J¼6.2 Hz, 2H), 3.48 (dt, J¼6.7, 6.2 Hz, 2H),
2.84 (br d, J¼4.1 Hz, 1H), 2.21 (t, J¼7.5 Hz, 2H), 2.03 (dt, J¼6.9,
6.4 Hz, 2H), 1.81 (tt, J¼7.8, 7.5 Hz, 2H), 1.69–1.96 (m, 4H), 1.62 (tt,
J¼6.6, 6.4 Hz, 2H),1.46 (m, 2H),1.24–1.37 (m, 34H), 0.87 (t, J¼6.9 Hz,
ethyl acetate in hexane) gave 24 (1.718 g, 84%) as a colorless solid.
24.0
[a
]
þ28.8 (c 1.01, CHCl3); IR (KBr disk, cmꢁ1) 3483, 2982, 1788,
D
1381, 1091, 779; 1H NMR (CDCl3, 400 MHz)
d
6.08 (ddd, J¼17.4, 10.5,
6.9 Hz, 1H), 5.57 (ddd, J¼17.1, 1.1, 1.1 Hz, 1H), 5.48 (ddd, J¼10.5, 0.9,
0.9 Hz, 1H), 5.03 (dddd, J¼8.0, 6.9, 1.1, 1.1 Hz, 1H), 4.31 (ddd, J¼7.6,
3.7, 3.7 Hz, 1H), 3.96 (ddd, J¼12.1, 6.6, 4.1 Hz, 1H), 3.78 (ddd, J¼12.1,
5.5, 3.4 Hz, 1H), 2.19 (dd, J¼6.6, 5.5 Hz, 1H), 1.56 (s, 9H); 13C NMR
3H); 13C NMR (CDCl3, 100 MHz)
d 174.4, 144.3, 144.0, 143.9, 136.6,
134.3, 128.4, 123.7, 98.4, 75.2, 65.14 (d, JC–P¼5.7 Hz, 1/2C), 65.11 (d,
JC–P¼6.7 Hz, 1/2C), 54.5 (d, JC–P¼4.4 Hz), 52.5 (d, JC–P¼7.7 Hz), 44.0,
36.8, 32.2, 31.9, 30.3 (d, JC–P¼6.7 Hz), 29.7, 29.6, 29.5, 29.4, 29.3,
29.2, 29.1, 29.0, 28.9, 28.4, 26.8, 25.8, 22.7, 22.0 (d, JC–P¼1.9 Hz), 21.9
(d, JC–P¼145.6 Hz), 14.1; ESI-HRMS m/z calcd for C40H69BrN5O8PNa
(MþNa)þ 880.3965, found 880.3957.
(CDCl3, 100 MHz) d 151.8, 149.7, 130.1, 121.6, 84.3, 78.8, 59.7, 59.6,
27.9; ESI-HRMS m/z calcd for C11H17NO5Na (MþNa)þ 266.1004,
found 266.1003.
3.5.2. N-[(3R)-(tert-Butyloxycabonyloxy)-(2S)-(tert-butyloxy-
cabonylamino)pent-4-enyl]-4-nitrobenzenesulfonamide (26)
To a solution of 24 (0.500 g, 2.06 mmol) in toluene (10.3 ml)
were added N-Boc nosylamide (0.934 g, 3.09 mmol), triphenyl-
phosphine (0.810 g, 3.09 mmol), and DIAD (0.60 ml, 3.09 mmol) at
room temperature. After the reaction mixture was stirred for 16 h
at the same temperature, it was concentrated in vacuo to give the
crude products. Column chromatography on silica gel (from 5% to
50% ethyl acetate in hexane) gave the roughly purified 25. To a so-
lution of 25 in methanol (4.1 ml) was added cesium carbonate
(0.805 g, 2.47 mmol) at room temperature. After the reaction
mixture was stirred for 1 h at the same temperature, saturated
aqueous NH4Cl solution was added and the resulting mixture was
extracted with ethyl acetate. The organic layers were combined,
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo to give the crude products. Column chromatography on silica
3.4.3. Fluorescence-labeled SM methylene analogue 4
To a solution of 23 (0.186 g, 0.217 mmol) in methanol (2 ml) was
added trimethylamine (0.6 ml) at room temperature. After the re-
action mixture was stirred for 48 h at the same temperature, the
solvent was removed in vacuo to give the crude products. Column
chromatography on silica gel (from 9% methanol in chloroform to
14% water and 43% methanol in chloroform) gave 4 (0.109 g, 61%) as
25.5
a brownish foam. [
a
]
ꢁ7.3 (c 0.4, MeOH); IR (KBr disk, cmꢁ1
)
D
3289, 2916, 2849, 1489, 1327, 1184, 968; 1H NMR (CDCl3, 400 MHz)
d
8.49 (d, J¼8.7 Hz, 1H), 6.63 (d, J¼8.9 Hz, 1H), 5.67 (dt, J¼15.3,
7.1 Hz, 1H), 5.44 (dd, J¼15.3, 7.1 Hz, 1H), 4.29 (br m, 2H), 3.89 (dd
J¼6.9, 7.1 Hz, 1H), 3.76–3.80 (m, 1H), 3.64–3.67 (m, 2H), 3.43–3.56
(m, 2H), 3.23 (s, 9H), 2.18 (t, J¼7.6 Hz, 2H), 2.01 (dt, J¼6.6, 6.7 Hz,
2H), 1.77 (tt, J¼7.3, 7.3 Hz, 2H), 1.24–1.67 (m, 42H), 0.88 (t, J¼7.1 Hz,
gel (from 2% to 3% methanol in chloroform) gave 26 (0.903 g, 87%,
24.0
3H); 13C NMR (CDCl3, 100 MHz)
d
176.2, 146.6, 145.8, 145.5, 138.6,
two steps) as a colorless solid. [
a
]
ꢁ2.9 (c 1.04, CHCl3); IR (KBr
D
134.4, 131.3, 122.8, 99.6, 76.1, 67.8, 58.7 (d, JC–P¼4.8 Hz), 57.1, 56.1,
54.78, 54.75, 54.71, 44.8, 37.4, 33.4, 33.1, 30.8, 30.75, 30.66, 30.61,
30.57, 30.47, 30.43, 30.35, 29.3, 28.1, 27.3, 25.6 (d, JC–P¼2.9 Hz), 24.6
(d, JC–P¼137.0 Hz), 23.7, 14.5; ESI-HRMS m/z calcd for
C42H75N6O8PNa (MþNa)þ 845.5282, found 845.5915.
disk, cmꢁ1) 3371, 2980, 1695, 1348, 1159, 856; 1H NMR (CDCl3,
400 MHz)
d
8.33–8.38 (m, 2H), 8.01–8.06 (m, 2H), 5.73 (ddd, J¼16.9,
10.5, 6.0 Hz, 1H), 5.62 (m, 1H), 5.35 (ddd, J¼12.6, 2.3, 1.1 Hz, 1H),
5.31 (ddd, J¼6.2, 2.3, 1.1 Hz, 1H), 5.07 (dddd, J¼6.6, 4.8, 1.4, 1.4 Hz,
1H), 4.87–4.89 (m, 1H), 3.80–3.86 (m, 1H), 3.30 (ddd, J¼11.0, 7.3,
3.7 Hz, 1H), 3.01–3.07 (m, 1H), 1.46 (s, 9H), 1.44 (s, 9H); 13C NMR
3.5. Synthesis of SM aza analogue 2
(CDCl3, 100 MHz) d 155.9, 152.6, 150.0, 145.8, 132.0, 128.42, 124.4,
119.6, 83.4, 80.7, 77.1, 52.3, 43.8, 28.3, 27.7; ESI-HRMS m/z calcd for
3.5.1. (4S)-(Hydroxymethyl)-3-(tert-butyloxycarbonyl)-(5R)-
vinyloxazolidinone (24)
C21H51N3O9SNa (MþNa)þ 524.1679, found 524.1674.
To a solution of 5a (2.734 g, 10.62 mmol) in dichloromethane
(53.1 ml) were added DMAP (0.649 g, 5.31 mmol), triethylamine
(2.96 ml, 231.2 mmol), and Boc2O (2.92 ml,15.9 mmol) at 0 ꢀC. After
the reaction mixture was stirred for 1 h at the same temperature,
saturated aqueous NH4Cl solution was added and the resulting
mixture was extracted with chloroform. The organic layers were
combined, washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo to give the crude products. Column chro-
matography on silica gel (from 5% to 16% ethyl acetate in hexane)
3.5.3. N-[(3R)-(tert-Butyloxycabonyloxy)-(2S)-(tert-butyloxy-
cabonylamino)octadec-(4E)-enyl]-4-nitrobenzenesulfonamide (27)
To a solution of 26 (1.000 g, 1.990 mmol) in dichloromethane
(20 ml) were added 1-pentadecene (2.15 ml, 7.980 mmol) and
second generation Grubbs catalyst (0.051 g, 0.060 mmol). After the
reaction mixture was stirred for 7 h under reflux, it was concen-
trated in vacuo to give the crude products. Column chromatography
on silica gel (from 3% to 20% ethyl acetate in hexane) gave 27
23.5
(0.983 g, 72%) as a colorless solid. [
a
]
D
ꢁ14.5 (c 1.12, CHCl3); IR
gave the Boc-protected oxazolidinone (3.536 g, 93%) as a colorless
(KBr disk, cmꢁ1) 3373, 2924, 1693, 1342, 1159, 734; 1H NMR (CDCl3,
400 MHz)
24.0
solid. [
a]
þ27.9 (c 0.9, CHCl3); IR (KBr disk, cmꢁ1) 2955, 1792,
d
8.34–8.36 (m, 2H), 8.03–8.05 (m, 2H), 5.78 (td, J¼14.4,
D
1373, 1089, 835; 1H NMR (CDCl3, 400 MHz)
7.7 Hz, 1H), 5.48 (ddd, J¼18.5, 1.1, 1.1 Hz, 1H), 5.44 (ddd, J¼10.5, 0.9,
0.9 Hz, 1H), 4.94 (dd, J¼7.8, 7.8 Hz, 1H), 4.19 (ddd, J¼7.6, 3.2, 1.4 Hz,
d
6.12 (ddd, J¼18.1, 10.3,
6.6 Hz, 1H), 5.66 (m, 1H), 5.32 (dd, J¼15.3, 7.3 Hz, 1H), 4.97 (dd,
J¼6.4, 6.4 Hz, 1H), 4.83 (m, 1H), 3.77–3.79 (m, 1H), 3.29 (ddd,
J¼13.0, 7.6, 3.7 Hz, 1H), 2.98–3.04 (m, 1H), 2.01 (dt, J¼6.6, 6.6 Hz,

T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
11657
2H), 1.44 (s, 9H), 1.44 (s, 9H), 1.21–1.36 (m, 2H), 1.26 (s, 20H), 0.88 (t,
Column chromatography on silica gel (from 0% to 9% methanol in
24.0
J¼7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d
155.9, 152.7, 150.0, 145.9,
chloroform) gave 29 (0.317 g, 90%) as a colorless oil. [
1.0, CHCl3); IR (KBr disk, cmꢁ1) 3298, 2932,1639, 1468,1074, 744; 1H
NMR (CDCl3, 400 MHz)
a]
D
ꢁ13.0 (c
137.9, 128.2, 124.4, 123.4, 83.1, 80.5, 52.5, 44.0, 32.3, 31.9, 29.7, 29.6,
29.4, 29.3, 29.1, 28.7, 28.3, 27.7, 22.7, 14.1; ESI-HRMS m/z calcd for
C34H57N3O9SNa (MþNa)þ 706.3713, found 706.3681.
d
6.21 (d, J¼8.0 Hz, 1H), 5.67 (td, J¼15.3,
6.9 Hz, 1H), 5.42 (dd, J¼15.3, 6.6 Hz, 1H), 4.28 (dd, J¼6.2, 3.9 Hz, 1H),
3.83–3.89 (m, 1H), 2.97 (dd, J¼13.0, 5.7 Hz, 1H), 2.81 (dd, J¼13.0,
4.3 Hz, 1H), 2.20 (dt, J¼10.8, 2.7 Hz, 2H), 2.02 (dt, J¼6.9, 6.9 Hz, 2H),
1.63 (tt, J¼7.1, 7.1 Hz, 2H), 1.22–1.35 (m, 48H), 0.94 (t, J¼7.8 Hz, 9H),
0.88 (t, J¼6.6 Hz, 6H), 0.58 (q, J¼7.7 Hz, 6H); 13C NMR (CDCl3,
3.5.4. N-[(3R)-Hydroxy-(2S)-1-(oxohexadecylamino)octadec-(4E)-
enyl]-4-nitrobenzenesulfonamide (28)
To a solution of 27 (2.031 g, 2.970 mmol) in dichloromethane
(15.0 ml) was added dropwise trifluoroacetic acid (6.00 ml) at 0 ꢀC.
After the reacting mixture was stirred for 3.5 h at the same tem-
perature, it was poured into a mixture of saturated aqueous NaHCO3
solution and chloroform at 0 ꢀC, and the resulting mixture was
stirred for another 5 min at the same temperature. To this mixture
were added potassium carbonate (4.105 g, 29.70 mmol) and pal-
mitoyl chloride (1.00 ml, 3.267 mmol) at 0 ꢀC, and the resulting
mixture was stirred for 30 min. Saturated aqueous NH4Cl solution
was added and the mixture was extracted with chloroform. The
organic layers were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the crude prod-
100 MHz)
d 173.1, 133.2, 129.7, 75.2, 54.8, 40.9, 37.0, 32.2, 31.9, 29.7,
29.7, 29.5, 29.4, 29.4, 29.4, 29.2, 29.1, 25.8, 22.7, 14.1, 6.9, 4.9; ESI-
HRMSm/z calcdforC40H82N2O2Si(MþH)þ 651.6224, found 651.6238.
3.5.6. 2-Bromoethyl dimethyl phosphite (30)
To a solution of methyl dichlorophosphite (5.0 ml, 55.0 mmol) in
THF (275 ml) were added diisopropylethylamine (28.7 ml,
165 mmol) and 2-bromoethanol (3.90 ml, 55.0 mmol) at ꢁ78 ꢀC.
After the reaction mixture was stirred for 1 h, methanol (2.23 ml,
55.0 mmol) was added to the reaction mixture at ꢁ78 ꢀC. After the
reaction mixture was stirred for 30 min at the same temperature, it
was slowly warmed to room temperature. Diethyl ether was added
and the precipitate was removed by filtration through Celite, and
then the filtrate was concentrated in vacuo. The residue was dis-
tilled under reduced pressure (bp 65 ꢀC at 7 mmHg) to give 30
(7.57 g, 64%) as a colorless liquid. IR (neat, cmꢁ1) 2948, 2838, 1456,
ucts. Column chromatography on silica gel (from 0% to 2% methanol
24.0
in chloroform) gave 28 (1.800 g, 84%) as a colorless solid. [a]
D
ꢁ13.0 (c 0.97, CHCl3); IR (KBr disk, cmꢁ1) 3294, 2920, 1649, 1350,
1161, 738; 1H NMR (CDCl3, 400 MHz)
d 8.33–8.37 (m, 2H), 8.01–8.05
(m, 2H), 6.06 (d, J¼8.0 Hz,1H), 5.98 (dd, J¼6.6, 5.3 Hz,1H), 5.73 (dtd,
J¼14.6, 6.6, 0.9 Hz, 1H), 5.38 (tdd, J¼15.3, 6.4, 0.9 Hz, 1H), 4.23 (dd,
J¼5.5, 5.5 Hz, 1H), 3.91–3.96 (m, 1H), 3.27 (ddd, J¼13.0, 7.6, 3.7 Hz,
1H), 3.16 (ddd, J¼13.0, 6.4, 5.0 Hz,1H), 2.18 (t, J¼7.1 Hz, 2H), 2.00 (dt,
J¼7.1, 7.1 Hz, 2H), 1.62 (m, 2H), 1.20–1.36 (m, 48H), 0.88 (t, J¼6.9 Hz,
1287, 1181, 1005; 1H NMR (CDCl3, 400 MHz)
d
4.10 (dt, J¼7.6, 6.6 Hz,
2H), 3.55 (d, J¼10.7 Hz, 6H), 3.49 (t, J¼6.6 Hz, 2H); 13C NMR (CDCl3,
100 MHz)
d
62.0 (JC–P¼11.6 Hz), 49.3 (JC–P¼10.8 Hz), 31.2 (JC–P¼
4.1 Hz); 31P NMR (CDCl3, 300 MHz)
d 141.2.
6H); 13C NMR (CDCl3, 100 MHz)
d 174.4, 150.0, 145.9, 135.2, 128.2,
127.9, 124.3, 77.2, 73.6, 52.6, 43.7, 36.8, 32.3, 31.9, 29.7, 29.7, 29.5,
29.5, 29.4, 29.3, 29.3, 29.1, 25.7, 22.7, 14.1; ESI-HRMS m/z calcd for
C40H71N3O6SNa (MþNa)þ 744.4961, found 744.4931.
3.5.7. 2-Bromoethyl methyl [(3R)-hydroxy-(2S)-(1-
oxohexadecylamino)octadec-(4E)-enyl]phosphoramidate (32)
To a mixture of 4 Å molecular sieves (0.300 g) and carbon tet-
rabromide (0.612 g, 1.844 mmol) in dichloromethane (3.0 ml) was
added 2-bromoethyl dimethyl phosphite 30 (0.28 ml, 1.844 mmol)
at 0 ꢀC. After the reaction mixture was stirred for 20 min, a solution
of 29 (0.300 g, 0.461 mmol) in dichloromethane and 2,6-lutidine
(0.02 ml, 0.184 mmol) were added to the reaction mixture at the
same temperature. After the reaction mixture was stirred for 1.5 h
at the same temperature, chloroform was added and the precipitate
was removed by filtration. Saturated aqueous NaHCO3 solution was
added and the resulting mixture was extracted with chloroform.
The organic layers were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (from 9% to 66%
ethyl acetate in hexane containing 3% triethylamine) gave the
roughly purified 31. To a solution of 31 obtained above in methanol
(5 ml) was added Dowex 50W (pH¼5) at room temperature. After
the reaction mixture was stirred for 5 min at the same temperature,
methanol was added and the precipitate was removed by filtration.
The filtrate was concentrated in vacuo to give the crude products.
Column chromatography on silica gel (from 0% to 3% methanol in
3.5.5. N-[1-Amino-(3R)-triethylsilyloxyoctadec-(4E)-en-(2S)-
yl]hexadecanamide (29)
To a solution of 28 (0.500 g, 0.693 mmol) in DMF (4.0 ml) were
added imidazole (0.283 g, 4.158 mmol) and chlorotriethylsilane
(0.29 ml, 1.663 mmol) at room temperature. After the reaction
mixture was stirred for 5 min at the same temperature, saturated
aqueous NaHCO3 solution was added and the resulting mixture was
extracted with diethyl ether. The organic layers were combined,
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo to give the crude products. Column chromatography on silica
gel (from 5% to 16% ethyl acetate in hexane containing 3% trie-
thylamine) gave the corresponding TES product (0.573 g, 99%) as
24.0
a colorless oil. [
a
]
ꢁ16.7 (c 1.08, CHCl3); IR (neat, cmꢁ1) 3287,
D
2924, 1649, 1348, 1167, 736; 1H NMR (CDCl3, 400 MHz)
d 8.34–8.37
(m, 2H), 8.00–8.04 (m, 2H), 6.11 (m, 1H), 5.96 (d, J¼7.8 Hz, 1H), 5.62
(dtd, J¼14.9, 6.9, 0.9 Hz, 1H), 5.23 (tdd, J¼15.6, 6.6, 1.4 Hz, 1H), 4.27
(dd, J¼6.0, 3.4 Hz, 1H), 3.82–3.88 (m, 1H), 3.25 (dd, J¼12.6, 3.4 Hz,
1H), 3.11 (dd, J¼12.6, 5.7 Hz, 1H), 2.15 (dt, J¼7.3, 2.7 Hz, 2H), 2.00
(dt, J¼7.1, 7.1 Hz, 2H), 1.61 (tt, J¼7.3, 7.3 Hz, 2H), 1.20–1.33 (m, 48H),
0.94 (t, J¼7.8 Hz, 9H), 0.88 (t, J¼6.6 Hz, 6H), 0.59 (q, J¼7.6 Hz, 6H);
chloroform) gave 32 (0.185 g, 54%, two steps) as a colorless solid.
24.0
[a
]
ꢁ9.3 (c 0.79, CHCl3); IR (KBr disk, cmꢁ1) 3277, 2953, 1713,
D
13C NMR (CDCl3, 100 MHz)
d
173.7, 149.9, 145.9, 134.2, 128.3, 128.2,
1643, 1545, 1466, 1364; 1H NMR (CDCl3, 400 MHz)
d 6.45 (br d,
124.2, 77.2, 75.0, 52.5, 46.1, 43.7, 36.7, 32.1, 31.9, 29.7, 29.6, 29.5,
29.4, 29.3, 29.3, 29.2, 29.0, 25.5, 22.7, 14.1, 6.7, 4.8; ESI-HRMS m/z
calcd for C46H85N3O6SSiNa (MþNa)þ 858.5826, found 858.5805.
To a solution of the TES product obtained above (0.452 g,
0.540 mmol) in DMF (10.8 ml) were added dodecylmercaptane
(1.08 ml, 1.081 mmol) and 60% sodium hydride (0.058 g,
2.430 mmol) at 0 ꢀC. After the reaction mixture was stirred for 1 h at
the same temperature, saturated aqueous NH4Cl solution was added
and the resulting mixture was extracted with diethyl ether. The or-
ganic layers were combined, washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo to give the crude products.
J¼6.4 Hz, 1H), 5.75 (dt, J¼15.3, 6.6 Hz, 1H), 5.47 (dd, J¼15.1, 6.6 Hz,
1H), 4.27 (m, 2H), 4.20 (m, 1H), 3.96 (m, 1H), 3.74 (d, J¼11.8 Hz, 3/
2H), 3.73 (d, J¼11.2 Hz, 3/2H), 3.54 (dt, J¼6.2, 2.3 Hz, 2H), 3.08–3.26
(m, 3H), 2.21 (t, J¼7.5 Hz, 2H), 2.04 (dt, J¼7.1, 7.1 Hz, 2H), 1.63 (tt,
J¼7.3, 7.3 Hz, 2H), 1.26–1.36 (m, 48H), 0.88 (t, J¼6.9 Hz, 6H); 13C
NMR (CDCl3, 100 MHz)
d
174.1, 134.2, 128.6, 73.8, 65.9 (d, JC–P
¼
4.8 Hz, 1/2C), 65.8 (d, JC–P¼5.7 Hz, 1/2C), 54.3 (1/2C), 54.2 (1/2C),
53.55 (d, JC–P¼5.7 Hz, 1/2C), 53.52 (d, JC–P¼5.7 Hz, 1/2C), 41.3, 36.8,
32.4, 31.9, 29.70, 29.66, 29.56, 29.55, 29.45, 29.40, 29.36, 29.31, 29.2,
25.8, 22.7, 14.1; ESI-HRMS m/z calcd for C37H74BrN2O5PNa (MþNa)þ
759.4416, found 759.4415.

11658
T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
3.5.8. 2-Bromoethyl methyl [(3R)-triethylsilyloxy-(2S)-(1-
and concentrated in vacuo to give the crude Weinreb amide 33a as
a colorless solid. To a solution of 33a obtained above in THF (96 ml)
was added a solution of vinylmagnesium bromide in THF, which was
prepared with Mg (2.342 g, 96.34 mmol) and vinyl bromide (14.73 g,
144.5 mmol) in THF (96 ml) under reflux, at room temperature. After
the reaction mixture was stirred for 10 min at the same temperature,
it was poured into 2 N HCl and ice at 0 ꢀC. The resulting mixture was
then extracted with hexane. The organic layers were combined,
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo to give the crude products. Column chromatography on silica
gel (25% ethyl acetate in hexane) gave the roughly purified corre-
oxohexadecylamino)octadec-(4E)-enyl]phosphoramidate (31)
To a solution of 32 (0.262 g, 0.356 mmol) in DMF (5.0 ml) were
added imidazole (0.145 g, 0.288 mmol) and chlorotriethylsilane
(0.14 ml, 0.854 mmol) at room temperature. After the reaction
mixture was stirred for 5 min at the same temperature, saturated
aqueous NaHCO3 solution was added and the resulting mixture was
extracted with diethyl ether. The organic layers were combined,
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo to give the crude products. Column chromatography on silica
gel (from 9% to 50% ethyl acetate in hexane containing 3% trie-
24.0
thylamine) gave 31 (0.288 g, 95%) as a colorless oil. [
a
]
ꢁ9.6 (c
sponding vinyl ketone 33b (9.033 g, 87%, two steps) as a pale yellow
D
24.0
0.8, CHCl3); IR (KBr disk, cmꢁ1) 3278, 2924, 1655, 1460, 1240; 1H
NMR (CDCl3, 400 MHz)
6.22 (dd, J¼8.2, 6.6 Hz, 1H), 5.67 (dtd,
oil. [
a
]
þ4.1 (c 1.19, CHCl3); IR (neat, cmꢁ1) 3376, 2982, 1682, 1510,
D
d
1277, 1163; 1H NMR (CDCl3, 400 MHz)
d
7.28 (m, 5H), 6.44 (dd, J¼17.3,
J¼15.1, 6.6, 0.9 Hz, 1H), 5.42 (dd, J¼15.3, 6.6 Hz, 1H), 4.30–4.34 (m,
1H), 4.22–4.29 (m, 2H), 3.84–3.91 (m, 1H), 3.74 (d, J¼11.0 Hz, 3/2H),
3.72 (d, J¼11.2 Hz, 3/2H), 3.54 (dt, J¼6.2, 3.9 Hz, 2H), 3.21 (m, 1H),
3.00–3.14 (m,1H), 2.17–2.21 (m, 2H), 2.03 (dt, J¼6.9, 6.9 Hz, 2H),1.63
(tt, J¼7.3, 7.3 Hz, 2H), 1.22–1.36 (m, 48H), 0.95 (t, J¼7.8 Hz, 9H), 0.88
(t, J¼6.6 Hz, 6H), 0.58 (q, J¼8.0 Hz, 6H); 13C NMR (CDCl3, 100 MHz)
10.5 Hz, 1H), 6.33 (dd, J¼17.3,1.2 Hz,1H), 5.84 (dd, J¼10.2,1.2 Hz,1H),
5.37–5.46(brm,1H), 4.75(m,1H), 3.73 (s, 2H), 2.89(dd, J¼13.7, 5.6 Hz,
1H), 2.67 (dd, J¼13.9, 6.1 Hz, 1H), 14.6 (s, 9H); 13C NMR (CDCl3,
100 MHz)
d 196.7, 155.0, 137.5, 132.7, 130.2, 128.8, 128.4, 127.0, 79.8,
56.3, 36.4, 32.6, 28.1; ESI-HRMS m/z calcd for C17H23NO3SNa
(MþNa)þ 344.1296, found 344.1295.
d
173.1 (1/2C), 173.1 (1/2C), 132.8, 129.7 (1/2C), 129.6 (1/2C), 77.2,
74.4 (1/2C), 74.2 (1/2C), 65.4 (d, JC–P¼4.8 Hz, 1/2C), 65.3 (d, JC–P
4.8 Hz, 1/2C), 53.2 (d, JC–P¼6.7 Hz, 1/2C), 53.0 (d, JC–P¼5.7 Hz, 1/2C),
¼
3.6.2. 1-Benzylthio-(2S)-(tert-butyloxycarbonylamino)pent-4-en-
(3R)-ol (33c)
52.3 (1/2C), 52.1 (1/2C), 47.8 (d, JC–P¼5.7 Hz, 1/2C), 47.8 (d, JC–P
¼
To a solution of 33b (8.38 g, 26.08 mmol) in ethanol (130 ml)
and THF (30.0 ml) was added lithium tri-tert-butoxyaluminohy-
dride (14.58 g, 57.37 mmol) at ꢁ78 ꢀC. After the reaction mixture
was stirred at the same temperature for 15 min, 2 N HCl was added.
The resulting mixture was then extracted with ethyl acetate. The
organic layers were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (from 9% to 33%
6.7 Hz,1/2C), 36.9, 33.6 (d, JC–P¼3.8 Hz,1/2C), 33.5 (d, JC–P¼3.8 Hz,1/
2C), 32.1, 31.9, 30.0, 29.9, 29.6, 29.5, 29.4, 29.3, 29.1, 29.1, 25.5, 22.7,
14.0, 6.8, 4.9; ESI-HRMS m/z calcd for C44H90BrN2O5PSiNa (MþNa)þ
887.5438, found 887.5431.
3.5.9. SM aza analogue 2
To a solution of 31 (0.147 g, 0.173 mmol) in toluene (3.0 ml) in
a sealed tube was added trimethylamine (2.5 ml) at ꢁ78 ꢀC. After
the reaction mixture was stirred for 48 h at 60 ꢀC, trimethylamine
was removed in vacuo. To a solution of the residue in methanol
(2.0 ml) was added Dowex 50W (pH¼4) at room temperature. After
the reaction mixture was stirred for 5 min at the same temperature,
methanol was added to the reaction mixture, Dowex 50W was
removed by filtration, and then the filtrate was concentrated in
vacuo to give the crude products. Column chromatography on silica
ethyl acetate in hexane) gave 33c (8.28 g, 98%) as a colorless solid.
24.0
[a
]
ꢁ44.0 (c 1.20, CHCl3); IR (KBr disk, cmꢁ1) 3351, 2982, 1686,
D
1528, 1292, 1173, 1013; 1H NMR (CDCl3, 400 MHz)
d 7.28 (m, 5H),
5.79 (ddd, J¼17.3, 10.5, 5.6 Hz, 1H), 5.30 (ddd, J¼17.3, 1.5, 1.5 Hz, 1H),
5.20 (ddd, J¼10.5, 1.5, 1.5 Hz,1H), 4.77–4.90 (br m, 1H), 4.27 (m, 1H),
3.82 (m, 1H), 3.73 (s, 2H), 2.75–2.83 (br m, 1H), 2.63 (dd, J¼13.9,
4.9 Hz, 1H), 2.54 (dd, J¼13.9, 7.8 Hz, 1H), 1.46 (s, 9H); 13C NMR
(CDCl3, 100 MHz) 156.3, 137.9, 136.6, 128.9, 128.5, 127.1, 116.9, 79.9,
74.5, 54.0, 36.4, 31.6, 28.3; ESI-HRMS m/z calcd for C17H25NO3SNa
(MþNa)þ 346.1453, found 346.1450.
gel (from 9% methanol in chloroform to 3% water and 27% methanol
24.0
in chloroform) gave 2 (0.037 g, 30%) as a colorless foam. [
a]
ꢁ1.5
D
(c 0.8, CHCl3); IR (KBr disk, cmꢁ1) 3387, 2919, 1655, 1200, 1053, 968;
1H NMR (CD3OD, 400 MHz)
d
5.68 (td, J¼15.1, 6.6 Hz, 1H), 5.44 (dd,
3.6.3. (3R)-Hydroxy-(2R)-(tert-butyloxycarbonylamino)pent-4-
ene-1-thiol (33d)
J¼15.3, 7.3 Hz, 1H), 4.17 (m, 2H), 4.02 (dd, J¼7.6, 7.6 Hz, 1H), 3.74–
3.79 (m, 2H), 3.60 (m, 2H), 3.22 (s, 9H), 3.08 (m, 2H), 2.19 (t,
J¼6.9 Hz, 2H), 2.02 (dt, J¼6.9, 6.9 Hz, 2H), 1.58 (m, 2H), 1.29 (s, 48H),
To a solution of lithium (0.63 g, 90.4 mmol) in liquid ammonia
(338.9 ml) was added a solution of 33c (7.31 g, 22.59 mmol) in THF
(113 ml) at ꢁ78 ꢀC. After the reaction mixture was stirred for 3 h at
reflux, ammoniumchloridewasaddedatꢁ78 ꢀC. Afterammoniawas
removed at room temperature, water was added and the resulting
mixture was extracted with ethyl acetate. The organic layers were
combined, washed with brine, dried over MgSO4, filtered, and con-
centrated in vacuo to give the crude products. Column chromatog-
0.90 (t, J¼7.1 Hz, 3H); 13C NMR (CD3OD, 100 MHz)
d 176.2, 134.5,
131.2, 73.8, 67.7 (m), 59.5 (d, JC–P¼3.8 Hz), 56.6 (d, JC–P¼4.8 Hz),
54.75, 54.71, 54.67, 43.1, 37.6, 33.5, 33.1, 30.9, 30.74, 30.66, 30.53,
30.49, 27.2, 23.8, 14.5; ESI-HRMS m/z calcd for C39H80N3O5PNa
(MþNa)þ 724.5733, found 724.5732.
3.6. Synthesis of SM sulfur analogue 3
raphy on silica gel (from 9% to 33% ethyl acetate in hexane) gave
23.5
33d (5.22 g, 99%) as a colorless solid. [
a
]
ꢁ3.2 (c 1.05, CHCl3); IR
D
3.6.1. Benzyl (2S)-(tert-butyloxycarbonylamino)-3-oxopent-4-eny
sulfide (33b)
(neat, cmꢁ1) 3358, 2980, 1688, 1530, 1171; 1H NMR (CDCl3,
400 MHz)
d
5.90 (ddd, J¼17.3, 10.5, 5.6 Hz, 1H), 5.37 (ddd, J¼17.3,
To a solution of N-Boc-S-Bn-L-cystein 33 (10.00 g, 32.11) in
1.5, 1.5 Hz, 1H), 5.27 (ddd, J¼10.5, 1.5, 1.5 Hz, 1H), 4.88–4.99 (br m,
1H), 4.31 (m, 1H), 3.88 (m, 1H), 2.77 (dd, J¼8.5, 6.6 Hz, 1H), 2.75
(dd, J¼8.8, 4.7 Hz, 3/2H), 1.46 (s, 9H); 13C NMR (CDCl3, 100 MHz)
dichloromethane (64 ml) were added N-methylmorpholine (3.88 ml,
35.3 mmol) and N,O-dimethylhydroxylamine hydrochloride (3.759 g,
38.54 mmol), at ꢁ15 ꢀC. To the mixture was then added 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride (7.387 g,
38.54 mmol) over 30 min at the same temperature. After the
reaction mixture was stirred at the same temperature for 1 h, ice and
1 N HCl were added and the resulting mixture was extracted with
chloroform. The organic layers were combined, washed with satu-
rated aqueous NaHCO3 solution and brine, dried over MgSO4, filtered,
d
156.0, 137.0, 117.1, 79.9, 73.9, 56.0, 28.3, 25.2; ESI-HRMS m/z calcd
for C10H19NO3SNa (MþNa)þ 256.0983, found 256.0972.
3.6.4. (3R)-(tert-Butyldimethylsilyloxy)-(2R)-(tert-
butyloxycarbonylamino)pent-4-ene-1-thiol (34)
To a solution of 33d (1.92 g, 8.23 mmol) in DMF (41 ml) were
added triethylamine (2.87 ml, 20.57 mmol), DMAP (1.005 g,

T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
11659
8.229 mmol), and TBSCl (3.101 g, 20.57 mmol) at 0 ꢀC. After the
reaction mixture was stirred for 14 h at room temperature, ice was
added and the resulting mixture was extracted with hexane. The
organic layers were combined, washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo to give the crude O,S-
silylated product 33e. To a solution of crude 33e in THF (20.6 ml)
was added 1.0 M TBAF in THF solution (7.8 ml) at ꢁ78 ꢀC. After the
reaction mixture was stirred for 10 min at the same temperature,
saturated aqueous NH4Cl solution was added and the resulting
mixture was extracted with ethyl acetate. The organic layers were
combined, washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo to give the crude 34 containing with small
amount of disulfide 34. To a solution of crude 34 in CH3CN (41 ml)
and H2O (8.2 ml) was added tributylphosphine (2.06 ml,
8.23 mmol) at room temperature. After the reaction mixture was
stirred for 1 h at the same temperature, saturated aqueous NH4Cl
solution was added and the resulting mixture was extracted with
ethyl acetate. The organic layers were combined, washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo to give
the crude 34. Column chromatography on silica gel (from 1% to 9%
vacuo to give the crude products. Column chromatography on silica
gel (from 9% to 50% ethyl acetate in hexane) gave 36 (0.213 g, 83%)
26.0
as a colorless oil. [
a]
þ12.2 (c 0.93, CHCl3); IR (neat, cmꢁ1) 3403,
D
2978, 1701, 1524, 1248, 1171, 1017; 1H NMR (CDCl3, 400 MHz)
d 5.89
(ddd, J¼16.9, 10.6, 5.7 Hz, 1H), 5.38 (d, J¼17.2 Hz, 1H), 5.27 (d,
J¼10.6 Hz, 1H), 5.15–5.23 (br d, J¼8.2 Hz, 1H), 4.31–4.44 (m, 2H),
4.13 (m, 1H), 3.87 (m, 1H), 3.85 (d, J¼12.6 Hz, 3/2H), 3.83 (d,
J¼12.6 Hz, 3/2H), 3.563 (t, J¼6.1 Hz, 2/2H), 3.559 (t, J¼6.1 Hz, 2/
2H),3.04–3.21 (m, 2H), 1.451 (s, 9/2H), 1.447 (s, 9/2H); 13C NMR
(CDCl3, 100 MHz)
d 155.9, 136.6, 117.3, 80.00 (1/2C), 79.96 (1/2C),
74.0, 66.6 (m), 55.4, 54.4 (d, JC–P¼6.7 Hz), 31.2 (m), 29.2 (1/2C), 29.1
(1/2C), 28.4; ESI-HRMS m/z calcd for C13H25BrNO6PSNa (MþNa)þ
456.0221, found 456.0213.
3.6.7. O-(2-Bromoethyl) S-[(3R)-hydroxy-(2R)-(tert-butyloxy-
carbonylamino)octadec-(4E)-enyl] O-methyl thiophosphate (37)
To a solution of 36 (0.423 g, 0.974 mmol) in dichloromethane
(9.7 ml) were added 1-pentadecene (1.06 ml, 3.90 mmol) and
second generation Grubbs catalyst (3 mg, 0.003 mmol). After the
reaction mixture was stirred for 2 h under reflux, it was concen-
trated in vacuo to give the crude products. Column chromatog-
ethyl acetate in hexane) gave 34 (2.241 g, 78%, three steps) as
23.5
a colorless oil. [
a]
ꢁ26.2 (c 0.92, CHCl3); IR (neat, cmꢁ1) 3366,
raphy on silica gel (from 17% to 33% ethyl acetate in hexane) gave
D
2957, 1716, 1496, 1253, 1171; 1H NMR (CDCl3, 400 MHz)
d
5.80 (ddd,
37 (0.450 g, 74%) as a colorless oil. [
a]
þ2.0 (c 0.75, CHCl3); IR
27.0
D
J¼17.2, 10.3, 5.7 Hz, 1H), 5.28 (ddd, J¼17.2, 1.4, 1.4 Hz, 1H), 5.19 (ddd,
J¼10.3, 1.4, 1.4 Hz, 1H), 4.78 (br d, J¼8.2 Hz, 1H), 4.32 (m, 1H), 3.69
(m, 1H), 2.75 (ddd, J¼14.0, 7.1, 7.1 Hz, 1H), 2.67 (ddd, J¼14.0, 9.4,
4.6 Hz, 1H), 1.45 (s, 9H), 0.90 (s, 9H), 0.07 (s, 3H), 0.03 (s, 3H); 13C
(neat, cmꢁ1) 3289, 2919, 2851, 1647, 1541, 1252, 1013; 1H NMR
(CDCl3, 400 MHz)
d
5.76 (dt, J¼15.3, 6.7 Hz, 1H), 5.47 (dd, J¼15.3,
6.9 Hz, 1H), 5.13 (br m, J¼1H), 4.31–4.44 (m, 2H), 4.16 (m, 1H), 3.85
(d, J¼12.6 Hz, 3/2H), 3.83 (d, J¼13.0 Hz, 3/2H), 3.561 (t, J¼6.4 Hz,
2/2H), 3.557 (t, J¼6.4 Hz, 2/2H), 2.98–3.21 (m, 3H), 2.04 (dt, J¼6.9,
6.9 Hz, 2H), 1.447 (s, 9/2H), 1.442 (s, 9/2H), 1.37 (m, 2H), 1.26–1.32
(m, 20H), 0.88 (t, J¼6.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
NMR (CDCl3, 100 MHz)
d 155.5, 137.9, 116.5, 79.5, 74.6, 56.3, 28.4,
25.8, 24.6, 18.1, ꢁ4.4, ꢁ5.0; ESI-HRMS m/z calcd for C16H33NO3SSiNa
(MþNa)þ 370.1848, found 370.1844.
d
155.9, 135.1, 128.1, 79.9, 73.9 (m), 66.59 (d, JC–P¼4.8 Hz, 1/2C),
3.6.5. O-(2-Bromoethyl) S-[(3R)-(tert-butyldimethylsilyloxy)-
(2R)-(tert-butyloxycarbonylamino)pent-4-enyl] O-methyl
thiophosphate (35)
66.54 (d, JC–P¼5.7 Hz, 1/2C), 55.6, 54.4 (d, JC–P¼6.7 Hz), 32.4, 31.9,
31.5 (m), 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 28.4, 22.7, 14.1; ESI-
HRMS m/z calcd for C26H51BrNO6PSNa (MþNa)þ 638.2256, found
638.2253.
To a mixture of 4 Å molecular sieves (0.300 g) and carbon tet-
rabromide (0.612 g, 1.844 mmol) in dichloromethane (3.0 ml) was
added 2-bromoethyl dimethyl phosphite 30 (0.28 ml, 1.844 mmol)
at 0 ꢀC. After the reaction mixture was stirred for 20 min, a solution
of 34 (0.103 g, 0.296 mmol) in dichloromethane (0.58 ml) and 2,6-
lutidine (0.007 ml, 0.059 mmol) were added to the reaction mix-
ture at the same temperature. After the reaction mixture was
stirred for 2 h at the same temperature, the solvent was removed in
vacuo to give the crude products. Column chromatography on silica
3.6.8. O-(2-Bromoethyl) S-[(3R)-hydroxy-(2R)-(1-oxohexa-
decylamino)octadec-(4E)-enyl] O-methyl thiophosphate (38)
To a solution of 37 (73 mg, 0.118 mmol) in dichloromethane
(0.59 ml) was added TFA (0.24 ml) at 0 ꢀC. After the reaction mix-
ture was stirred for 2.5 h at the same temperature, chloroform and
saturated K2CO3 solution were added until the mixture turned
basic. To this mixture was added palmitoyl chloride (0.043 ml,
0.142 mmol) at 0 ꢀC, and the resulting mixture was stirred for
25 min. Saturated aqueous NH4Cl solution was added and the
mixture was extracted with chloroform. The organic layers were
combined, washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo to give the crude products. Column chro-
gel (from 5% to 17% ethyl acetate in hexane) gave 35 (96 mg, 61%)
26.0
as a colorless oil. [
a
]
D
ꢁ9.7 (c 0.90, CHCl3); IR (neat, cmꢁ1) 3327,
2957, 2857, 1711, 1522, 1254, 1017; 1H NMR (CDCl3, 400 MHz)
d
5.79 (ddd, J¼16.9, 10.6, 5.2 Hz, 1H), 5.30 (d, J¼17.2 Hz, 1H), 5.20
(d, J¼10.5 Hz, 1H), 5.17–5.09 (br m, 1H), 4.27–4.43 (m, 3H), 3.82 (d,
J¼12.6 Hz, 3/2H), 3.80 (d, J¼12.6 Hz, 3/2H), 3.76 (m, 1H), 3.54 (t,
J¼6.5 Hz, 2/2H), 3.52 (d, J¼5.5 Hz, 2/2H), 3.071 (ddd, J¼17.2, 13.7,
10.1 Hz, 1/2H), 3.065 (m, 1/2H), 2.935 (ddd, J¼16.2, 13.7, 10.1 Hz, 1/
2H), 2.935 (ddd, J¼17.2, 13.7, 10.1 Hz, 1/2H), 1.44 (s, 9/2H), 1.43 (s,
9/2H), 0.90 (s, 9H), 0.05 (s, 3H), 0.01 (s, 3H); 13C NMR (CDCl3,
matography on silica gel (from 25% to 50% ethyl acetate in hexane)
27.0
gave 38 (82 mg, 92%) as a colorless solid. [
a
]
D
þ1.3 (c 0.76, CHCl3);
IR (KBr disk, cmꢁ1) 3405, 2926, 2855, 1711, 1524, 1250, 1013; 1H
NMR (CDCl3, 400 MHz)
d
6.46 (t, J¼6.9 Hz, 1H), 5.75 (dt, J¼15.3,
6.6 Hz, 1H), 5.46 (dd, J¼15.3, 6.6 Hz, 1H), 4.29–4.42 (m, 2H), 4.12–
4.18 (m, 2H), 3.83 (d, J¼12.8 Hz, 3H), 3.553 (t, J¼6.2 Hz, 2/2H), 3.545
(t, J¼6.2 Hz, 2/2H), 3.46 (br d, J¼15.6 Hz, 1H), 3.03–3.19 (m, 2H),
2.22 (t, J¼7.3 Hz, 2H), 2.04 (dt, J¼7.1, 7.1 Hz, 2H), 1.63 (m, 2H), 1.26–
1.36 (m, 46H), 0.88 (t, J¼6.6 Hz, 6H); 13C NMR (CDCl3, 100 MHz)
100 MHz)
d 155.3, 137.4, 116.6, 79.4, 74.7 (1/2C), 74.6 (1/2C), 66.2
(d, JC–P¼4.5 Hz), 54.0 (d, JC–P¼5.4 Hz), 44.8, 30.3 (1/2C), 30.1 (1/
2C), 28.2, 25.7, 20.1 (m), 18.0, ꢁ4.6, ꢁ5.2; ESI-HRMS m/z calcd for
C19H39BrNO6PSSiNa (MþNa)þ 570.1086, found 570.1086.
d
174.4 (1/2C),174.3 (1/2C),135.0,127.9, 74.3 (1/2C), 74.2 (1/2C), 66.8
3.6.6. O-(2-Bromoethyl) S-[(3R)-hydroxy-(2R)-(tert-butyloxy-
carbonylamino)pent-4-enyl] O-methyl thiophosphate (36)
(d, JC–P¼4.5 Hz), 55.1 (m), 54.6 (d, JC–P¼6.7 Hz), 36.69 (1/2C), 36.67
(1/2C), 32.4, 31.9, 30.7 (m), 29.71, 29.67, 29.5, 29.41, 29.36, 29.28,
29.17, 25.7, 22.7, 14.1; ESI-HRMS m/z calcd for C37H73BrNO5PSNa
(MþNa)þ 776.4028, found 776.4033.
To a solution of 35 (0.326 g, 0.594 mmol) in methanol (5.9 ml)
was added 2 N HCl (1.2 ml) at room temperature. After the reaction
mixture was stirred for 4 h at the same temperature, saturated
aqueous NaHCO3 solution was added and the resulting mixture was
extracted with ethyl acetate. The organic layers were combined,
washed with brine, dried over MgSO4, filtered, and concentrated in
3.6.9. SM sulfur analogue 3
To a solution of 38 (48 mg, 0.064 mmol) in methanol (1.3 ml)
was added trimethylamine (0.64 ml) at room temperature. After

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T. Yamamoto et al. / Tetrahedron 64 (2008) 11647–11660
4. (a) Talbott, C. M.; Yappert, M. C. Biochim. Biophys. Acta 2000, 1467, 326; (b)
Niemela¨, P.; Hyvo¨nen, M. T.; Vattulainen, I. Biophys. J. 2004, 87, 2976; (c) Ait-
toniemi, J.; Niemela¨, P. S.; Hyvo¨nen, M. T.; Karttunen, M.; Vattulainen, I. Biophys.
J. 2007, 92, 1125.
the reaction mixture was stirred for 1 day at the same temperature,
the solvent was filtered and removed in vacuo to give the crude
products. Column chromatography on silica gel (from 9% methanol
5. (a) Huang, J.; Buboltz, J. T.; Feigenson, G. W. Biochim. Biophys. Acta 1999, 1417,
89; (b) Terova, B.; Heczko, R.; Slotte, J. P. Biophys. J. 2005, 88, 2661.
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S. J. Org. Chem. 2002, 67, 4839; (b) Hakogi, T.; Taichi, M.; Katsumura, S. Org. Lett.
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Med. Chem. Lett. 2005, 15, 2141; (d) Hakogi, T.; Yamamoto, T.; Fujii, S.; Ikeda, K.;
Katsumura, S. Tetrahedron Lett. 2006, 47, 2627.
7. (a) Grubbs, R. H. Handbook of Metathesis; Wiley-VCH: Germany, 2003; (b)
Connon, S.; Blechert, S. Angew. Chem., Int. Ed. 2003, 42, 1900 and references
cited therein.
8. On the synthesis of the functionalized sphingolipids including natural ones: (a)
Rai, A. N.; Basu, A. Org. Lett. 2004, 6, 2861; (b) Torssell, S.; Somfai, P. Org. Biomol.
Chem. 2004, 2, 1643; (c) Singh, O. V.; Kampf, D. J.; Han, H. Tetrahedron Lett.
2004, 45, 7239; (d) Nussbaumer, P.; Ettmayer, P.; Peters, C.; Rosenbeiger, D.;
Hoegenauer, K. Chem. Commun. 2005, 5086; (e) Chaudhari, V. D.; Kumar, K. S.
A.; Dhavale, D. D. Org. Lett. 2005, 7, 5805; (f) Dougherty, A. M.; McDonald, F. E.;
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baumer, P. J. Org. Chem. 2007, 72, 1842.
in chloroform to 4% water and 27% methanol in chloroform) gave 3
27.0
(23 mg, 50%) as a colorless foam. [
a
]
ꢁ8.3 (c 0.42, CH3OH); IR
D
(KBr disk, cmꢁ1) 3887, 2920, 1638, 1468, 1233, 1074; 1H NMR
(CD3OD, 400 MHz)
d
5.69 (dt, J¼15.3, 6.9 Hz, 1H), 5.44 (dd, J¼15.3,
6.9 Hz, 1H), 4.28 (m, 2H), 3.98 (m, 2H), 3.67 (m, 2H), 3.23 (s, 9H),
3.15 (m, 1H), 2.83 (ddd, J¼14.6, 13.7, 8.5 Hz, 1H), 2.19 (t, J¼7.1 Hz,
2H), 2.02 (dt, J¼7.1, 6.8 Hz, 2H), 1.59 (m, 2H), 1.28–1.36 (m, 46H),
0.90 (t, J¼6.6 Hz, 6H); 13C NMR (CD3OD, 100 MHz)
d 176.2, 134.9,
130.9, 75.2, 67.5 (m), 60.5 (d, JC–P¼5.7 Hz), 56.1 (m), 54.78, 54.74,
54.71, 37.4, 33.5, 33.1, 32.9, 30.9, 30.8, 30.7, 30.6, 30.52, 30.48, 27.3,
23.8, 14.5; ESI-HRMS m/z calcd for C39H79N2O5PSNa (MþNa)þ
741.5345, found 741.5333.
Acknowledgements
9. Hasegawa, H.; Yamamoto, T.; Hatano, S.; Hakogi, T.; Katsumura, S. Chem. Lett.
2004, 33, 1592.
10. (a) Yamamoto, T.; Hasegawa, H.; Hakogi, T.; Katsumura, S. Org. Lett. 2006, 8,
5569; (b) Yamamoto, T.; Hasegawa, H.; Hakogi, T.; Katsumura, S. Chem. Lett.
2008, 37, 188.
11. Ishizuka, T.; Kunieda, T. Tetrahedron Lett. 1987, 28, 4185.
12. (a) McKenna, C. E.; Schmidhauser, J. J. Chem. Soc., Chem. Commun. 1979, 739; (b)
Pergament, I.; Srebnik, M. Tetrahedron Lett. 1999, 40, 3895.
13. Thomas, B. N.; Corcoran, R. C.; Cotant, C. L.; Lindemann, C. M.; Kirsch, J. E.;
Persichini, P. J. J. Am. Chem. Soc. 1998, 120, 12178.
We thank Prof. J. Peter Slotte and Dr. Anders Bjo¨rkbom of åbo
Academi University for the experiments on membrane properties
of the synthetic SM analogues. This work was supported by a Grant-
in-Aid for Scientific Research on Priority Areas 16073222 from the
Ministry of Education, Culture, Sports, Science and Technology, Ja-
pan. This work was also supported by Maching Fund Subsidy for
a Private University.
14. Timperley, C. M.; Bird, M.; Holden, I.; Black, R. M. J. Chem. Soc., Perkin Trans. 1
2001, 26.
Supplementary data
15. Wen, X.; Zhang, P.; Liu, J.; Zhang, L.; Wu, X.; Ni, P.; Sun, H. Bioorg. Med. Chem.
Lett. 2006, 16, 722.
16. A small amount of the by-product 160 was detected.
1H and 13C NMR spectra of compounds 1–4. Supplementary data
associated with this article can be found in the online version, at
doi:10.1016/j.tet.2008.10.018.
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References and notes
P
O
OMe
N
O
PMB
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O
16'
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