Synthesis and biological activity of hydroxylated analogs of RCAI-80

Article abstract of DOI:10.1016/j.tet.2013.09.029

RCAI-80 is one of the ester analogs of KRN7000 (α-galactosylceramide) . This compound released mainly T helper 2 (Th2) cytokines, such as IL-4 rather than T helper 1 (Th1) cytokines, such as IFNγ from the invariant natural killer T (iNKT) cells. In addition, it has been known that some of the hydroxylated derivatives of KRN7000 make the cytokine secretion bias to Th2 by decreasing the IFNγ production to almost zero. This time, the three compounds having these two characteristic properties, namely an ester group and also some extra hydroxy groups existing on the ester side chain and/or on the 2-acyloxy-3,4-dihydroxyoctadecyl main chain of RCAI-80, were synthesized to examine the biological activities. As a result, it was found that these compounds made the cytokine secretion skew to Th2. Therefore, their effectiveness for experimental autoimmune encephalomyelitis (EAE) was examined. It was recognized that one of them showed moderate suppression of EAE symptom.

Full text of DOI:10.1016/j.tet.2013.09.029

Tetrahedron 69 (2013) 9710e9725
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and biological activity of hydroxylated analogs of RCAI-80
,
a
b
c
Masao Shiozaki a , Takuya Tashiro , Hiroyuki Koshino , Tomokuni Shigeura ,
*
Hiroshi Watarai c, Masaru Taniguchi c, Kenji Mori a
a Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
b Advanced Science Institute, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
c Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN, Yokohama Institute, Suehiro-cho 1-7-22, Tsurumi-ku,
Yokohama-shi, Kanagawa 230-0045, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2013
Received in revised form 4 September 2013
Accepted 5 September 2013
Available online 18 September 2013
RCAI-80 is one of the ester analogs of KRN7000 (
T helper 2 (Th2) cytokines, such as IL-4 rather than T helper 1 (Th1) cytokines, such as IFN
a
-galactosylceramide). This compound released mainly
from the
invariant natural killer T (iNKT) cells. In addition, it has been known that some of the hydroxylated
derivatives of KRN7000 make the cytokine secretion bias to Th2 by decreasing the IFN production to
g
g
almost zero. This time, the three compounds having these two characteristic properties, namely an ester
group and also some extra hydroxy groups existing on the ester side chain and/or on the 2-acyloxy-3,4-
dihydroxyoctadecyl main chain of RCAI-80, were synthesized to examine the biological activities. As
a result, it was found that these compounds made the cytokine secretion skew to Th2. Therefore, their
effectiveness for experimental autoimmune encephalomyelitis (EAE) was examined. It was recognized
that one of them showed moderate suppression of EAE symptom.
Keywords:
Glycolipids
Glycosylation
Ester derivatives of the
galactosylceramides
iNKT cells
a-
Ó 2013 Elsevier Ltd. All rights reserved.
Th1/Th2
EAE
1. Introduction1
the aglycon, and it is known that OCH induces strongly a Th2 bias.
By analogy from the hydrophilicity of OCH relatively increasing
Agelasphins are a series of glycosphingolipids isolated from the
compare to KRN7000, the more hydroxylated analogs of KRN7000
should become hydrophilic and may induce the secretion of Th2
cytokines from iNKT cells. Therefore, RCAI-147 and 160, hydroxyl-
ated analogs of KRN7000, had been synthesized and showed good
suppression of experimental autoimmune encephalomyelitis
(EAE)7,8 symptoms as reported in the previous paper.9 And these
marine sponge (Agelas mauritianus), and they show potent effects
on the immune system of mice.2 KRN7000,3 one of the
a-gal-
actosylceramides, was developed by Kirin Brewery Co. in the pro-
cess of synthetic studies on the agelasphins. Invariant natural killer
T (iNKT) cells4 produce immunoregulatory cytokines, such as both
IFNg
[T helper 1 (Th1) type] and IL-4 [T helper 2 (Th2) type] by the
compounds were found to inhibit mostly the IFNg production, and
recognition of glycolipid antigens, such as KRN7000 bound by
a major histocompatibility complex (MHC) class I-like protein, i.e.,
CD1d,5 which is an antigen presenting protein for the Tcell receptor
of iNKT cells. The glycolipids stimulating the secretion of mainly
Th1 type cytokines or of mainly Th2 type cytokines are expected to
be promising anticancer drugs or immunosuppressive agents and
autoimmune disease treatment medicine, respectively.
their IL-4 production ability still remained approximately more
than 55% of KRN7000. Accordingly, it is anticipated that the hybrid
compounds, having the mixed properties of the ester compound
RCAI-80 and the hydroxylated compounds RCAI-147/-160, behave
as inducing the secretion of Th2 cytokines from iNKT cells (Fig. 1).
However, there is no specific evidence that hydrophobicity/hy-
drophilicity is key. It may be just binding/recognition (and not
hydrophilicity per se) that effects biological activity.
RCAI-80,6 an ester analog of KRN7000, was found to induce
more selectively IL-4 than IFN
g
compared to KRN7000. Besides this,
For whatever reason, this time, three relevant compounds (13,
22, and 38) of RCAI-80’s hydroxylated analogs were synthesized to
evaluate their biological activities, because we were interested in
the biological activities (EAE) of hydroxylated analogs of KRN7000.9
(Schemes 1e6) (see Fig. 2).
the compound OCH, reported by Miyamoto et al.,7 has two trun-
cated chains both on the main chain and on the amide side chain of
At last, in the previous paper6 for the synthesis of RCAI-80, we
had to use the acidic condition (aqueous HF) to remove the
* Corresponding author. E-mail addresses: shiozaki@rcai.riken.jp, michael1621@
nifty.com (M. Shiozaki).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.09.029

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9711
OH
OH
O
OH
O
O
n-C22H45
HO
HO
HO
HO
HO
HO
n-C25H51
n-C25H51
O
O
O
OH
HN
O
HN
OH
OH
OH
HO
HO
HO
O
O
O
(CH2)11CH(CH3)2
n-C14H29
n-C14H29
OH
OH
OH
KRN7000
HO
Agelasphin-9b
RCAI-80
HO
O
HO
OH
OH
HO
OH
HO
n-C23H47
OH
n-C22H25
O
HO
HO
n-C23H47
OH
n-C5H11
O
O
O
O
OH
NH OH
HN
HO
HO
NH OH OH
HO
HO
HO
O
O
O
n-C12H25
n-C14H29
OH OH
RCAI-147
Fig. 1. Structures of agelasphin-9b, RCAI-80, KRN7000, OCH, RCAI-147, and RCAI-160.
OH
OH
OCH
RCAI-160
O
O
O
MeO
HO
OBn
a
OTBDMS
b
HO
OTBDMS
ref. 10
L-ribose
+
OBn
OBn
OBn
BnO
BnO
BnO
2'
2
1
OH OBn
TBDMSO
HO
OBn
OH OBn
c
d
TBDMSO
HO
n-C12H25
n-C12H25
n-C12H25
+
OBn
OBn
3'
OBn
3
4
OH
OBn
HO
n-C14H29
OBn
5
Scheme 1. Reagents and conditions: (a) (1) Ac2O, cat. H2SO4, 6e10 ꢀC, 1 h; (2) MeOH, cat. KOH, rt, 2 h, two steps 53%; (3) TBDMS-Cl, imidazole, CH2Cl2, 15e20 ꢀC, 2 h, 84% (three
steps 45%); (b) n-C13H27PPh3Br, n-BuLi, THF, ꢁ10 ꢀC, 30 min, then 2 and 20, ꢁ10 ꢀC, 10 min, and rt 16 h, 3 (54%), a mixture of 3 and 30 (6%), and 30 (4%); (c) TBAF, THF, rt, 30 min, 95%;
(d) H2, 10% Pd/C, hexane, rt, 25 min, 82%.
protecting groups. This condition caused to yield the acyl migrated
compound from C2eO to C4eO positions as a side reaction.
Therefore, an alternative route was attempted to prevent this mi-
gration and to improve the overall yield of RCAI-80 synthesis.
Esterification of alcohol 7 with carboxylic acid 89 using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC)
as a dehydrating agent in THFeCH2Cl2 (1:1) gave 9 (79%). Reaction of
9
with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in
dichloromethaneewater (CH2Cl2eH2O¼10:1) gave 10. Treatment of
10 with HF$pyridine in THFepyridine gave 11. The isopropylidene
group of 11 was deprotected with aqueous HF in CH2Cl2eMeCN (1:1)
to give 12. Two benzyl groups of 12 were hydrogenolyzed using
Pd(OH)2 on carbon [Pd(OH)2/C] in THF to give 13 (RCAI-166). The 2D
COSY analysis of 13 showed that the (20,30-dihydroxy)hexacosanoyl
group stayed on the C2eO position in the octadecyl chain. However,
silica gel column chromatography of 13 by elution with
CHCl3eMeOH (9:1) yielded gradually a lower Rf product, which may
be C4eO acyl migrated compound6 (Scheme 2).
2. Results and discussion
2.1. Synthesis
Firstly, compound 13 (RCAI-166), having two hydroxy groups on
the C20- and C30-positions in the hexacosanoyl ester side chain, was
synthesized via methyl 2,3,5-tri-O-benzyl-
b-L-ribofuranoside (1)
obtained easily from
L
-ribose.10 Reaction of 1 with Ac2O containing
a catalytic amount of concd H2SO4 gave a diacetyl intermediate,
whichwasfurtherconverted toa mixtureofdiols inMeOHcontaining
a catalytic amount of KOH, and then a mixture of silyl ethers 2 and 20
(84%) with tert-butyldimethylsilyl chloride (TBDMS-Cl) using imid-
azole as a base. Wittig reaction of this mixture (2 and 20) with the
phosphorane derived from n-tridecyltriphenylphosphonium bro-
mide and n-BuLi gave a mixture of 3 and 30 (64%), which was con-
verted to 4 (E,Z-mixture, 95%) by tetra-n-butylammonium fluoride
(TBAF) treatment. The E,Z-double bonds of 4 were reduced to 5 (82%)
by treatment with H2 at room temperature for 30 min using Pd on
carbon (Pd/C) as a catalyst (Scheme 1).
Secondly, compound 22, having three hydroxy groups on the
C20-, C30- and C40-positions in the hexacosanoyl ester side chain,
was synthesized via compound 7 and carboxylic acid 18, which was
obtained from compound 2. Wittig reaction of a mixture of 2 and 20
with
the
phosphorane
derived
from
n-henicosyl-
triphenylphosphonium bromide (n-C21H43PPh3Br) and n-BuLi gave
14 (E,Z-mixture). The double bond of 14 was reduced to 15 by
treatment with H2 at room temperature for 30 min using Pd/C as
a catalyst. The alcohol of 15 was benzylated with benzyl bromide
using NaH as a base to give 16, which was converted to 17 by
treatment with TBAF. The primary alcohol of 17 was oxidized to
carboxylic acid 18 by using Jones reagent (Scheme 3).
The diol 5 was treated with the imidate 66 using silver tri-
fluoromethanesulfonate (AgOTf) to yield
a-anomer 7 (73%).

9712
M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
t-Bu
Si
t-Bu
Si
t-Bu
Si
t-Bu
t-Bu
t-Bu
O
O
O
O
O
O
O
O
c
O
a
n-C23H47
OBn
NH
CCl3
O
O
O
b
PMBO
PMBO
PMBO
OH
O
OBn
O
PMBO
PMBO
PMBO
O
O
n-C14H29
n-C14H29
OBn
OBn
6
7
9
t-Bu
Si
O
t-Bu
O
O
O
O
HO
O
n-C23H47
O
OH
HO
OH
HO
O
O
O
n-C23H47
OBn
n-C23H47
OBn
OH
O
O
O
HO
HO
HO
d
e
O
O
OBn
HO
HO
HO
O
O
O
n-C14H29
n-C14H29
n-C14H29
OBn
OBn
OBn
12
10
11
HO
n-C23H47
OH
HO
n-C23H47
HOOC
O
OH
O
f
HO
O
OH
O
O
HO
O
n-C14H29
OH
8
13 (RCAI-166)
Scheme 2. Reagents and conditions: (a) 5, MS 4 A, AgOTf, CH2Cl2, 0 ꢀC, 2 h, then rt 1 h, 73%; (b) 8, DMAP, EDAC, THFeCH2Cl2 (1:1), rt, 5 h, 79%; (c) DDQ, CH2Cl2eH2O (10:1), rt, 3 h,
57%; (d) HFepyridine, pyridine, THF, rt, 1.5 h; (e) aq 46% HF, CH2Cl2eMeCN (1:1), rt, 1.5 h, two steps 72%; (f) H2, Pd(OH)2/C, THF, rt, 5 h, 80%.
ꢀ
O
OH
OBn
HO
OH
OBn
n-C22H45
OTBDMS
a
b
c
TBDMSO
TBDMSO
n-C20H41
OBn
BnO
OBn
OBn
14
15
2
OBn
OBn
n-C22H45
OBn
OBn
OBn
OBn
n-C22H45
e
d
TBDMSO
HO
HOOC
n-C22H45
OBn
OBn
OBn
18
16
17
Scheme 3. Reagents and conditions: (a) n-C21H43PPh3Br, n-BuLi, THF, ꢁ10 ꢀC, 30 min, then 2, THF, ꢁ10 ꢀC, 20 min, then rt, 3 h, 53%; (b) H2, 10% Pd/C, hexane, rt, 25 min, 90%; (c)
BnBr, NaH, DMF, 5 ꢀC, 16 h; (d) TBAF, THF, rt, 2 h, two steps 77%; (e) Jones reagent, acetone, 0 ꢀC, 5 min, then rt, 30 min, 72%.
t-Bu
Si
t-Bu
Si
t-Bu
Si
BnO
BnO
t-Bu
t-Bu
t-Bu
O
O
O
BnO
O
BnO
O
n-C22H45
n-C22H45
O
O
O
O
O
O
OBn
OBn
PMBO
HO
PMBO
OH
O
OBn
n-C14H29
OBn
n-C14H29
O
OBn
n-C14H29
a
b
PMBO
HO
PMBO
O
O
O
OBn
OBn
OBn
7
20
19
HO
BnO
OH
HO
O
OH
BnO
O
n-C22H45
n-C22H45
HO
HO
O
O
OH
OH
O
OBn
HO
HO
O
OBn
n-C14H29
HO
O
c
HO
d
O
n-C14H29
OH
OBn
22 (RCAI-169)
21
Scheme 4. Reagents and conditions: (a) 18, DMAP, EDAC, THFeCH2Cl2 (1:1), rt, 3.5 days, 76%; (b) DDQ, CH2Cl2eH2O (10:1), rt, 2.5 h; (c) HFepyridine, pyridine, THF, rt, 2 h, two steps
26%; (d) H2, Pd(OH)2/C, THF, rt, 21 h, 78%.

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9713
OTBDMS
O
OH
OH
OBn
OBn
BnO
BnO
OH
OH
a
TBDMSO
TBDMSO
(S)
(R)
n-C12H25
n-C12H25
+
OH
OBn
OBn
OBn
BnO
23
OBn
24
24'
O
O
O
O
O
(S)
F3C
Ph
MeO
Ph
Ph
Ph
(R)
(S)
(R)
b
b
O
OBn O
OBn O
MeO
OMe
CF3
OMe
F3C
CF3
24
24
TBDMSO
TBDMSO
(S)
(S)
n-C12H25
n-C12H25
OBn OBn
OBn OBn
24R
24S
OH
O
O
OBn OH
OBn OH
n-C12H25
OBn OBn
c
d
e
HO
O
O
24
n-C12H25
n-C12H25
OBn
OBn
OBn
OBn
OBn
OBn
25
26
27
OH
OH
O
OBn
OBn OBn
n-C12H25
OBn
OBn
OBn
n-C12H25
f
g
h
HO
TBDMSO
TBDMSO
n-C12H25
OBn
OBn
OBn
OBn
OBn
OBn
30
29
28
j
OH
i
OBn OBn
n-C12H25
OH
OBn OBn
n-C12H25
31
TBDMSO
29
HO
+
OBn
OBn
OBn
OBn
31
32
Scheme 5. Reagents and conditions: (a) n-C12H25MgBr, Et2O, reflux, 24 (44%) and 240 (38%); (b) 24 (5 mg), (þ)-MTPA chloride or (ꢁ)-MTPA chloride, pyridine, DMAP, THF, rt, 15 min,
24R (5 mg) or 24S (5 mg); (c) TBAF, THF, rt, 1 h, 71%; (d) 2,2-dimethoxypropane, p-TsOH$H2O, rt, 1 h; (e) BnBr, NaH, DMF, rt, 1 h, two steps 93%; (f) aq 46% HF, CH2Cl2eMeCN (1:1), rt,
30 min, 99%; (g) TBDMS-Cl, imidazole, catalytic DMAP, rt, 16 h, 93%; (h) DesseMartin periodinane, rt, 2 h, 91%; (i) NaBH4, EtOH, rt, 30 min, 31 (34%) and 29 (52%); (j) TBAF, THF, rt,
1 h, 86%.
t-Bu
t-Bu
t-Bu
t-Bu Si
t-Bu Si
t-Bu Si
O
O
O
O
O
O
O
O
a
b
O
n-C23H47
OBn
NH
O
O
O
PMBO
PMBO
PMBO
OH
O
OBn
OBn
n-C12H25
OBn
n-C12H25
O
PMBO
PMBO
PMBO
CCl3
O
O
OBn
OBn
OBn
OBn
6
33
34
t-Bu
t-Bu Si
O
O
O
O
O
OH
HO
e
c
d
O
O
O
n-C23H47
OBn
n-C23H47
OBn
O
O
HO
HO
O
O
OBn
n-C12H25
OBn
n-C12H25
HO
HO
O
O
OBn
OBn
OBn
OBn
35
36
HO
O
HO
OH
HO
OH
n-C23H47
OH
n-C23H47
OH
f
HO
O
O
O
O
HO
HO
O
OBn
OH
OBn
OH
HO
HO
O
O
n-C12H25
n-C12H25
OBn
OH
OBn
OH
37
38 (RCAI-170)
ꢀ
Scheme 6. Reagents and conditions: (a) 32, AgOTf, MS 4 A, CH2Cl2, rt, 1 h, 38%; (b) 8, DMAP, EDAC, CH2Cl2eTHF (1:1), 5 h, 63%; (c) DDQ, CH2Cl2eH2O (10:1), rt, 2.5 h; (d)
HFepyridine, pyridine, THF, rt, 1.5 h, two steps 74%; (e) aq 46% HF, CH2Cl2eMeCN (1:1), rt, 1.5 h, 87%; (f) H2, Pd(OH)2/C, THF, rt, 16 h, 74%.

9714
M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
was tried to yield 32. DesseMartin oxidation of C2-secondary al-
(a)
pg/m l
18000
IFNγ
cohol of 29 gave a ketone 30, which was reduced to a mixture of 29
(52%) and 31 (34%) using sodium borohydride (NaBH4). Treatment
of 31 with TBAF afforded 1,2-diol 32, i.e., a C2eOH epimer of 28
(Scheme 5).
KRN7000
16000
14000
12000
10000
8000
6000
4000
2000
0
13 (RCAI-166)
22 (RCAI-169)
38 (RCAI-170)
Compound 38, having two hydroxy groups on the C20 and C30-
positions in the hexacosanoyl ester side chain, and four hydroxy
groups on the C3, C4, C5, and C6-positions in the octadecyl aglycone
main chain, was synthesized through alcohol 33 obtained by the
coupling reaction with imidate 6 and diol 32.
The glycosylation reaction of glycosyl acceptor 32 with glycosyl
donor 6 was performed by use of AgOTf to construct a-anomer 33.
The C2eOH of 33 was esterified with carboxylic acid 8 in the
presence of dehydrating agent, EDAC, to form 34. Compound 34
was converted to 38 (RCAI-170) in four steps via intermediates 35,
36, and 37 by the same procedure to convert 9 to 13 through in-
termediates 10, 11, and 12. It was obvious by 2D COSY analysis that
the (20,30-dihydroxy)hexacosanoyl group of 38 was on the C2eO
position on the octadecane chain (Scheme 6).
0
12
24
36
48
60
72
(b)p g / m l
IL-4
600
500
400
300
200
100
0
Finally, an alternative route other than going through compound
7 for the synthesis of RCAI-80 was attempted.
KRN7000
L
-Ribose was converted to benzylidene compound 39 according
13 (RCAI-166)
22 (RCAI-169)
38 (RCAI-170)
to the reported method.12 The primary alcohol of this compound
was protected as a silyl ether with TBDMS-Cl and imidazole to yield
a 10:2:1 mixture of 40
Anomeric configuration at C1 of 40
vicinal coupling constant values JH1,H2 of 0 Hz and 4.1 Hz, re-
spectively. Relative stereochemistry of benzylidene acetal was de-
termined by NOESY data.
b, 40a
, and 41 (from 1H NMR analysis).
b
and 40a was determined by
3
The Wittig reaction of this mixture (40b, 40a, and 41) with
a
phosphorane (Ph3P]CHC12H25 obtained from n-tridecyl-
)
triphenylphosphonium bromide and n-BuLi afforded 42EZ (a
mixture of E- and Z-isomers) and 43EZ (a mixture of E- and Z-
isomers). These compounds 42EZ and 43EZ were separable chro-
matographically. The double bond of 42EZ was reduced under H2
atmosphere using Pd/C as a catalyst in hexane to furnish 44, which
was treated with TBAF to give diol 45 (Scheme 7).
0
12
24
36
48
60
72
Fig. 2. The concentrations of IFNg (panel a) and IL-4 (panel b) in sera of mice (2 mg/
mouse, in vivo) upon administration of the RCAI-80 variants (ester analogs of
KRN7000), namely three compounds 13, 22 and 38 (the structures shown in Table 1).
The glycosylation reaction of glycosyl acceptor 45 with glycosyl
donor 6 was performed by use of AgOTf as a catalyst to construct a-
Esterification of alcohol 7 with carboxylic acid 18 using EDAC as
a dehydrating agent in THFeCH2Cl2 (1:1) afforded 19. Reaction of 19
with DDQ in dichloromethaneewater (CH2Cl2eH2O¼10:1) gave 20
as a mixture with 4-methoxybenzaldehyde. This mixture was
treated with HF$pyridine in THFepyridine to give 21 (two steps
26%). Five benzyl groups of 21 were hydrogenolyzed using Pd(OH)2/
C in THF to give 22 (RCAI-169) (Scheme 4).
anomer 46. The C2eOH of 46 was esterified with n-hexacosanoic
acid in the presence of dehydrating agent, EDAC, to form 47, which
was further converted to 48 using DDQ in CH2Cl2eH2O (10:1).
Treatment of 48 with HF$pyridine in THFepyridine yielded 49
(80%). Hydrogenolysis of 49 in THF under H2 atmosphere using
Pd(OH)2/C as a catalyst gave 50 (RCAI-80) without occurring the
acyl migration from C2eO to C4eO (Scheme 8). This synthetic route
was improved in both total yield and steps as compared to the
previously reported method.6
Thirdly, compound 32, constructing the aglycon part of 38, was
obtained in nine steps (4% yield) from 1-O-tert-butyldimethylsilyl-
2,3,4-tri-O-benzyl-
-galactose (23).9 Grignard reaction of 23 with
D
dodecylmagnesium bromide gave a mixture of 24 and 240.
The absolute configuration of C6eOH of 24 was determined by
2.2. Biological activity
Mosher’s method using MTPA
(a-methoxy-a-trifluoromethyl
phenylacetyl)diesters forC2andC6hydroxygroups.11 CompleteNMR
assignments of (R)-MTPA ester (24R) and (S)-MTPA ester (24S) were
confirmed by 2D DQF-COSY, NOESY, HSQC, and HMBC experiments
(see Experimental).
The cytokine-producing [IFNg (Th1 response) and IL-4 (Th2
response)] activity from iNKT cells of mice in vivo of the three
synthetic compounds 13 (RCAI-166), 22 (RCAI-169) and 38 (RCAI-
170) was investigated using KRN7000 as a positive control as
Compound 24 was desilylated by treatment with TBAF to yield
triol 25, in which 1- and 2-hydroxy groups were distinguished from
C6-hydroxy group by construction of 1,2-O-isopropylidene using p-
toluenesulfonic acid monohydrate (TsOH$H2O) as a catalyst in 2,2-
dimethoxypropane to afford 26. The remaining C6eOH group was
benzylated with BnBr and NaH as a base to yield 27. The iso-
propylidene group of 27 was deprotected with aqueous HF to give
1,2-diol 28. The primary alcohol of 28 was protected selectively
with TBDMS-Cl using imidazole as a base to form 29. Mitsunobu
inversion at the C2eOH position of 29 was attempted. However, the
target product could not be obtained. Therefore, another approach
shown in Fig. 2 [IFNg (panel a) and IL-4 (panel b)], and the struc-
tures of these compounds were listed in Table 1.
The results of biological activity are described below.
Both compounds 13 and 38 have consecutively (2R,3R)-dihy-
droxy groups in the immediate vicinity of ester carbonyl of RCAI-80.
Furthermore, the compound 38 is attached on the extra (5S,6S)-
dihydroxy groups on the aglycon main chain of RCAI-80. And the
compound 22 has consecutively (2R,3R,4R)-trihydroxy groups in
the immediate vicinity of ester carbonyl of RCAI-80.
The IFN
g production ability of compound 13 (RCAI-166) de-
creased to approximately 11.4% of KRN7000, and yet the IL-4

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9715
HO
ref. 12.
O
TBDMSO
TBDMSO
O
TBDMSO
+
O
OH
O
OH
OH
OH
a
L-ribose
+
O
O
O
O
O
O
O
O
Ph
Ph
Ph
Ph
39
40β
40α
41
OH
O
OH
TBDMSO
TBDMSO
b
n-C12H25
n-C12H25
40β + 40α + 41
O
O
+
O
Ph
42EZ
Ph
43EZ
OH
OH
TBDMSO
HO
n-C14H29
n-C14H29
c
d
42EZ
O
O
O
O
Ph
Ph
45
44
Scheme 7. Reagents and conditions: (a) TBDMS-Cl, imidazole, CH2Cl2, rt, 45 min, 74% (40
H2, 10% Pd/C, hexane, rt, 30 min, 96%; (d) TBAF, THF, rt, 45 min, 68%.
b:40
a
:41¼10:2:1); (b) n-C13H27Ph3PBr, n-BuLi, THF, ꢁ10 ꢀC, 71% (42EZ) and 6% (43EZ); (c)
t-Bu
Si
t-Bu
Si
t-Bu
Si
t-Bu
t-Bu
O
t-Bu
O
O
O
O
O
O
O
NH
O
O
n-C25H51
n-C14H29
c
a
b
PMBO
PMBO
OH
O
PMBO
O
O
PMBO
O
PMBO
PMBO
CCl3
n-C14H29
O
O
O
O
6
46
47
Ph
Ph
t-Bu
Si
t-Bu
O
OH
OH
O
HO
HO
HO
HO
O
O
O
O
O
O
n-C25H51
n-C25H51
n-C25H51
n-C14H29
HO
O
O
OH
O
d
e
HO
HO
HO
O
n-C14H29
O
O
n-C14H29
O
O
O
O
OH
50 (RCAI-80)
48
Ph
49
Ph
ꢀ
Scheme 8. Reagents and conditions: (a) 45, MS 4 A, AgOTf, CH2Cl2, rt, 4 h, 87%; (b) n-C25H51COOH, DMAP, EDAC, THFeCH2Cl2 (1:1), rt, 4 days, 95%; (c) DDQ, CH2Cl2eH2O (10:1), rt,
2.5 h, 85%; (d) HFepyridine, pyridine, THF, rt, 2.5 h, 80%; (e) H2, Pd(OH)2/C, THF, rt, 4 h, 87%.
Table 1
Structures of newly synthesized three compounds. The extra hydroxy groups added to RCAI-80 are painted in red
n-C22H45
HO
OH
OH
OH
n-C14H29
HO
O
n-C23H47
OH
HO
O
OH
OH
HO
n-C23H47
OH
HO
HO
HO
HO
O
O
O
O
OH
OH
O
HO
O
O
OH OH
HO
HO
HO
O
O
O
n-C14H29
n-C12H25
OH
22 (RCAI-169)
OH
OH OH
38 (RCAI-170)
13 (RCAI-166)
production amount remained approximately more than 45.4% of
KRN7000. Therefore, compound 13 biased to Th2 response (almost
4.0 times) as compared to that of KRN7000.
that of KRN7000. Therefore, compound 38 biased to Th2 re-
sponse (about 9.9 times) as compared to that of KRN7000
(Fig. 2).
The IFN
g
production ability of compound 22 (RCAI-169) was
These compounds induced Th2 cytokine (IL-4) rather than Th1
reduced to almost zero, and its IL-4 production amount remained
merely 4.6% of that of KRN7000. However, compound 22 biased to
Th2 response.
cytokine (IFNg) from iNKT cells compared to KRN7000. Therefore,
these three compounds 13, 22, and 38 were tested for the sup-
pression activity of the symptoms associated with experimental
autoimmune encephalomyelitis (EAE),7,8 an animal (mouse) model
of multiple sclerosis, as shown in Fig. 3. In addition, the EAE test of
RCAI-80 was shown in Fig. 4.
The IFN
g production ability of compound 38 (RCAI-170)
decreased to approximately 2.5% of that of KRN7000, and its
IL-4 production amount still remained approximately 24.8% of

9716
Clinical score
M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
IFNg production to almost zero may be important for the pre-
5
4
3
2
1
0
ventive effects against EAE.
DC + OCH
DC + 13 (RCAI-166)
DC + 22 (RCAI-169)
DC + 38 (RCAI-170)
4. Experimental
4.1. General
IR spectra were measured with a Jasco FT/IR-460 plus spec-
trometer. 1H NMR spectra (TMS as an internal standard) and 13C
NMR spectra were recorded with a Varian VNMRS-500 spectrom-
eter using tetramethylsilane (TMS) as an internal standard. Mass
spectra and High Resolution MS were recorded with Jeol JMS-
SX102A or Bruker BioAPEX II 70e FT-ICR mass spectrometers.
Separation by HPLC was performed by use of Hitachi L7110 appa-
ratus. Optical rotation values were measured with a Jasco P-1010
polarimeter. Column chromatography was carried out using silica
gel 60N (70e230 mesh ASTM, using ca. 10e50 times the weight of
substrate if there is no indication) under a slightly elevated pres-
sure for elution. Preparative TLC was carried out on a PLC plate
(Merck, Silica gel 60 F254, 0.5 mm).
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Days after immunization
Fig. 3. Clinical score of compounds 13, 22, and 38 compared to OCH for EAE
suppression.
Clinical score
5
DC + KRN7000
DC + RCAI-80
4
3
2
1
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Days after immunization
Fig. 4. Clinical score of RCAI-80 compared to KRN7000 for EAE suppression. RCAI-80 showed better clinical score than that of KRN7000.
Compound 13 (RCAI-166) showed moderate clinical score.
However, its intensity is a little bit weaker than that of OCH. Nev-
ertheless, the IL-4 production of compound 22 (RCAI-169) was
merely 4.6% of KRN7000, it showed a weak depression tendency for
EAE effectiveness. It may be due to no production of IFNg. The EAE
test of compound 38 (RCAI-170) was disappointing. It is not known
4.1.1. 2,3-Di-O-benzyl-5-O-tert-butyldimethylsilyl-
and 2,3-Di-O-benzyl-4-O-tert-butyldimethylsilyl-
L
-ribofuranose (2)
-ribopyranose
L
(20). (i) A solution of 1 (2.17 g, 4.99 mmol) in Ac2O (125 mL) con-
taining concd H2SO4 (0.055 mL) was stirred for 1 h at 6e10 ꢀC, and
diluted with EtOAc (250 mL), which was washed with aq saturated
NaHCO3 (three times), and brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to give an oil. Toluene was
added to this oil, and concentrated in vacuo to remove Ac2O at 60 ꢀC
of bath temperature. This procedure was repeated three times to
give a mixture of diacetates.
exactly why.
3. Conclusion
(ii) The above obtained mixture was dissolved in MeOH
(350 mL) containing KOH (200 mg), stirred for 2 h at 15e20 ꢀC to
give alcohols from the diacetates, and concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (1:1, then 1:2), gave a mixture (0.88 g,
The cytokine secretion ability of 13 (RCAI-166), 22 (RCAI-169),
and 38 (RCAI-170) was biased to the Th2 response comparing to
that of KRN7000, because they inhibited mostly the IFN
g pro-
duction (approximately 88.6%, 100%, 97.5%, respectively), and their
IL-4 production ability still remained approximately 45.4%, 4.6%,
and 24.8% of that of KRN7000, respectively. However, compound 13
showed only moderate suppressive effects against EAE symptoms,
and compound 22 showed the weak tendency for EAE effective-
ness. Compound 38 did not show effectiveness against the EAE
suppression. It may be difficult to deduce the structureeactivity
relationship of this series of hydroxylated RCAI-80 analogs, because
of difficulty of finding out the rational and systematic regularity
from the above mentioned results. However the balance of the
hydroxy groups on the sphingosine main chain and the ester side
chain may be important for EAE suppression. In addition, not only
making the cytokine secretion bias to Th2 but also decreasing the
53%) of 2,3-di-O-benzyl-L-ribofuranose and 2,3-di-O-benzyl-L-
ribopyranose as an oil. ESI MS: m/z 353.14 [MþNa]þ. HRESI MS:
calcd for C19H22O5Na: 353.1365; observed 353.1370.
(iii)
A solution of the above obtained mixture (0.88 g,
2.67 mmol), imidazole (545 mg, 8.01 mmol) and tert-butyldime-
thylsilyl chloride (643 mg, 4.27 mmol) in CH2Cl2 (70 mL) was stir-
red for 2 h at 15e20 ꢀC, and diluted with CH2Cl2, which was washed
with aq saturated NaHCO3 (three times) and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a mixture, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (9:1, then 9:2), gave an inseparable

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9717
mixture of 2 and 20 [1.00 g, 84% (three steps 45%)] as an oil.
Rf¼0.383 (hexaneeEtOAc¼3:1). IR nmax (KBr) 3509 (br), 2929, 2856,
[MþNa]þ. HRESI MS: calcd for C32H48O4Na: 519.3450; observed,
519.3441. Physical data of E-isomer of 4: [Rf¼0.263
(hexaneeEtOAc¼2:1)]; IR nmax (KBr) 3415 (br), 2925, 2853, 1456,
1455, 1253 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d
ꢁ0.02 to 0.05 (3H,
m), 0.83e0.89 (9H, m), 3.33e4.40 (5H, m), 4.60e4.74 (4H, m),
5.26e5.29 (1H, m), 7.28e7.40 (10H, m). ESI MS: m/z 467.2 [MþNa]þ.
HRESI MS: calcd for C25H36O5SiNa: 467.2230; observed: 467.2219.
1271 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d
0.88 (3H, t, J¼6.9 Hz), 1.26
(16H, br s), 1.38e1.43 (2H, m), 1.57e1.60 (2H, m), 2.10e2.14 (2H, m),
3.24 (1H, br, OH), 3.58 (1H, t, J 6.5 Hz), 3.70e3.74 (2H, m), 3.78 (1H,
m), 4.00 (1H, dd, J 6.0, 8.4 Hz), 4.36, 4.64 (2H, AB-q, J 11.7 Hz), 4.59,
4.68 (2H, AB-q, J 10.9 Hz), 5.48 (1H, dd, J 8.4, 15.6 Hz), 5.80 (1H, td, J
6.7, 15.6 Hz), 7.28e7.36 (10H, m). ESI MS: m/z 519.3 [MþNa]þ. HRESI
MS: calcd for C32H48O4Na: 519.3450; observed, 519.3439.
4.1.2. (2S,3S,4R,5EZ)-1-tert-Butyldimethylsilyloxy-3,4-dibenzyloxy-
5-octadecen-2-ol (3) and (2S,3R,4R,5EZ)-2-tert-Butyldimethylsily-
loxy-3,4-di-benzyloxy-5-octadecen-1-ol (30). To a solution of n-tri-
decyltriphenylphosphonium bromide (1.17 g, 2.22 mmol) in dry
THF (3 mL) was added a solution of n-BuLi (1.65 M in hexane,
1.80 mL, 2.97 mmol, after the lasting phosphorane red color) at
ꢁ10 ꢀC under argon with stirring. After stirring for 30 min at ꢁ10 ꢀC,
to this red-colored solution was added a solution of a mixture of 2
and 20 (327 mg, 0.99 mmol) in THF (2 mL). This mixture was stirred
for 10 min at ꢁ10 ꢀC, and then overnight at room temperature, and
diluted with EtOAc. The solution was washed with water and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in
vacuo to give a residue, which was chromatographed on a silica gel
column. Elution with hexaneeEtOAc (19:1) gave an E,Z-mixture of 3
(325 mg, 54%) as an oil, a mixture of 3 and 30 (37 mg, 6%), and an
E,Z-mixture of 30 (26 mg, 4%) as an oil.
4.1.4. (2S,3S,4R)-3,4-(Dibenzyloxy)octadecane-1,2-diol (5). A solu-
tion of 4 (516 mg, 1.04 mmol) in hexane (80 mL) was stirred for
25 min at 16e18 ꢀC under hydrogen using 10% Pd/C (200 mg) as
a catalyst, and filtered. The filtrate was concentrated in vacuo to
give crude 5, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (3:1, then 3:2) gave 5 (425 mg, 82%) as
an oil. IR nmax (KBr) 3853 (br), 2921, 2853, 1518, 1263 cmꢁ1. 1H NMR
(500 MHz, CDCl3)
d 0.88 (3H, t, J 6.8 Hz), 1.26 (20H, br s), 1.33e1.44
(4H, m), 1.61e1.74 (2H, m), 1.90 (2H, br, OH), 3.61 (1H, dd, J 4.6,
6.3 Hz), 3.73 (1H, m), 3.75e3.77 (2H, m), 3.82 (1H, m), 4.61 (2H, s),
4.62, 4.69 (2H, AB-q, J 11.3 Hz), 7.30e7.37 (10H, m). ESI MS: m/z
521.4 [MþNa]þ. HRESI MS: calcd for C32H50O4Na: 521.3607; ob-
served, 521.3606.
The E,Z-mixture of 3 (10 mg) was separated on a silica gel TLC
plate by development with hexaneeEtOAc (9:1) to give Z-isomer of
3 (4 mg) and E,Z-mixture (3 mg), and also the E,Z-mixture of 30
(10 mg) was separated on a silica gel TLC plate by development
with hexaneeEtOAc (9:1) to give Z-isomer of 30 (2 mg) and E,Z-
mixture of 30 (5 mg). Physical data of Z-isomer of 3: Rf¼0.634
(hexaneeEtOAc¼6:1). IR nmax (KBr) 3567, 2925, 2854, 1517, 1457,
4.1.5. (2S,3S,4R)-3,4-Dibenzyloxy-2-hydroxyoctadecyl 2,3-di-O-(4-
methoxybenzyl)-4,6-O-(di-tert- butyl)silylene-
(7). To solution of imidate [obtained from 2,3-di-O-(4-
methoxybenzyl)-4,6-O-(di-tert-butyl)silylene- -galactopyranose
a-D-galactopyranoside
a
6
a-D
(311 mg, 0.555 mmol) and CCl3CN (801 mg, 5.55 mmol) using
Cs2CO3 (266 mg) as a base in CH2Cl2 (6 mL) at room temperature
over night] and diol 5 (166 mg, 0.333 mmol) in dry CH2Cl2 (15 mL)
1255 cmꢁ1 1H NMR (500 MHz, CDCl3)
. d 0.05 (6H, s), 0.88 (3H, t,
J¼6.8 Hz), 0.89 (9H, s), 1.25 (16H, br s), 1.31e1.37 (2H, m), 1.50e1.60
(2H, m), 2.05 (1H, m), 2.11 (1H, m), 3.57 (1H, m), 3.66e3.70 (2H, m),
3.76 (1H, dd, J 3.4, 10.0 Hz), 4.38, 4.65 (2H, AB-q, J 12.0 Hz), 4.59 (1H,
m), 4.62, 4.87 (2H, AB-q, J 11.2 Hz), 5.55 (1H, t, J 11.0 Hz), 5.77 (1H,
m), 7.27e7.34 (10H, m). ESI MS of E,Z-mixture of 3: m/z 633.4
[MþNa]þ. HRESI MS: calcd for C58H62O4SiNa: 633.4315; observed,
633.4309. Physical data of Z-isomer of 30: [Rf¼0.488
(hexaneeEtOAc¼6:1)]; IR nmax (KBr) 3566, 2925, 2854, 1456,
ꢀ
was added 4 A MS (dry powder, 1.0 g). After stirring for 30 min at
room temperature, the mixture was cooled at 0 ꢀC, and to this
mixture was added AgOTf (110 mg, 0.428 mmol). After stirring for
2 h at 0 ꢀC and for 1 h at room temperature, the mixture was fil-
tered, and the filter cake was washed with CH2Cl2. The combined
filtrate was washed with satd aq NaHCO3 and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a mixture, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (6:1, then 4:1) gave 7 (253 mg, 73%) as
an oil. IR nmax (KBr) 3442, 2925, 2854, 1511, 1249 cmꢁ1. 500 MHz 1H
1252 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d 0.05 (3H, s), 0.09 (3H, s),
0.88 (3H, t, J¼7.1 Hz), 0.90 (9H, s), 1.26 (18H, m), 1.57 (2H, m), 1.92
(1H, m), 1.98 (1H, m), 3.60 (1H, m), 3.67e3.74 (2H, m), 3.99 (1H, m),
4.35 (1H, m), 4.36, 4.61 (2H, AB-q, J 11.9 Hz), 4.66, 4.73 (2H, AB-q, J
11.4 Hz), 5.43 (1H, t, J 11.0 Hz), 5.72 (1H, m), 7.30e7.33 (10H, m). ESI
MS: m/z 633.4 [MþNa]þ. HRESI MS: calcd for C58H62O4SiNa:
633.4315; observed, 633.4300.
NMR (CDCl3)
d 0.88 (3H, t, J 6.8 Hz), 0.99 (9H, s), 1.05 (9H, s),
1.25e1.32 (24H, m), 1.53e1.70 (2H, m), 3.45 (1H, dd, J 8.6, 10.6 Hz),
3.60 (1H, s), 3.64 (1H, dd, J 3.1, 6.6 Hz), 3.71 (1H, td, J 3.4, 8.1 Hz),
3.75e3.81 (7H, m, containing two 3H singlets at 3.76 and
3.81 ppm), 3.88 (1H, m), 3.96 (1H, dd, J 2.7, 10.5 Hz), 3.98 (1H, dd, J
2.7, 10.5 Hz), 4.46 (1H, d, J 3.0 Hz), 4.52 (1H, d, J 11.5 Hz, benzylic H),
4.59e4.68 (5H, m, benzylic 5H), 4.71 (1H, d, J 3.6 Hz, anomeric H),
4.74 (1H, d, J 11.5 Hz, benzylic H), 4.79 (1H, d, J 11.2 Hz, benzylic H),
6.80 (2H, d, J 8.8 Hz), 6.88 (2H, d, J 8.8 Hz), 7.24e7.35 (14H, m). ESI
MS: m/z 1063.6 [MþNa]þ. HRESI MS: calcd for C62H92O11SiNa,
1063.6307; observed, 1063.6307.
4.1.3. (2S,3S,4R,5EZ)-3,4-Dibenzyloxy-5-octadecene-1,2-diol (4). To
a solution of a mixture of 3 and 30 (360 mg, 0.59 mmol) in dry THF
(6 mL) was added tetra-n-butylammonium fluoride (1.0 M solution
in THF, 3.0 mL). After stirring for 30 min at room temperature, the
reaction mixture was diluted with EtOAc, which was washed with
water and brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a residue, which was chromato-
graphed on a silica gel column. Elution with hexaneeEtOAc (6:1,
then 2:1) gave 4 (279 mg, 95%) as an oily mixture of E- and Z-iso-
mers. The E,Z-mixture of 4 (10 mg) was separated on a silica gel TLC
plate by development with hexaneeEtOAc (2:1) to give Z-isomer of
4 (4 mg) and E,Z-mixture of 4 (4 mg). Physical data of Z-isomer of 4:
Rf¼0.338 (hexaneeEtOAc¼2:1). IR nmax (KBr) 3411 (br), 2925, 2853,
4.1.6. (2S,3S,4R)-3,4-Dibenzyloxy-2-[(20R,30R)-20,30-(iso-
propylidenedioxy)hexacosanoyloxy]octadecyl 2,3-di-O-(4-
methoxybenzyl)-4,6-O-(di-tert-butyl)silylene-a-D-galactopyranoside
(9). To a solution of 7 (242 mg, 0.232 mmol) and DMAP (452 mg,
3.70 mmol) in dry THFeCH2Cl2 (1:1, 24 mL) were added (2R,3R)-
2,3-(isopropylidenedioxy)hexacosanoic
acid
8
(289
mg,
1456 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d
0.88 (3H, t, J¼6.8 Hz), 1.25
0.617 mmol) and EDAC (590 mg, 3.08 mmol). The solution was
stirred for 5 h at room temperature, concentrated in vacuo, and
diluted with CH2Cl2. The solution was washed with satd aq NaHCO3
and brine, dried over MgSO4, and filtered. The filtrate was con-
centrated in vacuo to give a mixture, which was chromatographed
on a silica gel column. Elution with hexaneeEtOAc (19:1, then 9:1)
(16H, s), 1.30e1.37 (2H, m), 1.57e1.62 (2H, m), 2.01 (1H, m), 2.08
(1H, m), 2.20 (1H, br, OH), 3.24 (1H, br, OH), 3.59 (1H, t, J 6.5 Hz),
3.72e3.76 (2H, m), 3.79 (1H, m), 4.35, 4.64 (2H, AB-q, J 11.6 Hz), 4.43
(1H, dd, J 6.3, 9.4 Hz), 4.58, 4.70 (2H, AB-q, J 11.0 Hz), 5.47 (1H, t, J
11.0 Hz), 5.81 (1H, m), 7.27e7.36 (10H, m). ESI MS: m/z 519.3

9718
M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
gave 9 (275 mg, 79%) as an oil. IR nmax (KBr) 2925, 2854, 1734, 1612,
1512, 1464, 1248 cmꢁ1. 1H NMR (500 MHz, CDCl3):
0.88 (6H, t, J
MS: m/z 1093.79 [MþNa]þ. HRESI MS: calcd for C64H110O12Na:
d
1093.7895; observed: 1093.7908.
7.0 Hz), 0.98 (9H, s), 1.04 (9H, s), 1.20e1.38 (62H, m, containing 3H
singlet at 1.34 ppm), 1.42e1.68 (4H, m, containing 3H singlet at
1.53 ppm), 3.55 (1H, m), 3.60 (1H, s), 3.53e4.01 (11H, m, containing
two 3H singlets at 3.74 and 3.77 ppm), 4.26 (1H, m), 4.40e4.46 (3H,
m), 4.54e4.74 (9H, m, benzylic 8H and anomeric H), 5.36 (1H, m),
6.75 (2H, d, J 8.7 Hz), 6.84 (2H, d, J 8.7 Hz), 7.21 (2H, d, J 8.8 Hz),
7.27e7.33 (12H, m). ESI MS: m/z 1514.03 [MþNa]þ. HRESI MS: calcd
for C91H146O14SiNa: 1514.0380; observed: 1514.0377.
4.1.10. (2S,3S,4R)-3,4-Dihydroxy-2-[(20R,30R)-20,30-(dihydroxy)hex-
acosanoyloxy]octadecyl
a-D-galactopyranoside (13) (RCAI-166). A
solution of 12 (48 mg, 1.04 mmol) in THF (40 mL) was stirred for 5 h
at room temperature under hydrogen using 20% Pd(OH)2/C (wet,
water ca. 50%, 52 mg) as a catalyst, and the reaction mixture was
filtered. The catalyst was washed with THF, and combined THF
solution was concentrated in vacuo to give a white powder, which
was washed with EtOAc (3 mL). The powder was dried with a pump
to give 13 (32 mg, 80%) as a powder. It was obvious by 2D COSY
analysis that the (20,30-dihydroxy)hexacosanoyl group of 13 was on
the C2eO position on the octadecane chain. However, silica gel
column chromatography of 13 by elution with CHCl3eMeOH (9:1)
yields gradually C4eO acyl migrated compound. Physical data of
4.1.7. (2S,3S,4R)-3,4-Dibenzyloxy-2-[(20R,30R)-20,30-(iso-
propylidenedioxy)hexacosanoyloxy]octadecyl 4,6-O-(di-tert-butyl)si-
lylene-a-D-galactopyranoside (10). To a solution of 9 (270 mg,
0.181 mmol) in CH2Cl2eH2O (10:1, 24 mL) was added DDQ (270 mg,
1.19 mmol). The mixture was stirred for 3 h at room temperature,
and diluted with CH2Cl2. The solution was washed twice with aq
satd NaHCO3 and brine, dried over MgSO4, and filtered. The filtrate
was concentrated in vacuo to give a residue, which was chromato-
graphed on a silica gel column. Elution with hexaneeEtOAc (9:1, 6:1,
then 4:1) gave 10 (128 mg, 57%) as an oil. IR nmax (KBr) 3470, 2925,
13: IR nmax (KBr) 3431 (br), 2919, 2850, 1732, 1468 cmꢁ1. [
a
]
19 þ42.2
D
(c 0.83, pyridine). 1H NMR (500 MHz, pyridine-d5)
d
0.88 (3H, t, J
6.8 Hz), 1.21e1.44 (62H, m), 1.58e1.68 (2H, m), 1.80e1.94 (3H, m),
2.02 (1H, m), 2.15 (1H, m), 2.23 (1H, m), 4.28 (1H, m), 3.73 (1H, m),
3.37e4.56 (8H, m), 4.64 (1H, dd, J 3.4, 9.8 Hz), 4.69 (1H, d, J 6.2 Hz),
5.51 (1H, d, J 3.6 Hz, anomeric H), 6.38 (1H, m). 13C NMR (125 MHz,
2854, 1734, 1466 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (6H, t, J
7.0 Hz),1.01 (9H, s),1.04 (9H, s),1.25 (62H, br s),1.37 (3H, s),1.42e1.66
(7H, m, containing 3H singlet at 1.59 ppm), 3.54e3.60 (2H, m), 3.63
(1H, s), 3.72e3.77 (3H, m), 3.98 (1H, dd, J 1.6,12.7 Hz), 4.05 (1H, dd, J
3.0,11.3 Hz), 4.08 (1H, dd, J 2.2,12.7 Hz), 4.29e4.36 (2H, m), 4.53 (1H,
d, J 6.6 Hz, C20-H), 4.54, 4.58 (2H, AB-q, J 11.5 Hz), 4.65, 4.70 (2H, AB-
q, J 11.3 Hz), 4.86 (1H, d, J 3.9 Hz, anomeric H), 5.42 (1H, m, C2eH),
7.29e7.34 (10H, m). ESI MS: m/z 1273.92 [MþNa]þ. HRESI MS: calcd
for C75H130O12SiNa: 1273.9229; observed: 1273.9233.
pyridine-d5) d 14.34, 22.99, 26.43, 29.66, 29.67, 29.97, 29.98, 30.05,
30.06, 30.08, 30.10, 30.15, 30.20, 30.38, 32.18, 34.11, 34.55, 62.68,
67.04, 70.49, 71.04, 71.57, 72.40, 72.83, 73.64, 74.00, 75.40, 76.85,
101.08, 174.22. ESI MS: m/z 913.70 [MþNa]þ. HRESI MS: calcd for
C50H98O12Na: 913.6956; observed, 913.6960.
4.1.11. (2S,3S,4R,5EZ)-1-tert-Butyldimethylsilyloxy-3,4-dibenzyloxy-
5-hexacosen-2-ol (14). To solution of n-henicosyl-
a
triphenylphosphonium bromide (5.13 g, 8.04 mmol, 3 equiv) in dry
THF (24 mL) was added n-BuLi (1.65 M in hexane, 4.87 mL,
8.04 mmol, after appearance of red color) at ꢁ10 ꢀC under argon
with stirring. After stirring for 30 min at ꢁ10 ꢀC, a solution of 2
(884 mg, 2.68 mmol) in dry THF (11 mL) was added to the above
obtained phosphorane solution at ꢁ10 ꢀC with stirring. After stirring
for 20 min at ꢁ10 ꢀC, the mixture was stirred for 3 h at room tem-
perature, and diluted with CHCl3, then washed with aq 3% H2O2,
water and brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a mixture, which was chromato-
graphed on a silica gel column. Elution with hexaneeEtOAc (29:1,
then 19:1) gave 14 (1.03 g, 53%) as an oily E,Z-mixture. IR nmax (KBr)
4.1.8. (2S,3S,4R)-3,4-Dibenzyloxy-2-[(20R,30R)-20,30-(iso-
propylidenedioxy)hexacosanoyloxy]octadecyl
a-D-galactopyranoside
(11). To a solution of 10 (78 mg, 0.062 mmol) and in dry THF (6 mL)
containing dry pyridine (50 mg) was added HF$pyridine (HF:
w70%; pyridine: w30%, 25 mg) at room temperature. After stirring
for 1.5 h at room temperature, the reaction mixture was diluted
with CHCl3, washed with satd aq NaHCO3, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to give a residue
(69 mg), which was employed for the next reaction without puri-
fication. A part of this residue (3 mg) was purified on a preparative
TLC plate. Development with CHCl3eMeOH (7:1) gave 11 (2 mg) as
a gum. IR nmax (KBr) 3472 (br), 2924, 2853, 1732, 1466, 1456 cmꢁ1
1H NMR (500 MHz, CDCl3):
0.88 (6H, t, J 7.0 Hz), 1.20e1.30 (64H,
.
3630, 2925, 2853, 1457 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d 0.05 (6H,
d
s), 0.86e0.89 (12H, m, containing 9H singlet at 0.89 ppm),1.22e1.40
(34H, m, containing 30H singlet at 1.25 ppm), 1.50e1.55 (2H, m),
2.00e2.11 (2H, m), 2.65 (1H, br s, OH), 3.57e3.78 (4H, m), 4.35e4.40
(1H, m), 4.57e4.66 (3H, m), 4.84e4.88 (1H, m), 5.53e5.59 (1H, m),
5.75e5.78 (1H, m), 7.26e7.34 (10H, m). ESI MS: m/z 745.6 [MþNa]þ.
HRESI MS: calcd for C46H78O4SiNa: 745.5567; observed, 745.5554.
m), 1.38 (3H, s), 1.43e1.72 (9H, m, containing 3H singlet at
1.60 ppm), 3.59 (1H, td, J 4.4, 7.4 Hz), 3.64e3.82 (7H, m), 4.00 (1H, d,
J 2.3 Hz), 4.10 (1H, dd, J 2.3,10.8 Hz), 4.32 (1H, m), 4.56 (1H, m), 4.56,
4.59 (2H, AB-q, J 11.5 Hz), 4.65, 4.71 (2H, AB-q, J 11.4 Hz), 4.87 (1H, d,
J 3.7 Hz, anomeric H), 5.46 (1H, m, C2eH), 7.28e7.36 (10H, m). ESI
MS: m/z 1133.82 [MþNa]þ. HRESI MS: calcd for C67H114O12Na:
1133.8208; observed: 1133.8218.
4.1.12. (2S,3S,4R)-1-tert-Butyldimethylsilyloxy-3,4-(dibenzyloxy)hex-
acosan-2-ol (15). A solution of 14 (660 mg, 0.913 mmol) in hexane
(75 mL) was stirred for 25 min at room temperature under hy-
drogen using 10% Pd on carbon (260 mg), and filtered. The filtrate
was concentrated in vacuo to give 15, which was chromatographed
on a silica gel column. Elution with hexaneeEtOAc (9:1) gave 15
(596 mg, 90%) as an oil. IR nmax (KBr) 3570, 2925, 2853, 1517,
4.1.9. (2S,3S,4R)-3,4-Dibenzyloxy-2-[(20R,30R)-20,30-(dihydroxy)hex-
acosanoyloxy]octadecyl a-D-galactopyranoside (12). To a solution of
crude 11 (63 mg, 0.057 mmol) in CH2Cl2eMeCN (1:1, 30 mL) was
added aq 46% HF solution (2.7 mL). The mixture was stirred for 1.5 h
at room temperature, and diluted with CHCl3. The solution was
washed with satd aq NaHCO3 and brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to give a mixture,
which was chromatographed on a silica gel column. Elution with
CHCl3eMeOH (96:4, then 94:6) gave 12 (44 mg, two steps 72%) as
1464 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d 0.06 (6H, s), 0.88 (3H, t, J
6.9 Hz), 0.90 (9H, s), 1.20e1.30 (40H, m), 1.50e1.75 (2H, m), 2.64
(1H, d, J 4.2 Hz, OH), 3.62e3.68 (3H, m), 3.76 (1H, m), 3.80 (1H, dd, J
2.4, 9.7 Hz), 4.55, 4.81 (2H, AB-q, J 11.6 Hz), 4.60, 4.65 (2H, AB-q, J
11.5 Hz), 7.26e7.37 (10H, m). ESI MS: m/z 747.6 [MþNa]þ. HRESI MS:
calcd for C46H80O4SiNa: 747.5724; observed, 747.5737.
a powder. IR nmax (KBr) 3412 (br), 2923, 2852,1733,1516,1457 cmꢁ1
1H NMR (500 MHz, CDCl3):
0.88 (6H, t, J 7.0 Hz), 1.21e1.32 (64H,
.
d
m), 1.44e1.70 (6H, m), 3.59e3.80 (7H, m), 4.01 (1H, s), 4.08e4.13
(2H, m), 4.56 (2H, s), 4.62, 4.68 (2H, AB-q, J 11.5 Hz), 4.84 (1H, d, J
3.2 Hz, anomeric H), 5.66 (1H, m, HC2eO), 7.28e7.36 (10H, m). ESI
4.1.13. (2S,3S,4R)-1-tert-Butyldimethylsilyloxy-2,3,4-(tribenzyloxy)
hexacosane (16). To a solution of 15 (645 mg, 0.889 mmol) in dry

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9719
DMF (7 mL) were added NaH (60% oil dispersion, 175 mg,
4.32 mmol) and then BnBr (500 mg, 2.92 mmol) at 0 ꢀC. The
mixture was stirred for 16 h at 5 ꢀC, and diluted with EtOAc. The
whole was washed with ice water, and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (29:1, then 19:1) gave benzyl bro-
mide contaminated 16 (780 mg) as an oil. This mixture was
employed for the next reaction without further purification. IR
1760.1 [MþNa]þ, 1761.2, 1762.2. HRESI MS: calcd for
C
109H160O15SiNa: 1760.1424; observed: 1760.1426.
4.1.17. (2S,3R,4R)-2-[(20R,30R,40R)-20,30,40-(Tribenzyloxy)hex-
acosanoyloxy]-3,4-(dibenzyloxy)octadecyl 4,6-O-(di-tert-butyl)sily-
lene-a-D-galactopyranoside (20). To a solution of 19 (536 mg,
0.308 mmol) in CH2Cl2eH2O (10:1, 44 mL) was added DDQ (536 mg,
2.36 mmol). The mixture was stirred for 2.5 h at room temperature,
and diluted with CH2Cl2. The whole was washed twice with aq satd
NaHCO3 and brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a residue, which was chromato-
graphed on a silica gel column. Elution with hexaneeEtOAc (9:1,
6:1, then 4:1) gave a mixture (206 mg) of 20 and inseparable 4-
methoxybenzaldehyde as an oil. This mixture was employed for
the next reaction without further purification. IR nmax (KBr) 3530,
nmax (KBr) 2925, 2853, 1455 cmꢁ1
.
1H NMR (500 MHz, CDCl3)
d
0.05 (6H, s), 0.88 (3H, t, J 6.8 Hz), 0.91 (9H, s), 1.26 (40H, s), 1.42
(1H, m), 1.61 (1H, m), 3.60e3.70 (2H, m), 3.78e3.84 (2H, m), 3.97
(1H, m), 4.46e4.78 (6H, m), 7.26e7.37 (15H, m). ESI MS: m/z
837.6 [MþNa]þ. HRESI MS: calcd for C53H86O4SiNa: 837.6193;
observed, 837.6191.
2925, 2854, 1750 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (6H, t, J
4.1.14. (2S,3R,4R)-2,3,4-(Tribenzyloxy)hexacosanol (17). To a solu-
tion of 16 (780 mg) in dry THF (30 mL) was added tetra-n-buty-
lammonium fluoride (1.0 M solution in THF, 3.5 mL). After stirring
for 2 h at room temperature, the reaction mixture was diluted with
EtOAc, which was washed with water and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a residue,
which was chromatographed on a silica gel column. Elution with
hexaneeEtOAc (11:1, then 4:1) gave 17 (485 mg, two steps 77%) as
an oil. IR nmax (KBr) 3370 (br), 2919, 2850, 1496, 1469, 1454 cmꢁ1. 1H
7.0 Hz), 1.00 (9H, s), 1.02 (9H, s), 1.26 (64H, br s), 1.45e1.65 (4H, m),
2.37 (1H, d, J 9.7 Hz, OH), 2.66 (1H, d, J 11.5 Hz, OH), 3.43e4.04 (11H,
m, excluding methoxy protons of the contaminated inseparable 4-
methoxybenzalehyde at 3.90 ppm), 4.24 (1H, d, J 3.4 Hz), 4.41e4.59
(8H, m), 4.69e4.76 (4H, m), 5.48 (1H, m), 7.21e7.36 (25H, m). ESI
MS: m/z 1520.0 [MþNa]þ. HRESI MS: calcd for C93H144O13SiNa:
1520.0274; observed: 1520.0243.
4.1.18. (2S,3R,4R)-2-[(20R,30R,40R)-20,30,40-(Tribenzyloxy)hex-
NMR (500 MHz, CDCl3)
d
0.88 (3H, t, J 7.0 Hz), 1.20e1.35 (38H, s),
acosanoyloxy]-3,4-(dibenzyloxy)octadecyl
a-D-galactopyranoside
1.35e1.65 (4H, m), 2.46 (1H, dd, J 1.9, 5.4 Hz, OH), 3.59 (1H, m), 3.63
(1H, m), 3.77e3.87 (3H, m), 4.51, 4.62 (2H, AB-q, J 11.5 Hz), 4.53,
4.64 (2H, AB-q, J 11.6 Hz), 4.71, 4.77 (2H, AB-q, J 11.3 Hz), 7.26e7.35
(15H, m). ESI MS: m/z 723.5 [MþNa]þ. HRESI MS: calcd for
(21). To solution of mixture (206 mg) of 20 and 4-
a
a
methoxybenzaldehyde in dry THF (12 mL) containing dry pyri-
dine (110 mg) was added HF$pyridine (HF: w70%; pyridine: w30%,
55 mg) at room temperature. After stirring for 2 h at room tem-
perature, the reaction mixture was diluted with CHCl3, washed
with satd aq NaHCO3, dried over MgSO4, and filtered. The filtrate
was concentrated in vacuo to give a mixture, which was chroma-
tographed on a silica gel column. Elution with hexaneeEtOAc (1:1,
then 2:3) gave 21 (109 mg, two steps 26%) as a viscous oil. IR nmax
C
47H72O4Na: 723.5328; observed, 723.5314.
4.1.15. (2R,3R,4R)-2,3,4-(Tribenzyloxy)hexacosanoic acid (18). To
a solution of 17 (632 mg, 0.901 mmol) in acetone (30 mL) was
added Jones reagent (0.65 mL) at 0 ꢀC. After stirring for 5 min at
0
ꢀC, the mixture was stirred for 30 min at room temperature,
.
(KBr) 3420, 2924, 2853, 1749 cmꢁ1 1H NMR (500 MHz, CDCl3):
and diluted with CHCl3, which was washed with water and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in
vacuo to give a residue, which was chromatographed on a silica
gel column. Elution with hexaneeEtOAc (4:1, then 2:1) gave 18
(464 mg, 72%) as an oil. IR nmax (KBr) 3032 (br), 2917, 2850, 1754,
1720 (shoulder), 1497, 1471, 1455 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d 0.88 (6H, t, J 6.9 Hz), 1.20e1.40 (64H, m), 1.58e1.71 (4H, m), 1.95
(1H, d, J 2.9 Hz, OH), 2.16 (1H, br s, OH), 2.42 (1H, d, J 11.9 Hz, OH),
2.58 (1H, s, OH), 3.14 (1H, dt, J 9.7, 1.1 Hz), 3.48e3.58 (5H, m), 3.62
(1H, dd, J 3.7, 4.4 Hz), 3.68e3.73 (2H, m), 3.78 (1H, d, J 2.7 Hz), 3.87
(1H, dd, J 2.0, 8.6 Hz), 4.09 (1H, dd, J 2.2, 11.0 Hz), 4.42e4.82 (12H,
m), 5.55 (1H, dt, J 9.3, 2.9 Hz), 7.20e7.36 (25H, m). ESI MS: m/z
1379.9 [MþNa]þ. HRESI MS: calcd for C85H128O13Na: 1379.9253;
observed: 1379.9287.
d
0.88 (3H, t, J 6.9 Hz), 1.15e1.40 (40H, m), 1.50e1.58 (2H, m), 3.73
(1H, dd, J 5.5, 11.4 Hz), 3.89 (1H, dd, J 3.9, 4.6 Hz), 4.92 (1H, d, J
1.6 Hz), 4.55, 4.58 (2H, AB-q, J 11.7 Hz), 4.59, 4.75 (2H, AB-q, J
11.5 Hz), 4.61, 4.79 (2H, AB-q, J 12.0 Hz), 7.26e7.34 (15H, m). ESI
MS: m/z 737.5 [MþNa]þ. HRESI MS: calcd for C32H50O4Na:
737.5121; observed, 737.5121.
4.1.19. (2S,3R,4R)-2-[(20R,30R,40R)-20,30,40-(Trihydroxy)hex-
acosanoyloxy]-3,4-(dihydroxy)octadecyl
a-D-galactopyranoside (22)
(RCAI-169). A solution of 21 (50 mg, 3.68ꢂ10ꢁ2 mmol) in THF
(40 mL) was stirred for 21 h at room temperature under hydrogen
using 20% Pd(OH)2 on carbon (wet, water ca. 50%, 60 mg) catalyst,
and the reaction mixture was filtered. The catalyst was washed
with THF, and combined THF solution was concentrated in vacuo to
give a white powder, which was washed with Et2O (5 mLꢂ2). This
powder was dried with a pump to give 22 (26 mg, 78%) as a powder.
It was obvious by 2D COSY analysis that the (20,30,40-trihydroxy)
hexacosanoyl group of 22 was on the C2eO position on the octa-
decane chain. However, silica gel column chromatography of 22 by
elution with CHCl3eMeOH (9:1) yields gradually C4eO acyl mi-
4.1.16. (2S,3R,4R)-2-[(20R,30R,40R)-20,30,40-(Tribenzyloxy)hex-
acosanoyloxy]-3,4-(dibenzyloxy)octadecyl 2,3-di-O-(4-
methoxybenzyl)-4,6-O-(di-tert-butyl)silylene-a-D-galactopyranoside
(19). To a solution of 7 (393 mg, 0.377 mmol) and DMAP (919 mg,
7.52 mmol) in dry THFeCH2Cl2 (1:1, 40 mL) were added carboxylic
acid 18 (665 mg, 0.930 mmol) and EDAC (1.44 g, 7.51 mmol). The
mixture was stirred for 3.5 days at room temperature, and diluted
with CHCl3, which was washed with water and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a mixture, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (19:1, then 9:1) gave 19 (498 mg, 76%)
as an oil. IR nmax (KBr) 2924, 2853, 1749, 1615, 1515, 1457,
grated compound. IR nmax (KBr) 3390, 2919, 2851, 1734, 1468 cmꢁ1
.
[a
]
21 þ44.6 (c 0.92, pyridine). 1H NMR (500 MHz, pyridine-d5)
d 0.87
D
(6H, t, J 6.9 Hz), 1.20e1.42 (60H, m), 1.60e1.72 (2H, m), 1.82e2.02
(4H, m), 2.02e2.35 (2H, m), 4.27 (1H, m), 4.37e4.58 (8H, m),
4.60e4.66 (2H, m), 4.93 (1H, dd, J 3.4, 7.6 Hz), 5.19 (1H, d, J 4.5 Hz),
5.53 (1H, d, J 3.5 Hz, anomeric H), 6.40 (1H, m, HC2eOCO). 13C NMR
1249 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (6H, t, J 7.0 Hz), 0.98
(9H, s), 1.01 (9H, s), 1.25 (64H, br s), 1.53e1.58 (4H, m), 3.46e3.92
(17H, m, containing two 3H singlets at 3.69 and 3.75 ppm),
4.30e4.75 (17H, m), 5.47 (1H, dd, J 5.1, 9.7 Hz), 6.70 (2H, d, J
8.8 Hz), 6.81 (2H, d, J 8.6 Hz), 7.15e7.32 (29H, m). ESI MS: m/z
(125 MHz, pyridine-d5)
d 14.33, 22.98, 26.41, 29.66, 29.97, 30.05,
30.08, 30.18, 30.34, 32.17, 34.21, 34.73, 62.72, 66.73, 70.53, 71.06,

9720
M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
71.63, 71.91, 72.38, 72.81, 74.93, 75.35, 77.99, 101.01, 174.04. ESI MS:
m/z 503.3, 929.7 [MþNa]þ. HRESI MS: calcd for C50H98O12Na:
929.6905; observed, 929.6892.
OeBn), 4.32 (1H, d, J 11.9 Hz, H-5-OeBn), 4.51 (2H, s, H-4-OeBn),
4.59 (1H, d, J 11.9 Hz, H-3-OeBn), 4.63 (1H, d, J 11.9 Hz, H-3-OeBn),
5.35 (1H, ddd, J 6.5, 6.5, 1.8 Hz, H-6), 5.47 (1H, ddd, J 6.4, 5.9, 2.7 Hz,
H-2), 7.10e7.61 (25H, m). 13C NMR (150 MHz, CDCl3, ref. 77.00 ppm)
ꢁ5.65, ꢁ5.58, 14.11, 18.12, 22.68, 25.77 (ꢂ3), 26.15, 28.92, 29.35,
29.45, 29.50, 29.52, 29.60, 29.64, 29.67, 31.92, 55.31, 55.64, 61.14,
73.35, 73.66, 74.10, 75.74, 76.07, 79.15, 79.52, 80.79, 84.30, 84.80,
126.89 (ꢂ2), 127.06 (ꢂ2), 127.21, 127.30, 127.39, 127.56, 127.79,
127.83, 128.32, 128.44, 129.47, 129.58, 131.74, 132.25, 137.82, 138.18,
138.27, 166.36, 166.42. ESI MS: m/z 1189.6 [MþNa]þ. HRESI MS:
calcd for C65H84O10F6SiNa: 1189.5636; observed: 1189.5632.
4.1.20. (2R,3S,4R,5S,6S)-3,4,5-Tribenzyloxy-1-tert-butyldimethylsily-
loxyoctadecane-2,6-diol (24) and (2R,3S,4R,5S,6R)-3,4,5-
tribenzyloxy-1-tert-butyldimethylsilyloxyoctadecane-2,6-diol
(240). A solution of 1-bromododecane (20.0 g, 80.25 mmol) in dry
Et2O (100 mL) was gradually added to Mg turnings (2.43 g, 0.10
atom) containing catalytic amount of I2 in a flask equipped with
a reflux condenser at room temperature. After dropping the ether
solution of 1-bromododecane, and ceasing the reflux, the resulting
Grignard solution was left to stand for 30 min. To this solution was
added dropwise a solution of 23 (8.89 g, 15.77 mmol) in Et2O
(50 mL) at room temperature. After ceasing the vigorous reaction,
the mixture was stirred for 1 h at room temperature, and quenched
with satd aq NH4Cl carefully. The mixture was diluted with CHCl3
(1 L), and the whole was washed with water and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (9:1, then 4:1) gave 240 (4.44 g, 38%) as
an oil and 24 (5.09 g, 44%) as an oil. Physical data of 24: Rf¼0.415
(hexaneeEtOAc (4:1); IR nmax (KBr) 3551, 2925, 2854, 1456, 1251,
4.1.22. Mosher’s ester of 24 (24S). The same treatment of 24 (5 mg)
with (R)-(ꢁ)-MTPA chloride (10 mg) afforded 24S (5 mg, 63%) as an
oil. IR nmax (KBr) 2928, 2855, 1747, 1497, 1454, 1258, 1170, 1122, 1021,
838 cmꢁ1. 1H NMR (600 MHz, CDCl3):
d 0.008 (3H, s), 0.017 (3H, s),
0.86 (9H, s), 0.89 (3H, t, J 7.1 Hz), 0.98 (1H, m, H-8a), 1.11(1H, m, H-
8b), 1.09e1.29 (18H, m), 1.63 (1H, m, H-7a), 1.65 (1H, m, H-7b), 3.41
(3H, s), 3.54 (3H, s), 3.62 (1H, dd, J 7.8, 4.1 Hz, H-4), 3.75 (1H, dd, J
10.5, 6.4 Hz, H-1a), 3.76 (1H, dd, J 4.1, 2.7 Hz), 3.88 (1H, dd, J 10.5,
6.9 Hz, H-1b), 3.95 (1H, dd, J 7.8, 3.2 Hz, H-3), 4.27 (1H, d, J 11.0 Hz,
5-OeBn), 4.34 (1H, d, J 11.0 Hz, H-3-OeBn), 4.47 (1H, d, J 11.9 Hz, H-
3-OeBn), 4.53 (2H, d, J 11.0 Hz, H-3-OeBn and H-4-OeBn), 4.59
(1H, d, J 11.9 Hz, H-3-OeBn), 5.40 (1H, ddd, J 9.7, 2.7, 2.7 Hz, H-6),
5.43 (1H, ddd, J 6.9, 6.4, 3.2 Hz, H-2), 7.16e7.60 (25H, m). 13C NMR
(150 MHz, CDCl3, ref. 77.00 ppm) ꢁ5.52, ꢁ5.54, 14.11, 18.12, 22.68,
25.58, 25.76 (ꢂ3), 29.23, 29.33, 29.36, 29.43, 29.49, 29.62, 29.65,
29.67, 31.92, 55.40, 55.48, 61.05, 73.23, 73.59, 74.01, 76.07, 76.35,
77.89, 78.26, 80.24, 84.40, 84.50, 126.95, 127.31, 127.39, 127.43,
127.46, 127.50, 127.76, 128.16, 128.22, 128.32, 128.34, 129.49, 129.52,
131.74, 132.12 (ꢂ2), 137.89, 138.15 (ꢂ2), 166.15, 166.50. ESI MS: m/z
1189.6 [MþNa]þ. HRESI MS: calcd for C65H84O10F6SiNa: 1189.5636;
observed: 1189.5621.
1106, 1065 cmꢁ1. 1H NMR (600 MHz, CDCl3):
d 0.05 (6H, s), 0.88 (3H,
t, J 7.3 Hz), 0.89 (9H, s), 1.26 (18H, m), 1.28 (2H, m), 1.47 (2H, m), 3.60
(1H, dd, J 4.6, 5.5 Hz), 3.63 (1H, dd, J 7.3, 10.1 Hz), 3.69 (1H, dd, J 5.5,
10.1 Hz), 3.84 (1H, ddd, J 4.6, 5.3, 7.3 Hz), 3.90 (1H, dd, J 1.4, 5.6 Hz),
3.96 (1H, ddd, J 1.4, 5.9, 7.3 Hz), 3.99 (1H, dd, J 5.1, 5.5 Hz), 4.57, 4.60
(2H, AB-q, J 11.5 Hz), 4.63, 4.68 (2H, AB-q, J 11.5 Hz), 4.75, 4.80 (2H,
AB-q, J 11.0 Hz), 7.26e7.34 (15H, m). 13C NMR (150 MHz, CDCl3)
ꢁ5.42, ꢁ5.36, 14.11, 18.19, 22.68, 25.73, 25.91 (ꢂ3), 29.36, 29.42,
29.59, 29.60, 29.63, 29.66, 29.69, 31.92, 33.16, 63.49, 71.10, 71.25,
72.91, 73.79, 74.35, 76.57, 79.88, 80.94, 127.70, 127.82 (ꢂ2), 128.06,
128.17 (ꢂ2), 128.43 (ꢂ6), 128.81 (ꢂ2), 128.87 (ꢂ2), 137.94, 138.06,
138.26. ESI MS: m/z 757.48 [MþNa]þ. HRESI MS: calcd for
4.1.23. (2R,3S,4R,5S,6S)-3,4,5-(Tribenzyloxy)octadecane-1,2,6-triol
(25). To a solution of 24 (5.23 g, 7.11 mmol) in THF (140 mL) was
added TBAF (1.0 M in THF, 28 mL). After stirring for 1 h at room
temperature, the reaction mixture was diluted with EtOAc, washed
with water and brine, dried over MgSO4, and filtered. The filtrate
was concentrated in vacuo to give a residue, which was chroma-
tographed on a silica gel column. Elution with hexaneeEtOAc (3:1,
then 1:1) gave 25 (3.11 g, 71%) as an oil. IR nmax (KBr) 3465, 2925,
C
45H70O6SiNa: 757.4839; observed: 757.4841. Physical data of 240:
Rf¼0.537 hexaneeEtOAc (4:1); IR nmax (KBr) 3558, 2926, 2855,1457,
1253, 1108, 1067 cmꢁ1 1H NMR (600 MHz, CDCl3):
.
d
0.03 (3H, s),
0.04 (3H, s), 0.87 (9H, s), 0.88 (3H, t, J 7.3 Hz),1.20 (1H, m),1.26 (16H,
m), 1.28 (2H, m), 1.39 (1H, m), 1.40 (1H, m), 1.54 (1H, m), 3.53 (1H,
dd, J 2.3, 7.3 Hz), 3.60 (1H, ddd, J 2.3, 4.6, 7.8 Hz), 3.86 (1H, ddd, J 1.9,
5.9, 7.3 Hz), 3.90 (1H, dd, J 1.9, 3.2 Hz), 4.11 (1H, dd, J 3.2, 7.3 Hz),
4.59, 4.85 (2H, AB-q, J 11.0 Hz), 4.61, 4.79 (2H, AB-q, J 11.5 Hz), 4.71,
4.79 (2H, AB-q, J 11.0 Hz), 7.27e7.42 (15H, m). 13C NMR (150 MHz,
CDCl3) ꢁ5.41 (ꢂ2),14.11,18.17, 22.68, 25.90 (ꢂ3), 25.96, 29.36, 29.59
(ꢂ2), 29.65 (ꢂ2), 29.68 (ꢂ2), 31.92, 34.74, 63.10, 71.05, 71.96, 73.28,
75.01, 75.04, 76.66, 81.25, 81.50, 127.74, 127.84 (ꢂ2), 127.95 (ꢂ2),
127.98 (ꢂ2), 128.12 (ꢂ2), 128.39 (ꢂ2), 128.41 (ꢂ4), 138.09 (ꢂ2),
138.15. ESI MS: m/z 757.48 [MþNa]þ. HRESI MS: calcd for
2854, 1457, 1065 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (3H, t, J
6.8 Hz), 1.22e1.31 (18H, m), 1.45e1.55 (4H, m), 1.99 (1H, m, OH),
2.47 (1H, d, J 5.6 Hz, OH), 3.13 (1H, d, J 5.9 Hz, OH), 3.55e3.61 (2H,
m), 3.68e3.73 (2H, m), 3.84 (1H, m), 3.96e4.02 (2H, m), 4.52, 4.60
(2H, AB-q, J 11.6 Hz), 4.62, 4.66 (2H, AB-q, J 11.5 Hz), 4.77 (2H, s),
7.26e7.35 (15H, m). ESI MS: m/z 643.4 [MþNa]þ. HRESI MS: calcd
for C39H56O6Na: 643.3975; observed: 643.3953.
C
45H70O6SiNa: 757.4839; observed: 757.4826.
4.1.24. (2R,3S,4R,5S,6S)-3,4,5-Tribenzyloxy-1,2-(isopropylidenedioxy)
octadecan-6-ol (26). A solution of 25 (3.20 g, 5.15 mmol) in 2,2-
dimethoxypropane (60 mL) containing p-toluenesulfonic acid
monohydrate (60 mg) was stirred for 1 h at room temperature, and
diluted with EtOAc, which was washed with satd aq NaHCO3 and
brine, dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to give a crude 26 (3.46 g, quant.), which was employed for
the next reaction without purification. A part of the crude 26
(10 mg) was purified on a preparative TLC plate. Development with
hexaneeEtOAc (4:1) gave 26 (8 mg) as an oil. IR nmax (KBr) 3630,
4.1.21. Mosher’s ester of 24 (24R). (S)-(þ)-
a
-Methoxy-a-tri-
fluoromethylphenylacetyl (MTPA) chloride (10 mg) was added to
a solution of 24 (5 mg) and DMAP (5 mg) in THF (0.2 mL) containing
pyridine (30 mg). After stirring for 15 min at room temperature, the
reaction mixture was diluted with EtOAc, washed with water and
brine, dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to give a residue, which was chromatographed on a TLC
plate. Development with hexaneeEtOAc (9:1) gave 24R (5 mg, 63%)
as an oil. IR nmax (KBr) 2927, 2855, 1746, 1497, 1454, 1256, 1170, 1121,
1019, 838 cmꢁ1. 1H NMR (600 MHz, CDCl3):
d
ꢁ0.023 (3H, s), ꢁ0.015
2925, 2854, 1457, 1067 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (3H,
(3H, s), 0.84 (9H, s), 0.89 (3H, t J 7.1 Hz), 1.07 (1H, m, H-8a), 1.27 (1H,
m, H-8b), 1.12e1.29 (18H, m), 1.75(2H, m, H-7), 3.51 (3H, s), 3.53
(3H, s), 3.70 (1H, dd, J 10.6, 6.4 Hz, H-1a), 3.70 (1H, dd, J 7.4, 4.1 Hz,
H4), 3.79 (1H, dd, J 4.1, 1.8 Hz, H-5), 3.80 (1H, dd, J 10.6, 5.9 Hz, H-
1b), 4.03 (1H, dd, J 7.4, 2.7 Hz, H-3), 4.04 (1H, d, J 11.9 Hz, H-5-
t, J 7.0 Hz), 1.26 (18H, br s), 1.36 (3H, s), 1.41e1.50 (5H, m, containing
3H singlet at 1.44 ppm),1.52e1.60 (2H, m), 2.81 (1H, d, J 5.1 Hz, OH),
3.47 (1H, dd, J 2.9, 6.1 Hz), 3.68e3.73 (2H, m), 3.77 (1H, dd, J 3.0,
6.4 Hz), 3.90 (1H, m), 3.94 (1H, dd, J 6.4, 8.3 Hz), 4.40 (1H, dd, J 6.5,
13.6 Hz), 4.48, 4.77 (2H, AB-q, J 11.5 Hz), 4.55, 4.57 (2H, AB-q, J

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9721
11.6 Hz), 4.59, 4.68 (2H, AB-q, J 11.3 Hz), 7.27e7.33 (15H, m). ESI MS:
m/z 683.4 [MþNa]þ. HRESI MS: calcd for C42H60O6Na: 683.4288;
observed: 683.4289.
(3.30 g, 91%) as an oil. IR nmax (KBr) 2926, 2854, 1734, 1518, 1497,
1455 cmꢁ1. 1H NMR (500 MHz, CDCl3):
0.03 (3H, s), 0.04 (3H, s),
d
0.88e0.93 (12H, m), 1.18e1.45 (21H, m), 1.74 (1H, m), 3.58 (1H, td, J
2.9, 8.8 Hz), 3.92 (1H, dd, J 3.9, 7.1 Hz), 3.97 (1H, dd, J 3.1, 7.1 Hz), 4.12
(1H, d, J 3.9 Hz), 4.40e4.76 (10H, m), 7.26e7.36 (20H, m). ESI MS: m/
z 731.4, 845.5 [MþNa]þ. HRESI MS: calcd for C52H74O6SiNa:
845.5152; observed: 845.5135.
4.1.25. (2R,3S,4R,5S,6S)-3,4,5,6-Tetrabenzyloxy-1,2-(iso-
propylidenedioxy)octadecane (27). To a solution of crude 26 (3.40 g,
5.14 mmol) in dry DMF (30 mL) were added NaH (60% oil disper-
sion, 370 mg, 15.4 mmol) and BnBr (1.76 g, 10.3 mmol) at 0 ꢀC, and
the mixture was stirred for 1 h at room temperature. The reaction
mixture was diluted with EtOAc, which was washed with water and
brine, dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to give a residue, which was chromatographed on a silica
gel column. Elution with hexaneeEtOAc (19:1, then 9:1) gave 27
(3.61 g, 93%, two steps) as an oil. IR nmax (KBr) 2925, 2854, 1517,
4.1.29. (2S,3S,4R,5S,6S)-3,4,5,6-Tetrabenzyloxy-1-(tert-butyldime-
thylsilyloxy)octadecan-2-ol (31). To
a solution of 30 (3.30 g,
3.29 mmol) in dry EtOH (80 mL) was added NaBH4 (powder,
220 mg, 5.82 mmol). The mixture was stirred for 30 min at room
temperature. The reaction mixture was quenched with a few drops
of AcOH, and neutralized with satd aq NaHCO3, and concentrated in
vacuo to give a residue, which was dissolved in EtOAc. The solution
was washed with water, and dried over MgSO4, and filtered. The
filtrate was concentrated in vacuo to give a mixture of 29 and 31
(3.33 g), which was separated on a silica gel column in three parts.
Silica gel (111 g) was used for separation of the mixture (1.11 g).
Elution with hexaneeEtOAc (19:1) gave 31 (total amount of three
times: 1.12 g, 34%) as an oil [Rf¼0.402 (hexaneeEtOAc¼9/1)] and 29
(1.71 g, 52%) as an oil [Rf¼0.348 (hexaneeEtOAc¼9/1)]. Physical
1496, 1455, 1067 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (3H, t, J
6.9 Hz), 1.21e1.30 (18H, m), 1.34 (3H, s), 1.40e1.46 (5H, m, con-
taining 3H singlet at 4.21 ppm), 1.62e1.71 (2H, m), 3.62e3.66 (2H,
m), 3.71e3.75 (2H, m), 3.86 (1H, t, J 3.9 Hz), 3.92 (1H, dd, J 6.5,
8.5 Hz), 4.29 (1H, dd, J 6.4, 14.0 Hz), 4.47 (2H, s), 4.48, 4.67 (2H, AB-
q, J 11.5 Hz), 4.60, 4.73 (2H, AB-q, J 11.5 Hz), 4.60, 4.75 (2H, AB-q, J
11.6 Hz), 7.25e7.32 (20H, m). ESI MS: m/z 773.5 [MþNa]þ. HRESI
MS: calcd for C49H66O6Na: 773.4757; observed: 773.4745.
data of 31: IR nmax (KBr) 3550, 2925, 2854, 1455 cmꢁ1
.
1H NMR
d 0.05 (3H, s), 0.06 (3H, s), 0.88 (3H, t, J 6.8 Hz),
4.1.26. (2R,3S,4R,5S,6S)-3,4,5,6-(Tetrabenzyloxy)octadecane-1,2-diol
(28). To a solution of crude 27 (3.60 g, 4.79 mmol) in CH2Cl2eMeCN
(1:1, 200 mL) was added aq 46% HF (3.0 mL) at room temperature.
The mixture was stirred for 30 min at room temperature, diluted
with CH2Cl2, which was washed with satd aq NaHCO3 and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in
vacuo to give 28 (3.36 g, 99%) as an oil. IR nmax (KBr) 3540, 2925,
(500 MHz, CDCl3):
0.90 (9H, s), 1.20e1.31 (18H, m), 1.42e1.52 (3H, m), 1.80 (1H, m),
2.67 (1H, d, J 4.4 Hz, OH), 3.58 (1H, m), 3.62 (1H, dd, J 2.2, 7.5 Hz),
3.67 (1H, dd, J 6.3, 10.2 Hz), 3.84 (1H, dd, J 3.1, 10.2 Hz), 3.93e3.97
(2H, m), 4.11 (1H, dd, J 3.1, 7.0 Hz), 4.42, 4.56 (2H, AB-q, J 11.7 Hz),
4.51, 4.62 (2H, AB-q, J 11.8 Hz), 4.75, 4.79 (2H, AB-q, J 11.0 Hz), 4.76
(2H, s), 7.23e7.35 (20H, m). ESI MS: m/z 847.5 [MþNa]þ, 848.5.
HRESI MS: calcd for C52H76O6SiNa: 847.5300; observed: 847.5287.
2854, 1515, 1456 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (3H, t, J
7.0 Hz), 1.26 (18H, br s), 1.46e1.48 (2H, m), 1.72e1.78 (2H, m), 1.93
(1H, m, OH), 3.47e3.76 (4H, m, containing 1H doublet J 4.2 Hz at
3.48 ppm, OH), 3.66 (1H, m), 3.85 (1H, dd, J 3.5, 6.2 Hz), 3.88 (1H,
m), 3.94 (1H, dd, J 3.8, 6.5 Hz), 4.43, 4.54 (2H, AB-q, J 11.7 Hz), 4.50,
4.52 (2H, AB-q, J 11.8 Hz), 4.71, 4.79 (2H, AB-q, J 11.5 Hz), 4.73, 4.77
(2H, AB-q, J 11.4 Hz), 7.25e7.33 (20H, m). ESI MS: m/z 733.4
[MþNa]þ. HRESI MS: calcd for C46H62O6Na: 733.4444; observed:
733.4438.
4.1.30. (2S,3S,4R,5S,6S)-3,4,5,6-Tetrabenzyloxyoctadecane-1,2-diol
(32). To a solution of 31 (1.14 g, 7.11 mmol) in THF (50 mL) was
added TBAF (1.0 M in THF, 10 mL). After stirring for 1 h at room
temperature, the reaction mixture was diluted with EtOAc
(350 mL), washed with water and brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to give a residue,
which was chromatographed on a silica gel column. Elution with
hexaneeEtOAc (3:1, then 1:1) gave 32 (845 mg, 86%) as an oil. IR
nmax (KBr) 3450, 2925, 2853, 1456 cmꢁ1. 1H NMR (500 MHz, CDCl3):
4.1.27. (2R,3S,4R,5S,6S)-3,4,5,6-Tetrabenzyloxy-1-(tert-butyldime-
thylsilyloxy)octadecan-2-ol (29). To
a
solution of 28 (3.36 g,
d 0.88 (3H, t, J 6.9 Hz), 1.20e1.30 (19H, m), 1.44 (1H, m), 1.61 (1H, m),
4.70 mmol) in dry CH2Cl2 (200 mL) was added TBDMS-Cl (1.42 g,
9.40 mmol), imidazole (960 mg, 14.1 mmol) and a catalytic amount
of DMAP (10 mg). The mixture was stirred for 16 h at room tem-
perature. The reaction mixture was diluted with CH2Cl2, which was
washed with water and brine, dried over MgSO4, and filtered. The
filtrate was concentrated in vacuo to give a residue, which was
chromatographed on a silica gel column. Elution with hex-
aneeEtOAc (19:1, then 3:2) gave 29 (3.63 g, 93%) as an oil. IR nmax
1.75 (1H, m), 2.15 (1H, t, J 6.4 Hz, OH), 3.19 (1H, d, J 4.7 Hz, OH), 3.55
(1H, quintuplet, J 3.9 Hz), 3.64 (1H, dd, J 2.7, 7.4 Hz), 3.67 (1H, m),
3.76e3.82 (2H, m), 3.84 (1H, m), 3.94 (1H, dd, J 2.7, 5.4 Hz), 4.45,
4.48 (2H, AB-q, J 11.5 Hz), 4.49, 4.56 (2H, AB-q, J 11.5 Hz), 4.69e4.75
(4H, m), 7.23e7.33 (20H, m). ESI MS: m/z 733.4 [MþNa]þ. HRESI
MS: calcd for C46H62O6Na: 733.4444; observed: 733.4438.
4.1.31. (2S,3S,4S,5S,6S)-2-Hydroxy-3,4,5,6-tetrabenzyloxyoctadecyl
(KBr) 3648, 2928, 2855, 1454 cmꢁ1
.
1H NMR (500 MHz, CDCl3):
2,3-di-O-(4-methoxybenzyl)-4,6-O-(di-tert-butyl)silylene-
actopyranoside (33). To a solution of imidate 6 [obtained from 2,3-
di-O-(4-methoxybenzyl)-4,6-O-(di-tert-butyl)silylene- -gal-
a-D-gal-
d
0.04 (6H, s), 0.86e0.90 (12H, m), 1.26 (18H, br s), 1.40e1.52 (2H,
m), 1.73e1.77 (2H, m), 3.51e3.55 (2H, m), 3.63e3.70 (2H, m), 3.76
(1H, d, J 3.8 Hz, OH), 3.89e3.96 (3H, m), 4.46, 4.55 (2H, AB-q, J
11.5 Hz), 4.52 (2H, s), 4.68, 4.82 (2H, AB-q, J 11.4 Hz), 4.74, 4.77 (2H,
AB-q, J 11.0 Hz), 7.25e7.34 (20H, m). ESI MS: m/z 847.5 [MþNa]þ.
HRESI MS: calcd for C52H76O6SiNa: 847.5309; observed: 847.5297.
D
actopyranose (1.10 g, 1.97 mmol) and CCl3CN (2.84 g, 5.55 mmol)
using Cs2CO3 (943 mg) as a base in CH2Cl2 (20 mL) at room tem-
perature over night] and diol 32 (838 mg, 1.18 mmol) in dry CH2Cl2
ꢀ
(60 mL) was added MS 4 A (dry powder, 3.2 g). After stirring for
30 min at room temperature, AgOTf (400 mg, 1.56 mmol) was
added to this mixture. After stirring for 1 h at room temperature,
the reaction mixture was filtered, and the filter cake was washed
with CH2Cl2. The combined filtrate was washed with satd aq
NaHCO3, dried over MgSO4, and filtered. The filtrate was concen-
trated in vacuo to give a mixture, which was chromatographed on
a silica gel column. Elution with hexaneeEtOAc (9:1, then 6:1) gave
33 (568 mg, 38%) as an oil. IR nmax (KBr) 3465, 2926, 2855, 1612,
4.1.28. (3R,4R,5S,6S)-3,4,5,6-Tetrabenzyloxy-1-(tert-butyldimethylsi-
lyloxy)octadecan-2-one (30). A solution of 29 (3.62 g, 4.39 mmol) in
dry CH2Cl2 (80 mL) containing DesseMartin periodinane (3.80 g,
8.96 mmol) was stirred for 2 h at room temperature, and diluted
with CH2Cl2, which was washed with aq 10% Na2S2O3 and satd aq
NaHCO3, dried over MgSO4, and filtered. The filtrate was concen-
trated in vacuo to give a mixture, which was chromatographed on
a silica gel column. Elution with hexaneeEtOAc (19:1) gave 30
1514, 1249 cmꢁ1. 500 MHz 1H NMR (CDCl3)
d 0.88 (3H, t, J 7.0 Hz),

9722
M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
0.99 (9H, s), 1.05 (9H, s), 1.20e1.30 (19H, m), 1.46e1.49 (2H, m), 1.64
(1H, br s),1.74 (1H, m), 3.48 (1H, dd, J 8.1,10.5 Hz), 3.56 (1H, m), 3.62
(1H, s), 3.67 (1H, dd, J 2.9, 6.6 Hz), 3.70 (1H, s), 3.77e3.80 (4H, m,
containing 3H singlet at 3.79 ppm), 3.87 (1H, dd, J 3.0, 6.0 Hz),
3.94e3.97 (2H, m), 4.02e4.03 (2H, m), 4.10e4.15 (2H, m), 4.41, 4.51
(2H, AB-q, J 11.6 Hz), 4.43 (1H, d, J 3.0 Hz), 4.56e4.79 (11H, m,
containing anomeric H), 6.74 (2H, d, J 8.8 Hz), 6.86 (2H, d, J 8.8 Hz),
7.22e7.34 (24H, m). ESI MS: m/z 1275.7 [MþNa]þ. HRESI MS: calcd
for C76H104O13SiNa: 1275.7144; observed: 1275.7131.
then 19:1) gave 36 (168 mg, 74%, two steps) as an oil. IR nmax (KBr)
3446, 2925, 2853, 1733, 1456 cmꢁ1 1H NMR (500 MHz, CDCl3):
0.88 (6H, t, J 7.0 Hz), 1.20e1.32 (62H, m), 1.39 (3H, s), 1.45e1.57
(3H, m), 1.60 (3H, s), 1.69 (1H, m), 2.07 (1H, m, OH), 2.23 (1H, d, J
10.8 Hz, OH), 2.48 (1H, br s, OH), 2.72 (1H, br s, OH), 3.52e3.90 (11H,
m), 4.24 (1H, dd, J 3.0,11.3 Hz), 4.33 (1H, m), 4.41e4.77 (9H, m), 4.85
(1H, d, J 3.9 Hz, anomeric H), 5.61 (1H, td, J 1.2, 8.8 Hz, HC2OC]O),
7.25e7.34 (20H, m). ESI MS: m/z 1345.9 [MþNa]þ, 1346.9. HRESI
MS: calcd for C81H126O14Na: 1345.9045; observed: 1345.9019.
.
d
4.1.32. (2S,3S,4R,5S,6S)-2-[(20R,30R)-20,30-(Isopropylidenedioxy)hex-
acosanoyloxy]-3,4,5,6-(tetrabenzyloxy)octadecyl 2,3-di-O-(4-
4.1.35. (2S,3S,4R,5S,6S)-2-[(20R,30R)-20,30-(Dihydroxy)hex-
acosanoyloxy]-3,4,5,6-(tetrabenzyloxy)octadecyl
a-D-galactopyrano-
methoxybenzyl)-4,6-O-(di-tert-butyl)silylene-
a
-
D
-galactopyranoside
side (37). To a solution of crude 36 (165 mg, 0.125 mmol) in
CH2Cl2eMeCN (1:1, 50 mL) was added aq 46% HF solution (5.4 mL).
The mixture was stirred for 1.5 h at room temperature, and diluted
with CHCl3. The solution was washed with satd aq NaHCO3 and
brine, dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to give a mixture, which was chromatographed on a silica
gel column. Elution with CHCl3eMeOH (19:1) gave 37 (140 mg,
87%) as a gum. IR nmax (KBr) 3425, 2924, 2853, 1741, 1456 cmꢁ1. 1H
(34). To a solution of 33 (553 mg, 0.441 mmol) in dry CH2Cl2eTHF
(1:1, 50 mL) were added DMAP (1.23 g, 10.14 mmol), carboxylic acid
8 (620 mg, 1.32 mmol) and then EDAC (1.69 g, 8.82 mmol) at room
temperature. After stirring for 5 h at room temperature, the re-
action mixture was concentrated in vacuo to give a mixture, which
was dissolved into CH2Cl2. The solution was washed with water and
brine, dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to give a mixture, which was chromatographed on a silica
gel column. Elution with hexaneeEtOAc (14:1, then 9:1) gave 34
(472 mg, 63%) as an oil. IR nmax (KBr) 2925, 2854, 1735, 1614, 1513,
NMR (500 MHz, CDCl3):
d 0.88 (6H, t, J 7.0 Hz), 1.20e1.33 (62H, m),
1.40e1.70 (4H, m), 2.14 (1H, br s, OH), 3.12 (1H, br s, OH), 3.42 (1H,
m), 3.60 (1H, m), 3.56e3.96 (12H, m, containing four OH), 4.05 (1H,
m), 4.21 (1H, d, J 8.6 Hz), 4.31 (1H, t, J 11.4 Hz), 4.41 (1H, d, J 11.5 Hz,
benzylic H), 4.50 (2H, s, benzylic 2H), 4.60e4.73 (6H, m), 4.82 (1H,
d, J 3.5 Hz, anomeric H), 5.80 (1H, d, J 9.5 Hz), 7.21e7.33 (20H, m).
ESI MS: m/z 1305.9 [MþNa]þ, 1306.9. HRESI MS: calcd for
1468, 1457 cmꢁ1. 500 MHz 1H NMR (CDCl3)
d 0.878 (3H, t, J 7.0 Hz),
0.882 (3H, t, J 6.8 Hz), 0.95 (9H, s), 1.01 (9H, s), 1.18e1.27 (62H, m),
1.42e1.48 (3H, m), 1.68 (1H, m), 3.43 (1H, s), 3.59 (1H, m), 3.67 (1H,
dd, J 2.8, 10.1 Hz), 3.74 (1H, s), 3.78e3.90 (10H, m, containing 3H
singlet at 3.78 ppm), 4.07 (1H, dd, J 4.2, 11.3 Hz), 4.24e4.32 (2H, m),
4.35e4.77 (12H, m), 5.62 (1H, m, HC2eOC]O), 6.75 (2H, d, J
8.8 Hz), 6.84 (2H, d, J 8.5 Hz), 7.19e7.31 (24H, m). ESI MS: m/z 1726.1
[MþNa]þ, 1727.1, 1728.1. HRESI MS: calcd for C105H158O16SiNa:
1726.1217; observed: 1726.1215.
C78H122O14Na: 1305.8732; observed: 1305.8723.
4.1.36. (2S,3R,4R,5S,6S)-2-[(20R,30R)-20,30-(Dihydroxy)hex-
acosanoyloxy]-3,4,5,6-(tetrahydroxy)octadecyl -galactopyrano-
a-D
side (38) (RCAI-170). A solution of 37 (68 mg, 0.053 mmol) in THF
(40 mL) was stirred for 16 h at room temperature under hydrogen
using 20% Pd(OH)2 on carbon (wet, water ca. 50%, 74 mg) as a cat-
alyst, and the reaction mixture was filtered. The catalyst was
washed with THF, and combined THF solution was concentrated in
vacuo to give a white powder, which was washed twice with Et2O
(5 mL) by sucking the supernatant Et2O cleaning solution. The
powder was dried with a pump to give 38 (36 mg, 74%) as a powder.
It was obvious by 2D COSY analysis that the (20,30-dihydroxy)hex-
acosanoyl group of 38 was on the C2eO position on the octadecane
4.1.33. (2S,3S,4R,5S,6S)-2-[(20R,30R)-20,30-(Isopropylidenedioxy)hex-
acosanoyloxy]-3,4,5,6-(tetrabenzyloxy)octadecyl 4,6-O-(di-tert-bu-
tyl)silylene-a-D-galactopyranoside (35). To a solution of 34 (468 mg,
0.275 mmol) in CH2Cl2eH2O (10:1, 36.3 mL) was added DDQ
(470 mg, 2.07 mmol). The mixture was stirred for 2.5 h at room
temperature, and diluted with CH2Cl2. The solution was washed
twice with satd aq NaHCO3 and brine, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a residue,
which was chromatographed on a silica gel column. Elution with
hexaneeEtOAc (6:1, then 3:1) gave a 1:1 mixture (255 mg) of 35
and inseparable 4-methoxybenzaldehyde as an oil. IR nmax (KBr)
chain. IR nmax (KBr) 3400, 2920, 2851,1734,1468 cmꢁ1. [
a
]
21 þ40.1 (c
D
0.64, pyridine). 1H NMR (500 MHz, pyridine-d5)
d
0.87 (6H, t, J
7.0 Hz), 1.20e1.40 (58H, m), 1.55e1.68 (2H, m), 1.78e1.95 (3H, m),
2.00 (1H, m), 2.15 (1H, m), 2.26 (1H, m), 4.36 (1H, d, J 5.9 Hz),
4.38e4.45 (4H, m), 4.48e4.54 (4H, m), 4.61e4.65 (2H, m), 4.91 (1H,
dd, J 3.9, 11.2 Hz), 4.96 (1H, d, J 8.8 Hz), 5.00 (1H, dd, J 3.3, 8.4 Hz),
5.48 (1H, d, J 3.7 Hz, anomeric H), 6.40 (1H, quintuplet, J 3.6 Hz,
3567, 2925, 2853, 1734 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (6H,
t, J 7.0 Hz), 0.99 (9H, s), 1.02 (9H, s), 1.20e1.31 (62H, m), 1.38 (3H, s),
1.45e1.56 (3H, m), 1.59 (3H, s), 1.68 (1H, m), 2.26 (1H, d, J 10.0 Hz,
OH), 2.44 (1H, d, J 10.2 Hz, OH), 3.43 (1H, s), 3.52 (1H, dt, J 3.4,
10.1 Hz), 3.58 (1H, m), 3.73 (1H, dd, J 3.6, 9.9 Hz), 3.77e3.86 (4H, m),
3.89 (1H, dd, J 1.7,12.5 Hz), 3.94 (1H, dd, J 2.0,12.5 Hz), 4.18 (1H, dd, J
2.9, 11.2 Hz), 4.21 (1H, d, J 3.4 Hz), 4.32 (1H, m), 4.40e4.75 (9H, m),
4.84 (1H, d, J 3.9 Hz, anomeric H), 5.60 (1H, dd, J 2.3, 8.7 Hz,
HC2OC]O), 7.23e7.33 (20H, m). ESI MS: m/z 1486.0 [MþNa]þ.
HC2eOC]O). 13C NMR (125 MHz, pyridine-d5)
d 14.33, 22.98,
26.39, 29.49, 29.65, 29.96, 30.02, 30.05, 30.07, 30.10, 30.15, 30.17,
30.19, 30.37, 32.16, 34.15, 35.13, 62.64, 66.81, 70.56, 71.00, 71.10,
71.57, 72.36, 72.53, 72.73, 73.89, 73.92, 75.64, 76.90, 101.02, 174.30.
ESI MS: m/z 945.7 [MþNa]þ. HRESI MS: calcd for C50H98O14Na:
945.6854; observed: 945.6839.
HRESI MS: calcd for
1486.0056.
C89H142O14SiNa: 1486.0067; observed:
4.1.37. (S)-2,3-O-Benzylidene-5-O-tert-butyldimethylsilyl-
furanose (40 ), (S)-2,3-O-benzylidene-5-O-tert-butyldimethylsilyl-
-ribofuranose (40 and (R)-2,3-O-benzylidene-5-O-tert-butyldi-
methylsilyl- -ribofuranose (41). To a solution of (R,S)-2,3-O-ben-
zylidene- -ribofuranose (39, 239 mg, 1.00 mmol), obtained from
-ribose according to the reported procedure,12 in dry CH2Cl2
(10 mL) were added imidazole (82 mg, 1.20 mmol) and TBDMS-Cl
(181 mg, 1.20 mmol). This solution was stirred for 45 min at room
temperature, and diluted with CH2Cl2, washed with H2O, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column.
b-L-ribo-
4.1.34. (2S,3S,4R,5S,6S)-2-[(20R,30R)-20,30-(Isopropylidenedioxy)hex-
acosanoyloxy]-3,4,5,6-(tetrabenzyloxy)octadecyl -galactopyrano-
b
a-
a-D
L
a)
side (36). To a solution of 35 (250 mg, 0.170 mmol) in dry THF
(16 mL) containing dry pyridine (140 mg) was added HF$pyridine
(HF: w70%; pyridine: w30%, 70 mg) at room temperature. After
stirring for 1.5 h at room temperature, the reaction mixture was
diluted with CHCl3, washed with satd aq NaHCO3, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (1:1) and then CHCl3eMeOH (47:3,
b-L
b-L
L

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9723
Elution with hexaneeEtOAc (6:1, then 4:1) gave a 10:2:1 in-
NMR (500 MHz, CDCl3):
d
0.08 (3H, s), 0.11 (3H, s), 0.88 (3H, t, J
separable mixture of 40
b
, 40
a
, and 41 (261 mg, 74%) as an oil. IR
7.1 Hz), 0.89 (9H, s), 1.26 (18H, br s), 1.38e1.40 (2H, m), 2.03 (1H, br
s, OH), 2.09 (1H, m), 2.22 (1H, m), 3.71 (1H, m), 3.75 (1H, m), 3.95
(1H, m), 4.71 (1H, dd, J 6.1, 6.8 Hz), 5.05 (1H, dd, J 7.1, 9.1 Hz), 5.61
(1H, dd, J 9.2,10.9 Hz), 5.73 (1H, m), 5.81 (1H, m), 7.36e7.38 (3H, m),
7.48e7.50 (2H, m). ESI MS: m/z 541.37 [MþNa]þ. HRESI MS: calcd
for C31H54O4SiNa: 541.3689; observed: 541.3674. Physical data of
43E: Rf¼0.388 (hexaneeEtOAc¼6:1); IR nmax (KBr) 3628 (br), 2925,
nmax (KBr): 3375, 2960, 2933, 2860, 1463, 1414, 1254 cmꢁ1. ESI MS:
m/z 375.16 [MþNa]þ. HRESI MS: calcd for C18H28O5SiNa: 375.1604;
observed: 375.1592. 1H NMR of 40
b (600 MHz, CDCl3): d 0.16 (3H, s),
0.17 (3H, s), 0.95 (9H, s), 3.80 (1H, dd, J 11.0, 2.3 Hz), 3.83 (1H, dd, J
11.0, 1.8 Hz), 4.54 (1H, dd, J 2.3, 1.8 Hz), 4.61 (1H, d, J 6.4 Hz), 4.79
(1H, d, J 6.4 Hz), 4.87 (1H, d, J 12.0 Hz, OH), 5.45 (1H, d, J 12.0 Hz),
5.77 (1H, s), 7.39 (3H, m), 7.51 (2H, m). 13C NMR (150 MHz, CDCl3)
ꢁ5.67, ꢁ5.64, 18.26, 25.78, 64.86, 82.60, 86.67, 88.25, 103.14, 105.79,
2854,1459, 1250,1069 cmꢁ1; 1H NMR (500 MHz, CDCl3):
d 0.07 (3H,
s), 0.11 (3H, s), 0.88 (3H, t, J 6.9 Hz), 0.90 (9H, s), 1.26 (18H, br s),
1.38e1.41 (2H, m), 2.06e2.10 (3H, m, containing OH), 3.75e3.80
(2H, m), 3.95 (1H, m), 4.24 (1H, dd, J 6.8, 7.1 Hz), 4.69 (1H, dd, J 7.1,
7.3 Hz), 5.60 (1H, dd, J 8.1, 15.4 Hz), 5.82 (1H, m), 7.36e7.39 (3H, m),
7.47e7.50 (2H, m). ESI MS: m/z 541.37 [MþNa]þ. HRESI MS: calcd
for C31H54O4SiNa: 541.3689; observed: 541.3680.
126.96, 128.44, 129.87, 135.84. 1H NMR of 40
a (600 MHz, CDCl3):
d
0.08 (6H, s), 0.91 (9H, s), 3.71 (1H, dd, J 11.0, 2.3 Hz), 3.80 (1H, dd, J
11.0, 1.8 Hz), 3.85 (1H, d, J 11.9 Hz, OH), 4.38 (1H, dd, J 2.3, 1.8 Hz),
4.67 (1H, dd, J 6.4, 4.1 Hz), 4.83 (1H, d, J 6.4 Hz), 5.56 (1H, dd, J 11.9,
4.1 Hz), 5.92 (1H, s), 7.40 (1H, m), 7.42 (2H, m), 7.54 (1H, m). 13C
NMR (150 MHz, CDCl3) ꢁ5.67, ꢁ5.64, 18.26, 25.83, 65.56, 79.98,
80.73, 83.19, 98.21, 106.30,126.83, 128.65, 130.08, 135.59. 1H NMR of
4.1.39. (2S,3S,4R)-[(R)-3,4-Benzylidenedioxy]-1-(tert-butyldime-
thylsilyloxy)octadecan-2-ol (44). A solution of 42EZ (250 mg,
0.482 mmol) in hexane (50 mL) was stirred for 30 min under hy-
drogen using 10% Pd/C (200 mg) at room temperature. The reaction
mixture was filtered, and the catalyst was washed with a small
amount of EtOAc containing 1% Et3N, and the combined filtrate was
concentrated in vacuo to give 44 (240 mg, 96%, stored in a freezer at
ꢁ80 ꢀC) as an oil (at room temperature). IR nmax (KBr) 3558, 2925,
41 1H NMR (600 MHz, CDCl3):
d
3.83 (2H, m), 4.49 (1H, dd, J 2.3,
1.8 Hz), 4.64 (1H, d, J 5.5 Hz), 4.86 (1H, d, J 12.0 Hz, OH), 4.90 (1H, d, J
5.5 Hz), 5.42 (1H, d, J 12.0 Hz), 5.96 (1H, s), 7.39e7.54 (5H, m). 13
C
NMR (150 MHz, CDCl3) ꢁ5.67, ꢁ5.64, 18.26, 25.88, 64.91, 81.52,
86.53, 86.88, 103.01, 103.87, 126.81, 128.54, 129.64, 129.75.
4.1.38. (2S,3S,4R,5EZ)-[(R)-3,4-Benzylidenedioxy]-1-tert-butyldime-
thylsilyloxy-5-octadecen-2-ol (42EZ) and (2S,3S,4R,5EZ)-[(S)-3,4-
Benzylidenedioxy]-1-tert-butyldimethylsilyloxy-5-octadecen-2-ol
(43EZ). To a solution of n-tridecyltriphenylphosphonium bromide
(1.58 g, 3.00 mmol) in dry THF (6 mL) was added a solution of n-
BuLi (1.67 M in hexane, 2.4 mL, 4.01 mmol) at ꢁ10 ꢀC under argon
2854, 1461 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.065 (3H, s), 0.069
(3H, s), 0.88 (3H, t, J 7.1 Hz), 0.90 (9H, s), 1.25 (20H, br s), 1.30e1.90
(6H, m), 2.64 (1H, d, J 5.1 Hz, OH), 3.69 (1H, dd, J 5.7, 9.7 Hz), 3.76
(1H, m), 3.82 (1H, dd, J 3.2, 9.7 Hz), 4.00 (1H, dd, J 6.5, 9.2 Hz), 4.26
(1H, m), 5.76 (1H, s), 7.34e7.39 (3H, m), 7.40e7.47 (2H, m). ESI MS:
m/z 543.39 [MþNa]þ. HRESI MS: calcd for C31H56O4SiNa: 543.3846;
observed: 541.3843.
with stirring. After stirring for 30 min at ꢁ10 ꢀC, a mixture of 40
b,
41 and 40
a
(353 mg, 1.00 mmol) in THF (3 mL) was added at ꢁ10 ꢀC
to the resulting red-colored phosphorane solution. This mixture
was stirred for 20 min at ꢁ10 ꢀC, and then at room temperature for
16 h, and the whole was concentrated under reduced pressure to
one-fifth of the volume, and quenched with water. The mixture was
diluted with EtOAc, washed with water, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to give a mixture,
which was chromatographed on a silica gel column. Elution with
hexaneeEtOAc (19:1, then 9:1) at 8e12 ꢀC or containing 1% Et3N (v/
v) at room temperature gave a 5:6 mixture of 42Z and 42E (370 mg,
71%; or 359 mg, 69%; stored in a freezer at ꢁ80 ꢀC) and a 1:2
mixture of 43Z and 43E (30 mg, 6%). The mixture of 42EZ (15 mg)
was separated using a preparative silica gel TLC plate. Development
with hexaneeEtOAc (9:1) at 5 ꢀC gave Z-isomer (5 mg) and E-iso-
mer (6 mg). Physical data of 42Z: Rf¼0.488 (hexaneeEtOAc¼9:1);
4.1.40. (2S,3S,4R)-[(R)-3,4-Benzylidenedioxy]octadecane-1,2-diol
(45). To a solution of 44 (370 mg, 0.710 mmol) in dry THF (10 mL)
was added TBAF (1 M in THF, 1.40 mL) at room temperature. After
stirring for 45 min, the reaction mixture was concentrated in vacuo
to give a residue, which was diluted with EtOAc. The solution was
washed with H2O and brine, dried over Na2SO4, and filtered, and
the filtrate was concentrated in vacuo to give a residue, which was
chromatographed on a silica gel column. Elution with hex-
aneeEtOAc (6:1, then 3:2) at 8e12 ꢀC or containing 1% Et3N (v/v) at
room temperature gave 45 (280 mg, 97% or 196 mg, 68%, re-
spectively; stored in a freezer at ꢁ80 ꢀC; unstable for the long-term
storage at room temperature) as a solid. IR nmax (KBr) 3349, 2919,
2851, 1465 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (3H, t, J 7.0 Hz),
IR nmax (KBr) 3569 (br), 2924, 2853, 1460, 1254 cmꢁ1
;
1H NMR
1.25 (21H, br s), 1.36 (1H, m), 1.44 (1H, m), 1.63 (1H, m),1.69 (1H, m),
1.85 (1H, m), 1.87 (1H, m, OH), 2.33 (1H, J 5.9 Hz, OH), 3.76 (1H, m),
3.80e3.86 (2H, m), 4.08 (1H, dd, J 6.6, 8.1 Hz), 4.27 (1H, ddd, J 3.4,
6.6, 10.0 Hz), 5.76 (1H, s), 7.37e7.39 (3H, m), 7.44e7.46 (2H, m). ESI
MS: m/z 429.30 [MþNa]þ. HRESI MS: calcd for C25H42O4Na:
429.2981; observed: 429.2974.
(500 MHz, CDCl3): 0.06 (3H, s), 0.07 (3H, s), 0.88 (3H, t, J 7.0 Hz),
d
0.90 (9H, s), 1.25 (18H, br s), 1.38e1.42 (2H, m), 2.12e2.22 (2H, m),
2.52 (1H, d, J 4.9 Hz, OH), 3.72 (1H, dd, J 5.5, 9.7 Hz), 3.80 (1H, m,
C2eH), 3.82 (1H, dd, J 3.2, 9.8 Hz), 4.11 (1H, dd, J 6.7, 8.4 Hz), 5.11
(1H, m), 5.63 (1H, m), 5.76 (1H, td, J 7.5, 11.0 Hz), 5.82 (1H, s),
7.36e7.38 (3H, m), 7.47e7.48 (2H, m). HRESI MS: calcd for
C
31H54O4SiNa: 541.3689; observed: 541.3674. Physical data of 42E:
Rf¼0.418 (hexaneeEtOAc¼9:1); IR nmax (KBr) 3569 (br), 2925, 2854,
1458, 1254 cmꢁ1; 1H NMR (500 MHz, CDCl3):
0.070 (3H, s), 0.074
4.1.41. (2S,3S,4R)-[(R)-3,4-Benzylidenedioxy]-2-hydroxyoctadecyl
2,3-di-O-(4-methoxybenzyl)-4,6-O-(di-tert-butyl)silylene-
actopyranoside (46). To a solution of imidate 6 [obtained from 2,3-
di-O-(4-methoxybenzyl)-4,6-O-(di-tert-butyl)silylene- -gal-
a-D-gal-
d
(3H, s), 0.88 (3H, t, J 6.8 Hz), 0.91 (9H, s), 1.25 (18H, br s), 1.38e1.41
(2H, m), 2.10 (2H, q, J 6.8 Hz), 2.52 (1H, d, J 4.9 Hz, OH), 3.72e3.84
(3H, m), 4.09 (1H, dd, J 6.8, 8.5 Hz), 4.74 (1H, t, J 7.1 Hz), 5.69 (1H, dd,
J 7.5,15.4 Hz), 5.81 (1H, s), 5.88 (1H, td, J 6.8,15.4 Hz), 7.38e7.40 (3H,
m), 7.47e7.49 (2H, m). ESI MS: m/z 541.37 [MþNa]þ. ESI MS: m/z
541.37 [MþNa]þ. HRESI MS: calcd for C31H54O4SiNa: 541.3689;
observed: 541.3676. The mixture of 43EZ (30 mg) was separated
using a preparative silica gel TLC plate. Development with hex-
aneeEtOAc (6:1) at 5 ꢀC gave Z-isomer (5 mg) and E-isomer
(10 mg). Physical data of 43Z: Rf¼0.425 (hexaneeEtOAc¼6:1); IR
D
actopyranose (620 mg, 1.11 mmol) and CCl3CN using Cs2CO3 as
a base] and diol 45 (135 mg, 0.332 mmol) in dry CH2Cl2 (13 mL) was
added.
ꢀ
MS 4 A (dry powder, 1.3 g) under argon. After stirring for 30 min
at room temperature, the mixture was cooled at 0 ꢀC, and to this
solution AgOTf (100 mg, 0.389 mmol) was added. After stirring for
30 min at 0 ꢀC and for 4 h at room temperature, the solution was
filtered, and the filter cake was washed with CH2Cl2. The combined
filtrate was washed with satd aq NaHCO3 and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
nmax (KBr) 3564 (br), 2925, 2854, 1462, 1251, 1094, 1068 cmꢁ1 1H
;

9724
M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
a mixture, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (6:1, then 4:1) at 8e12 ꢀC or containing
1% Et3N (v/v) at room temperature gave 46 (275 mg, 87% or 284 mg,
90%, respectively; stored in a freezer at ꢁ80 ꢀC) as a gum. IR nmax
(KBr): 3440, 2927, 2855, 1737, 1613, 1513, 1465, 1250 cmꢁ1. 1H NMR
2919, 2850, 1732, 1469 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (6H,
t, J 7.0 Hz), 1.25 (66H, br s), 1.38e1.62 (6H, m), 2.10 (4H, br, OH),
2.29e2.33 (2H, m), 3.64 (1H, dd, J 5.6, 11.5 Hz), 3.71 (1H, dd, J 3.3,
11.8 Hz), 3.75e3.79 (3H, m), 3.85 (1H, m), 4.00 (1H, dd, J 2.2,
11.3 Hz), 4.05 (1H, d, J 2.9 Hz), 4.21 (1H, m), 4.28 (1H, dd, J 6.2,
8.2 Hz), 4.85 (1H, d, J 3.7 Hz, anomeric H), 5.10 (1H, m), 5.77 (1H, s),
7.38e7.40 (3H, m), 7.43e7.45 (2H, m). ESI MS: m/z 969.7 [MþNa]þ.
HRESI MS: calcd for C57H102O10Na: 969.7371; observed: 969.7361.
(500 MHz, CDCl3):
d 0.88 (3H, t, J 7.1 Hz), 0.98 (9H, s), 1.05 (9H, s),
1.26 (21H, br s), 1.40e1.84 (5H, m), 3.43 (1H, dd, J 8.0, 10.9 Hz), 3.61
(1H, s), 3.77e3.82 (8H, m), 3.87 (1H, m), 3.94e4.00 (2H, m), 4.05
(1H, dd, J 1.6, 12.5 Hz), 4.16 (1H, dd, J 2.0, 12.5 Hz), 4.20 (1H, m), 4.48
(1H, d, J 2.9 Hz), 4.60, 4.80 (2H, AB-q, J 11.3 Hz), 4.61, 4.67 (2H, AB-q,
J 11.3 Hz), 4.75 (1H, d, J 3.6 Hz, anomeric H), 5.72 (1H, s), 6.84 (2H, d J
8.6 Hz), 6.89 (2H, d J 8.6 Hz), 7.33e7.43 (4H, m). ESI MS: m/z 971.6
[MþNa]þ. HRESI MS: calcd for C55H84O11SiNa, 971.5681; observed,
971.5660.
4.1.45. (2S,3S,4R)-3,4-Dihydroxy-2-(hexacosanoyloxy)octadecyl
a-D-
galactopyranoside (50) (RCAI-80). To a solution of 49 (24 mg,
0.025 mmol) in THF (10 mL) was added Pd(OH)2/C (20 wt. %,
Degussa type, wet, w50%, 30 mg). After stirring for 4 h under hy-
drogen at room temperature, the catalyst was filtered, and washed
with THF, and then CHCl3eMeOH (5:1). The combined filtrate was
concentrated in vacuo to give a white powder, which was washed
with EtOAc to give 50 (19 mg, 87%), or the powder was chroma-
tographed on a silica gel (1 g) column. Elution with CHCl3eMeOH
(19:1, then 9:1) gave 50 (11 mg, 51%) as a powder. The 1H NMR data
of 50 were identical with those of previously reported data of RCAI-
80.6
4.1.42. (2S,3S,4R)-[(R)-3,4-Benzylidenedioxy]-2-(hexacosanoyloxy)
octadecyl 2,3-di-O-(4-methoxybenzyl)-4,6-O-(di-tert-butyl)silylene-
a-D
-galactopyranoside (47). To
a
solution of 46 (54 mg,
5.69ꢂ10ꢁ2 mmol) in THFeCH2Cl2 (1:1, 8 mL) were added DMAP
(160 mg, 1.31 mmol, 23 equiv), n-hexacosanoic acid (68 mg,
1.71ꢂ10ꢁ1 mmol, 3 equiv) and EDAC (218 mg, 1.14 mmol, 20 equiv).
The mixture was stirred for 4 days at room temperature, and di-
luted with CHCl3. The solution was washed with H2O and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in
vacuo to give a mixture, which was chromatographed on a silica gel
column. Elution with hexaneeEtOAc (9:1, then 4:1) gave 47 (72 mg,
95%) as a viscous oil. IR nmax (KBr) 2925, 2853, 1741, 1613, 1512,
4.2. Methods for measurement of biological activity
4.2.1. Bioassay (mouse in vivo).13 In vivo experiment. The stock
solutions (1.0 mg/mL in DMSO) of
were diluted to 10 g/mL in Dulbecco’s phosphate buffered saline
(Sigma, Product No. D8537) just before injection into mice. Each
glycolipid solution (10 g/mL, 200 L) was administered in-
aGalCer and synthesized samples
m
1466 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (6H, t, J 7.0 Hz), 0.97
(9H, s), 1.04 (9H, s), 1.25 (66H, br s), 3.73e3.77 (4H, m, containing
3H, singlet at 3.76 ppm), 3.81 (3H, s), 3.84 (1H, dd, J 2.4, 11.5 Hz),
3.91 (1H, dd, J 3.5, 10.1 Hz), 4.02 (1H, dd, J 1.4, 12.4 Hz), 4.12e4.18
(2H, m), 4.39 (1H, dd, J 6.4, 8.1 Hz), 4.44 (1H, d, J 2.9 Hz), 4.57, 4.72
(2H, AB-q, J 11.5 Hz), 4.64 (2H, s), 4.69 (1H, d, J 3.4 Hz, anomeric H),
5.05 (1H, m), 5.74 (1H, s), 6.78 (2H, d, J 8.8 Hz), 6.87 (1H, d, J 8.8 Hz),
7.26e7.46 (9H, m). ESI MS: m/z 1349.9 [MþNa]þ. HRESI MS: calcd
for C81H134O12SiNa: 1349.9542; observed: 1349.9542.
m
m
travenously. Peripheral blood was collected from the retro-orbital
plexus of mice at indicated time points, using heparin-coated
capillary tubes (Funakoshi Pharmaceutical, Japan), and plasma
was prepared.
4.2.2. Cytokine measurement. The cytokine concentrations in
plasma were quantified by cytometric bead array (CBA) system (BD
Biosciences) for mouse IFNg and IL-4 according to the manufac-
turer’s protocol.
4.1.43. (2S,3S,4R)-[(R)-3,4-Benzylidenedioxy]-2-(hexacosanoyloxy)
octadecyl
4,6-O-(di-tert-butyl)silylene-a-D-galactopyranoside
(48). To a solution of 47 (110 mg, 0.083 mmol) in CH2Cl2eH2O
(10:1,11 mL) was added DDQ (110 mg, 0.48 mmol). The mixture was
stirred for 2.5 h at room temperature, and diluted with CH2Cl2. The
solution was washed twice with aq satd NaHCO3 and brine, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo to
give a residue, which was chromatographed on a silica gel column.
Elution with hexaneeEtOAc (9:1, then 3:1) gave 48 (77 mg, 85%) as
a gum. IR nmax (KBr) 3535 (br), 2922, 2851, 1733, 1470, 1168 cmꢁ1. 1H
4.2.3. Experimental autoimmune encephalomyelitis (EAE) induction
-
by active immunization in C57BL/6 (B6) mice, and Clinical Score..7,8
EAE is induced in B6 female mice by immunization with an
emulsion of MOG35-55 peptide in complete Freund’s adjuvant
(CFA). MOG in CFA (Hooke Lab) solution (200
cutaneously injected at two sites on lower back (one injection over
each hip/base of tail) with 100 L of emulsion at each site on day 1.
5 ng/mL pertussis toxin (200 L in PBS) was intraperitoneally
mL) was sub-
m
m
NMR (500 MHz, CDCl3): d 0.88 (6H, t, J 6.8 Hz), 1.00 (9H, s), 1.04 (9H,
injected on day 1 and day 3. Mice were observed daily until day 30
for clinical score as 0e5 graduations with 0.5 for intermediate
scores. 0: no clinical signs, 1: flaccid tail, 2: hind limb weakness or
abnormal gait, 3: complete hind limb paralysis, 4: complete hind
limb paralysisþforelimb weakness or paralysis, 5: moribund or
deceased. In order to analyze the effect of glycolipid on EAE, each
glycolipd-pulsed GM-CSF induced DC (GM-DC) (5ꢂ105 cells/
mouse) were intravenously injected on day 5 and day 3. GM-DC
were induced by bone marrow by culturing with GM-CSF (10 ng/
mL) for 5 days, and then enriched by CD11c MACS beads (Miltenyi
Biotec). Each glycolipid (100 ng/mL) was pulsed by culturing GM-
DC (5ꢂ106/mL) for 24 h.
s), 1.25 (66H, s), 1.27e1.42 (2H, m), 1.50e1.63 (4H, m), 2.27e2.32
(2H, m), 3.62e3.68 (3H, m), 3.75 (1H, m, changed to dd, J 4.0, 9.8 Hz
on addition of D2O), 3.96 (1H, dd, J 2.2, 11.3 Hz), 4.08 (1H, dd, J 1.5,
12.5 Hz), 4.17e4.23 (2H, m), 4.27 (1H, dd, J 6.1, 8.8 Hz), 4.39 (1H, d, J
3.2 Hz), 4.39 (1H, d, J 3.2 Hz), 4.84 (1H, d, J 3.9 Hz, anomeric H), 5.09
(1H, m), 7.39e7.45 (5H, m). ESI MS: m/z 1109.8 [MþNa]þ. HRESI MS:
calcd for C65H118O10SiNa: 1109.8392; observed: 1109.8394.
4.1.44. (2S,3S,4R)-[(R)-3,4-Benzylidenedioxy]-2-(hexacosanoyloxy)
octadecyl
a-D-galactopyranoside (49). To a solution of 48 (75 mg,
0.069 mmol) in dry THF (7 mL) containing dry pyridine (60 mg) was
added HF$pyridine (HF: w70%; pyridine: w30%, 58 mg, ca.
2.03 mmol) under argon at room temperature. After stirring for
2.5 h, the reaction mixture was diluted with CHCl3, washed with
satd aq NaHCO3, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a mixture, which was chromato-
graphed on a silica gel column. Elution with hexaneeEtOAc (2:3)
and EtOAc gave 49 (52 mg, 80%) as a powder. IR nmax (KBr) 3440,
Acknowledgements
We thank Drs. Takemichi Nakamura and Yayoi Hongo, The In-
stitute of Physical and Chemical Research (RIKEN), for measure-
ments of mass spectra.

M. Shiozaki et al. / Tetrahedron 69 (2013) 9710e9725
9725
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