Synthesis and biological activity of hydroxylated analogues of KRN7000 (α-galactosylceramide)

Article abstract of DOI:10.1016/j.carres.2013.01.010

KRN7000 is one of the α-galactosylceramides, which has a 2-hexacosanoylamino-3,4-dihydroxyoctadecyl group. This compound, known as a ligand for the activation of CD1d mediated invariant natural killer T cells (iNKT cells) which release both T helper 1 (Th

Full text of DOI:10.1016/j.carres.2013.01.010

Carbohydrate Research 370 (2013) 46–66
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and biological activity of hydroxylated analogues of
KRN7000 (
a-galactosylceramide)
a
b
c
c
Masao Shiozaki a, , Takuya Tashiro , Hiroyuki Koshino , Tomokuni Shigeura , Hiroshi Watarai ,
⇑
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:
KRN7000 is one of the a-galactosylceramides, which has a 2-hexacosanoylamino-3,4-dihydroxyoctadecyl
Received 12 November 2012
Received in revised form 15 January 2013
Accepted 16 January 2013
Available online 4 February 2013
group. This compound, known as a ligand for the activation of CD1d mediated invariant natural killer T
cells (iNKT cells) which release both T helper 1 (Th1) cytokines such as IFN and Th2 cytokines such as
IL-4, has been anticipated as an antitumor drug, because of its strong secretion of IFN . This time, we
c
c
focused on the hydroxylated analogues of KRN7000 which could be thought of as increasing hydrophilic-
ity and showing bias to Th2 cytokine (IL-4) secretion. Therefore, they may become the drugs for autoim-
mune diseases for the following reasons: (i) compound OCH, one of the a-galactosylceramide analogues
Keywords:
CD1d
with a shorter sphingosine chain than KRN7000, increases hydrophilicity relative to KRN7000; and (ii)
OCH is known to induce much more Th2 cytokines (IL-4) than Th1 cytokines from iNKT cells compared
to KRN7000. Naturally, OCH has become one of the candidate drugs for autoimmune diseases. The more
hydroxylated derivatives of KRN7000 are anticipated to induce Th2 bias. Therefore, eight analogues with
1–4 excess hydroxyl groups on the lipid chain of KRN7000 were synthesized to examine if they behave in
the same way as OCH. As a result, three out of eight compounds biased largely to IL-4 secretion, and their
effectiveness for experimental autoimmune encephalomyelitis (EAE) was examined. It was recognized
that two compounds RCAI-147/-160 showed good suppression of EAE symptoms.
iNKT cells
Glycosylation
EAE
Th1/Th2
Agelasphins
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
also the ester analogue of KRN7000, namely RCAI-80,6 biased lar-
gely the cytokine secretion from iNKT cells to Th2. However, in
Agelasphins are a series of
a
-galactosylceramides isolated from
comparison to OCH, the induced Th2 bias of RCAI-80 was fairly
a marine sponge (Agelas mauritianus), and they showed potent ef-
lower than that of OCH.5
fects on the immune system of mice.1 The compound known as
The compound OCH has been a potential candidate agent for
the treatment of autoimmune diseases, immunosuppressant in
association with organ transplantation, therapy of inflammatory
conditions, etc. It will be considered that the lipophilicity of a trun-
cated analogue of KRN7000 is decreasing. If this is true, reversely,
this may mean that the hydrophilicity of OCH is increasing. It is
known that KRN7000 forms the hydrogen bonding between the
amide N–H hydrogen on the phytosphingosine and the oxygen
on the hydroxyl group of the adjacent threonine-156 residue of
mouse CD1d (mCD1d) (or threonine-154 residue of human
CD1d). And also we can recognize from the X-ray crystallographic
analysis7 that crucial hydrogen bonding interactions were formed
between mCD1d residue Arg79 and the 30-OH of KRN7000 phyto-
sphingosine, and residue Asp80 showed hydrogen bonds with both
30- and 40-OH of the phytosphingosine. X-ray crystallographic anal-
ysis reveals the presence of mCD1d residue Asp80 formed the same
hydrogen bond with both 30- and 40-OH of compound OCH in the
KRN70002 (one of the
a-galactosylceramides) was developed by
Kirin Brewery Co. through synthetic studies on agelasphins. Invari-
ant natural killer T cells (iNKT cells)3 produce immunoregulatory
cytokines such as IFNc (Th1 type) and IL-4 or IL-10 (Th2 type) by
the recognition of glycolipid antigens such as KRN7000 bound by
a major histocompatibility complex (MHC) class I-like protein,
CD1d.4 These cytokines secreted by iNKT cells hold the promise
of the therapeutic use for anticancer drugs or immunosuppressive
agents and autoimmune disease treatment.
This time, we were interested in the compound OCH reported
by Miyamoto et al.,5 a truncated analogue of KRN7000 as shown
in Figure 1, because it appears to induce a Th2 bias. In addition,
⇑
Corresponding author.
E-mail address: shiozaki@rcai.riken.jp (M. Shiozaki).
RCAI- is a code number.
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.01.010

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
47
OH
as a mixture of E- and Z-isomers. Reduction of the double bond
in 4 under hydrogen using Pd on carbon (Pd/C) in hexane yielded
alcohol 5. Treatment of 5 with diethyl azodicarboxylate (DEAD)
and triphenylphosphine (Ph3P) in tetrahydrofuran (THF), and then
with diphenylphosphoryl azide [(PhO)2P(O)N3] gave C2-azide 6
with inversion of the configuration from R to S at C2-OH position.
Deprotection of TBDMS group from 6 with n-tetrabutylammonium
fluoride (n-Bu4NF) in THF afforded primary alcohol 7 as a glycosyl
acceptor.
OH
O
O
C22H45
OH
HO
HO
HO
HO
C25H51
OH
O
O
HN
HN
OH
HO
HO
O
O
(CH2)11CH(CH3)2
C14H29
OH
OH
Agelasphin-9b
KRN7000 (αGalCer)
On the other hand, the reported compound 8,6 which was
converted into imidate of compound 9 as a glycosyl donor, was
benzylated with benzyl bromide (BnBr) and NaH as a base in
N,N-dimethylformamide (DMF) to afford 2,3-di-O-benzylated com-
pound of 8, and then the anomeric S-tolyl group was deprotected
with N-bromosuccinimide (NBS) in acetone to give 9, which was
converted into the imidate of 9 with trichloroacetonitrile (CCl3CN)
using cesium carbonate (Cs2CO3) as a base.
OH
OH
O
O
O
HO
HO
HO
HO
C23H47
OH
C5H11
C25H51
OH
O
O
HN
HO
HO
O
O
C14H29
OH
OH
OCH
RCAI-80
The glycosylation reaction of glycosyl acceptor 7 with this imi-
date of 9 was performed by use of silver trifluoromethanesulfonate
Figure 1. Structures of agelasphin-9b, KRN7000 (aGalCer), OCH, and RCAI-80.
(AgOTf) as a catalyst to give a-anomer 10 quantitatively. The azide
of 10 was reduced to amine 11 by treatment with trimethylphos-
phine (Me3P) in THF, then with aqueous 1 M NaOH. A condensation
reaction of amine 11 with hexacosanoic acid (n-C25H51COOH)
using N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride (EDAC) as a dehydrating agent in THF–CH2Cl2 (1:1) gave
the amide 12. Treatment of 12 with HFꢀpyridine in THF–pyridine
gave 13 (92%). Five benzyl groups of 13 were hydrogenolyzed using
Pd(OH)2 on carbon [Pd(OH)2/C] in THF to give 14 (RCAI-104) in 47%
yield.
ternary TCR/OCH/CD1d complex.8 Introduction of an additional or
multiple hydroxyl groups on the phytosphingosine main chain
and/or amide side chain of KRN7000, may influence to simply in-
crease the hydrophilicity of the compound without hydrogen
bonding. Do these hydroxylated KRN7000 analogues bias toward
Th2 cytokine secretion compared to KRN7000?
Therefore, eight hydroxylated KRN7000 analogues were synthe-
sized to verify this hypothesis.
Secondly, we synthesized compound 30 (RCAI-120), which has
another (6S)-hydroxyl group on the main-chain carbon adjacent
to the C5-hydroxyl group of compound 14 as shown in Schemes
2–4.
2. Results and discussion
2.1. Synthesis
The known starting material 15,10 converted into the glycosyl
acceptor 23 in eight steps, was changed to diol 16 (63% yield,
unstable at room temperature) by osmium tetroxide (OsO4)
oxidation in acetone–H2O (4:1) containing each 1.2 equiv of
4-methylmorpholine N-oxide and p-toluenesulfonic acid (TsOH).
Treatment of 16 with tert-butyldimethylsilyl trifluoromethanesul-
fonate (TBDMSOTf) using 2,6-lutidine as a base gave 17 (96%, sta-
ble at room temperature).
Stereochemistry of the syn-diol portion of 16 was determined
by detailed NMR analysis of tri-TBDMS lactone derivative 17. Com-
plete 1H and 13C NMR assignments were carried out by analysis of
2D DQF-COSY, 1H–13C HSQC, and HMBC, and also 1H–29Si HMBC
data of 17. Relative stereochemistry of the oxygenated oxepan-
2-one ring was confirmed by NOE correlations between an isopro-
pylidene methyl at 1.48 ppm and silyloxyl methines (H-3 and H-4),
Firstly, we synthesized compound 14, which has another (5S)-
hydroxyl group on the phytosphingosine main-chain carbon adja-
cent to the C4-hydroxyl group of KRN7000. It should be possible to
synthesize 14 via compound 10 obtained by coupling reaction of
alcohol 7 and imidoyl derivative of 9 as shown in Schemes 1 and 2.
Compound 1, which was converted into the primary alcohol 7
as a glycosyl accepter, was treated with acetic anhydride contain-
ing catalytic amount of conc. sulfuric acid to give 1,6-diacetate of
2.9 The diacetate was further converted into 2 in 95% yield by
transesterification reaction in MeOH using KOH as a catalyst. Com-
pound
2 was treated with tert-butyldimethylsilyl chloride
(TBDMS-Cl) using imidazole as a base to give TBDMS ether 3. Wit-
tig reaction of 3 with the phosphorane derived from n-dodecyltri-
phenylphosphonium bromide [Ph3P(Br)C12H25] and n-BuLi gave 4
OBn
BnO
OH
OTBDMS
BnO
BnO
BnO
BnO
OH OBn
a
b
c
O
O
O
TBDMSO
C11H23
BnO
OH
OH
BnO
OMe
BnO
BnO
BnO
OBn
4
1
2
3
N3 OBn
OH OBn
N3 OBn
d
e
f
TBDMSO
C13H27
TBDMSO
C13H27
HO
C13H27
BnO
OBn
BnO
OBn
BnO
OBn
5
6
7
Scheme 1. Reagents and conditions: (a) (1) Ac2O, concd H2SO4 (catalytic amount), rt, 1 h, (2) KOH (catalytic amount), MeOH, rt, 1 h, two steps 95%; (b) TBDMSCl, imidazole,
CH2Cl2, rt, 1.5 h, 78%; (c) Ph3P(Br)C12H25, n-BuLi (1.6 M in hexane), THF, ꢁ10 °C, then 3, ꢁ10 °C ? rt, 16 h, 45%; (d) H2, Pd/C, hexane, rt, 50 min, 71%; (e) DEAD, Ph3P,
(PhO)2P(O)N3, THF, Ar, ꢁ10 ? 0 °C, 30 min, then rt, 3 h, 78%; (f) n-Bu4NF, THF, rt, 30 min, 93%.

48
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
t-Bu
t-Bu
t-Bu
Si
Si
Si
t-Bu
t-Bu
O
O
t-Bu
O
t-Bu
O
O
O
O
a
b
c
O
O
O
HO
S
BnO
BnO
N3 OBn
OH
BnO
BnO
HO
O
C13H27
9
8
10
BnO
OBn
t-Bu
t-Bu
Si
Si
t-Bu
O
OH
HO
O
O
O
O
d
e
O
O
O
C25H51
OBn
C25H51
OBn
BnO
BnO
BnO
NH2 OBn
HN
HN
BnO
BnO
BnO
O
O
O
C13H27
C13H27
C13H27
BnO
OBn
BnO
OBn
BnO
OBn
11
12
13
OH
HO
O
f
O
C25H51
OH
HO
HN
HO
O
C13H27
HO
OH
14 (RCAI-104)
Scheme 2. Reagents and conditions: (a) (1) BnBr, NaH, DMF, Ar, 0 °C, 15 min, then rt, 15 min, quant, (2) NBS, acetone, ꢁ20 °C, 40 min, then quenched with satd aq NaHCO3,
65%; (b) (1) CCl3CN, Cs2CO3, CH2Cl2, rt, 16 h, (2) 7, MS 4 Å, CH2Cl2, rt, 30 min, then 0 °C, AgOTf, 1 h, and rt, 16 h, quant; (c) Me3P, THF, rt, 2 h, then aq 1 M NaOH, rt, 2 h, 77%; (d)
n-C25H51COOH, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 1.5 h, 70%; (e) HFꢀpyridine, pyridine, THF, Ar, rt, 1 h, 92%; (f) H2, Pd(OH)2/C, THF, rt, 24 h, 47%.
O
O
O
OH
OH
OTBDMS
OTBDMS
O
O
O
O
a
b
c
TBDMSO
TBDMSO
TBDMSO
O
O
O
O
O
15
16
17
OH
OH
OTBDMS
OMs
O
OTBDMS
OTBDMS
OTBDMS
O
O
C10H21
C10H21
TBDMSO
TBDMSO
d
e
TBDMSO
O
O
O
OTBDMS
OTBDMS
O
19
20
18
OMs
OTBDMS
OMs
O
OTBDMS
N3
OTBDMS
C12H25
C10H21
C12H25
HO
HO
HO
f
g
h
O
O
O
O
OTBDMS
O
OTBDMS
OTBDMS
21
22
23
Scheme 3. Reagents and conditions: (a) OsO4, 4-methylmorpholine N-oxide, p-TsOH, 0 °C, 15 min, then rt, 16 h, 16 (63%) and recovery 15 (11%); (b) t-BuMe2SiOTf, 2,6-
lutidine, CH2Cl2, rt, 16 h, 96%; (c) DIBAH, toluene, ꢁ78 °C, 45 min, 96%; (d) 4 equiv Ph3P(Br)C11H23, THF, ꢁ30 °C, then LiN(SiMe3)2, ꢁ30 °C ? ꢁ10 °C over 20 min, ꢁ10 °C,
15 min, then rt, 3 h, 54%; (e) (MeSO2)2O, pyridine, CH2Cl2, rt, 45 min, 77%; (f) HFꢀpyridine, pyridine, THF, 0 °C, 5 min, and rt, 5 h, 62%; (g) H2, Pd/C, EtOAc, rt, 1 h, 100%; (h)
NaN3, DMF, 100 °C, 14 h, 76%.
and also between another methyl at 1.36 ppm and methines at-
tached with the isopropylidene group (H-5 and H-6).
The lactone carbonyl of 17 was reduced to hemiacetal 18
with diisobutylaluminum hydride (DIBAH) in toluene at ꢁ78 °C
for 45 min in 96% yield. Wittig reaction of 18 with the phosphorane
derived from n-undecyltriphenylphosphonium bromide [Ph3P
(Br)C11H23] and lithium bis(trimethylsilyl)amide gave 19 as a mix-
ture of E- and Z-isomers. Mesylation of 19 with methanesulfonic
anhydride in dichloromethane (CH2Cl2) using pyridine as a base
gave 20. Selective desilylation of primary silyl ether of 20 by

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
49
t-Bu
t-Bu
t-Bu
Si
Si
Si
t-Bu
O
t-Bu
O
t-Bu
O
O
O
O
a
b
O
O
O
PMBO
PMBO
PMBO
N3
OTBDMS
C12H25
NH2
O
OTBDMS
C12H25
PMBO
PMBO
PMBO
OH
O
O
24
O
OTBDMS
O
O
OTBDMS
25
26
t-Bu
Si
t-Bu
Si
O
t-Bu
O
t-Bu
O
O
O
O
c
d
e
C25H51
OTBDMS
C12H25
C25H51
OTBDMS
C12H25
O
O
PMBO
HO
HN
HN
PMBO
HO
O
O
O
O
O
OTBDMS
O
OTBDMS
27
28
OH
HO
OH
HO
O
O
f
C25H51
OTBDMS
C12H25
C25H51
OH OH
O
O
HO
HO
HN
HN
HO
HO
O
O
C12H25
O
O
OH
OH
OTBDMS
29
30 (RCAI-120)
Scheme 4. Reagents and conditions: (a) (1) CCl3CN, Cs2CO3, CH2Cl2, rt, 16 h, (2) 23, MS 4 Å, CH2Cl2, rt, 30 min, then 0 °C, AgOTf, 1 h, and rt, 1.5 h, 78%; (b) Me3P, THF, rt, 2 h,
then aq 1 M NaOH, rt, 2 h, 77%; (c) n-C25H51COOH, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 3 h, 86%; (d) DDQ, CH2Cl2–H2O (10:1), rt, 2 h, 85%; (e) HFꢀpyridine, pyridine, THF, rt, 1 h,
85%; (f) aq 46% HF, CH2Cl2–MeCN (1:1), H2O, rt, 16 h, 92%.
a
b
c
BnOOC
C20H41
HOOC
O
OH
O
BnOOC
BnOOC
C20H41
O
O
O
32
OH
33
34
31 (97% ee)
d
e
BnOOC
HOOC
C24H49
C24H49
OTBDMS
OTBDMS
35
36 (97.9% ee)
O
O
H
H
N
NH2
NH2
N
C24H49
OTBDMS
OTBDMS
f
f
40
38
37 (98% ee)
39 (98% ee)
Scheme 5. Reagents and conditions: (a) BnOH, EDAC, CH2Cl2, rt, 1.5 h, 70%; (b) BH3, 2-methyl-2-butene, THF, 0 °C, 75 min, then 32, and rt, 15 h, 51%; (c) Ph3P(Br)C21H43
LiN(SiMe3)2, THF, 0 °C, 1 h, and rt, 2 h, 61%; (d) TBDMSOTf, 2,6-lutidine, CH2Cl2, rt, 1.5 h, 79%; (e) H2, Pd(OH)2/C, THF, quant; (f) 36, EDAC, CH2Cl2, rt, 2 h, 70%.
,
HFꢀpyridine gave 21. The double bond of 21 was reduced as
described for the formation of 5 from 4 to give 22 (quantitatively).
Treatment of 22 with sodium azide in DMF at 100 °C for 14 h gave
azide 23 accompanied by inversion of the configuration.
TBDMS and acetonide were removed with aqueous HF in
CH2Cl2–MeCN (1:1) to give 30 (RCAI-120).
Thirdly, we synthesized compound 43 (RCAI-129), which has
another (2R)-hydroxyl group on the amide side-chain of compound
14 as shown in Schemes 5 and 6.
The
a
-imidoyl compound of 24,6 prepared by the same proce-
dure from 9, was treated with alcohol 23 according to the proce-
dure from 9 to 10 to give 25. Compound 25 was converted into
27 via amine 26 according to the same procedure from 10 to 12.
Treatment of 27 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) in dichloromethane–water (CH2Cl2–H2O) (10:1) gave 28.
The silylene group of 28 was deprotected to give 29 with HFꢀpyri-
dine according to the procedure from 12 to 13. In the last step, two
(R)-2-(tert-Butyldimethylsilyloxy)hexacosanoic acid (36) was
synthesized from the commercially available (R)-5-oxo-2-tetra-
hydrofurancarboxylic acid (31, 97% ee) in five steps (12%). The car-
boxylic acid of 31 was esterified with benzyl alcohol using EDAC as
a dehydrating agent to give benzyl ester 32. The lactone carbonyl
of 32 was reduced to a hemiacetal 33 using borane tetrahydrofuran
complex (1.0 M in THF)/2-methyl-2-butene (2 M in THF).11 Wittig

50
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
t-Bu
t-Bu
Si
Si
t-Bu
O
O
t-Bu
OH
OTBDMS
OH
O
O
HO
BnO
O
O
O
O
C24H49
O
C24H49
a
b
BnO
BnO
NH2 OBn
HN
OBn
HN
OBn
BnO
BnO
BnO
O
O
O
C13H27
C13H27
OBn
C13H27
OBn
BnO
OBn
BnO
BnO
11
41
42
OH
OH
HO
HO
O
O
C24H49
c
HN
OH
HO
O
C13H27
HO
OH
43 (RCAI-129)
Scheme 6. Reagents and conditions: (a) 36, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 3 h, 54%; (b) (1) aq 46% HF, CH2Cl2–MeCN (1:1), rt, 3 h, (2) HFꢀpyridine, pyridine, THF, rt, 1.5 h,
two steps 68%; (c) H2, Pd(OH)2/C, THF, 48 h, 52%.
t-Bu
Si
O
t-Bu
Si
O
t-Bu
Si
O
t-Bu
O
t-Bu
O
t-Bu
O
OTBDMS
C24H49
OTBDMS
C24H49
O
O
O
O
O
PMBO
a
PMBO
b
HO
NH2
O
OTBDMS
C12H25
HN
OTBDMS
HN
OTBDMS
PMBO
PMBO
HO
O
C12H25
O
C12H25
O
OTBDMS
O
O
OTBDMS
O
O
OTBDMS
O
26
44
45
OH
OH
OTBDMS
OH
HO
HO
HO
HO
O
O
O
C24H49
O
C24H49
c
d
OH OH
HN
OTBDMS
HN
HO
HO
C12H25
O
O
C12H25
O
OH OH
(RCAI-130)
OTBDMS
O
46
47
Scheme 7. Reagents and conditions: (a) 36, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 16 h, quant; (b) DDQ, CH2Cl2–H2O (10:1), rt, 3 h, 60%; (c) HFꢀpyridine, pyridine, THF, rt, 1 h,
68%; (d) aq 46% HF, CH2Cl2–MeCN (3:2), H2O, rt, 16 h, 65%.
reaction of 33 with the phosphorane derived from n-henicosyltri-
phenylphosphonium bromide [n-C21H43P(Br)Ph3] and LiN(SiMe3)2
gave 34 as a mixture of E- and Z-isomers. Silylation of this mixture
with TBDMSOTf and 2,6-lutidine afforded 35, which was reduced
to 36 quantitatively using Pd(OH)2/C as a catalyst.
This carboxylic acid 36 was converted into amides 38 and 40 to
determine the enantiomeric excess of 36. Reaction of 36 with both
(R)- and (S)-1-(1-naphthyl)ethylamines (98% ee) using EDAC as a
condensing agent gave amides 38 [(R,R)-isomer] and 40
[(S,R)-isomer], respectively. The diastereomeric excess of crude
sample of 40 was 97.9% as determined by HPLC analysis, and the
enantiomeric excess of starting 31 was 97%. Therefore, no
epimerization occurred during the conversion from 31 to 36.
Condensation of amine 11 and carboxylic acid 36 using EDAC as
a dehydrating agent gave amide 41. The TBDMS and silylene
groups of 41 were deprotected successively by aqueous HF and
then HFꢀpyridine to give 42. Hydrogenolysis of five benzyl groups
of 42 using Pd(OH)2/C as a catalyst gave 43 (RCAI-129) in 53% yield.
Fourthly, amine 26 and carboxylic acid 36 were converted into
47 (RCAI-130) in four steps with an overall yield of 21% via com-
pounds 44, 45, and 46 according to almost the same procedure
from 26 to 30 via 27, 28, and 29 as shown in Scheme 7.
O
OH
O
OH
C20H41
HO
C23H47
O
HOOC
O
C23H47
O
HO
a
b
c
d
O
HO
O
O
O
OH
O
52
48
49
50
51
Scheme 8. Reagents and conditions: (a) 2,2-dimethoxypropane (1.5 equiv), Amberlyst 15, DMF, rt, 18 h, 50%; (b) Ph3P(Br)C21H43, LiN(SiMe3)2, THF, 0 °C, 1 h, and rt, 2 h, 50%;
(c) H2, Pd/C, hexane, rt, 2 h, 98%; (d) RuCl3ꢀnH2O, NaIO4, CCl4–MeCN–H2O (2:2:3), rt, 3 h, 80%.

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
51
t-Bu
t-Bu
Si
Si
O
O
O
O
OH
HO
HO
t-Bu
t-Bu
C23H47
OH
O
O
O
O
a
O
O
O
C23H47
OBn
b
BnO
BnO
BnO
NH2 OBn
HN
HN
OBn
BnO
BnO
BnO
O
O
O
C13H27
C13H27
C13H27
BnO
BnO
OBn
BnO
OBn
OBn
11
53
54
HO
OH
HO
C23H47
OH
O
O
c
HO
HN
OH
HO
O
C13H27
HO
OH
55 (RCAI-142)
Scheme 9. Reagents and conditions: (a) 52, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 16 h, 68%; (b) (1) aq 46% HF, CH2Cl2–MeCN (1:1), rt, 3 h, (2) HFꢀpyridine, pyridine, THF, rt,
1.5 h, two steps 69%; (c) H2, Pd(OH)2/C, THF, 16 h, 52%.
Fifthly, the compound 55 (RCAI-142), which has two consecu-
tive (2R,3R)-hydroxy groups adjacent to the carbonyl group on
the amide side-chain of 43, was synthesized as shown in Scheme 8.
(2R,3R)-2,3-O-Isopropylidenehexacosanoic acid (52) was pre-
Sixthly, the compound 59 (RCAI-147), which has two consecu-
tive (2R,3R)-hydroxyl groups on the amide side-chain and four suc-
cessive (3S,4S,5S,6S)-hydroxyl groups on the main-chain carbon
adjacent to C2-amino group, was synthesized in four steps with
an overall yield of 32% via 56, 57, and 58 from amine 26 and car-
boxylic acid 52 according to almost the same procedure from 26
to 47 via 44, 45, and 46 as shown in Scheme 10.
Seventh, compound 63 (RCAI-151), which has two (2R,3R)-
hydroxyl groups on the amide side-chain of KRN7000, was synthe-
sized. Reaction of amine 6013 and carboxylic acid 52 with EDAC as
a dehydrating agent gave amide 61. Hydrogenolysis of four benzyl
groups of 61 in THF using Pd(OH)2/C as a catalyst gave 62, and the
isopropylidene and two TBDMS groups of 62 were removed by
treatment of aqueous HF to give 63 (RCAI-151) in 63% yield as
shown in Scheme 11.
pared from 2-deoxy-L-ribose (48). Compound 48 was treated with
2,2-dimethoxypropane in DMF using strongly acidic ion exchange
resin (Amberlyst 15) as a catalyst to afford 49. Wittig reaction of
49 with the phosphorane derived from n-C21H43P(Br)Ph3 and
LiN(SiMe3)2 gave 50 as a mixture of E- and Z-isomers, which was
hydrogenated to 51 using Pd(OH)2/C as a catalyst. The primary
alcohol of 51 was oxidized to carboxylic acid 52 using
ruthenium(III) chloride hydrate (RuCl3ꢀnH2O) and sodium
periodate (NaIO4) in carbon tetrachloride–acetonitrile–water
(CCl4–MeCN–H2O) (2:2:3).12
Amine 11 and carboxylic acid 52 were converted into 55 (RCAI-
142) in three steps with 24% yield via 53 and 54 according to al-
most the same procedure from 11 to 43 via 41 and 42 as shown
in Scheme 9.
Finally, compound 73 (RCAI-160), which has three (2R,3R,4R)-
hydroxyl groups on the amide side-chain of KRN7000, was synthe-
sized as shown in Schemes 12 and 13.
t-Bu
t-Bu
t-Bu
Si
Si
Si
t-Bu
O
t-Bu
O
O
O
t-Bu
O
O
O
O
O
O
O
O
O
O
C23H47
C23H47
O
O
O
PMBO
PMBO
HO
a
b
NH2
O
OTBDMS
C12H25
NH
OTBDMS
NH
OTBDMS
PMBO
PMBO
HO
C12H25
O
O
C12H25
O
OTBDMS
O
OTBDMS
OTBDMS
O
O
57
56
26
HO
OH
HO
C23H47
OH
O
OH
O
HO
HO
O
O
C23H47
OTBDMS
C12H25
O
O
HO
c
d
NH OH OH
NH
HO
HO
O
O
C12H25
OH OH
OTBDMS
O
O
59 (RCAI-147)
58
Scheme 10. Reagents and conditions: (a) 52, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 16 h, 90%; (b) DDQ, CH2Cl2–H2O (10:1), rt, 2 h, 94%; (c) HFꢀpyridine, pyridine, THF, rt, 1 h,
78%; (d) aq 46% HF, CH2Cl2–MeCN (1:1), H2O, rt, 16 h, 48%.

52
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
O
O
O
O
OBn
OBn
OH
BnO
BnO
BnO
BnO
HO
HO
O
O
C23H47
C23H47
O
O
O
NH2 OTBDMS
NH OTBDMS
NH OTBDMS
a
b
BnO
BnO
HO
O
O
O
C14H29
OTBDMS
C14H29
C14H29
OTBDMS
OTBDMS
61
62
60
HO
C23H47
OH
OH
HO
HO
O
O
NH OH
c
HO
O
C14H29
OH
63 (RCAI-151)
Scheme 11. Reagents and conditions: (a) 52, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 16 h, 78%; (b) H2, Pd(OH)2/C, THF, rt, 16 h, 77%; (c) aq 46% HF, CH2Cl2–MeCN (1:1), H2O, rt,
16 h, 63%.
O
OH
OH
OH
OTBDMS
C22H45
TBDMSO
C22H45
a
TBDMSO
b
TBDMSO
TBDMSO
C20H41
c
O
O
O
O
O
O
O
O
64
65
66
67
OH
OTBDMS
OTBDMS
C22H45
C22H45
C22H45
HO
HO
HOOC
d
e
O
O
O
O
O
O
67
68
+
+
69
68
70
Scheme 12. Reagents and conditions: (a) 3 equiv Ph3P(Br)C21H43, 3 equiv n-BuLi, THF, ꢁ10 °C, 30 min, then 64, 4 h, rt, 63%; (b) H2, Pd(OH)2/C, EtOAc, rt, 1 h, quant; (c)
TBDMSOTf, 2,6-lutidine, CH2Cl2, rt, 1 h, 90%; (d) HFꢀpyridine, pyridine, THF, rt, 16 h, 67 (recovery 32%), 68 (47%), and 69 (15%); (e) RuCl3ꢀnH2O, NaIO4, CCl4–MeCN–H2O (2:2:3),
rt, 3 h, 77%.
Preparation of carboxylic acid 70 was performed from 5-O-tert-
butyldimethylsilyl-2,3-O-isopropylidene-
-ribofuranose 646 as
2.2. Biological activity
L
shown in Scheme 12. Wittig reaction of 64 with the phosphorane
derived from Ph3P(Br)C21H43 and n-BuLi gave 65 as a mixture of
E- and Z-isomers. The double bond reduction of 65 under hydrogen
using Pd(OH)2/C as a catalyst in ethyl acetate (EtOAc) gave alcohol
66. Treatment of 66 with TBDMSOTf using 2,6-lutidine as a base
gave 67. Selective cleavage of C1-primary TBDMS ether of 67 was
performed using HFꢀpyridine to give 68 (47%) accompanying recov-
ered 67 (32%) and 1,2-diol 69 (15%). Oxidation of the primary alco-
hol of 68 using RuCl3ꢀnH2O and NaIO4 in CCl4–MeCN–H2O (2:2:3)12
afforded carboxylic acid 70.
The cytokine-producing [IFNc (Th1 response) and IL-4 (Th2 re-
sponse)] activity from iNKT cells of mice in vivo of the eight syn-
thetic compounds was investigated using KRN7000 as a positive
control as shown both in Figure 2 [IFN
b), compounds 14, 30, 43, and 47] and in Figure 3 [IFN
c
(panel a) and IL-4 (panel
(panel a)
c
and IL-4 (panel b), compounds 55, 59, 63, and 73], and the struc-
tures of newly synthesized eight compounds are listed in Table 1.
Three compounds 59, 63, and 73 out of these eight compounds
strongly induced Th2 cytokine (IL-4) rather than Th1 cytokine
(IFNc) from iNKT cells compared to KRN7000. Therefore, these
Reaction of amine 6013 and carboxylic acid 70 with EDAC as a
dehydrating agent gave amide 71 (38%) as shown in Scheme 13.
Hydrogenolysis of four benzyl groups of 71 in THF using
Pd(OH)2/C as a catalyst gave 72, and the isopropylidene as well
as three TBDMS groups of 72 were removed by treatment of aque-
ous HF to give 73 (RCAI-160) in 48% yield.
three compounds 59, 63, and 73 were tested for the suppression
activity of symptoms associated with experimental autoimmune
encephalomyelitis (EAE)14 as shown in Figure 4.
The results of biological activity are described below.
Both compounds 14 and 43 are holding another (5S)-hydroxyl
group in the immediate vicinity of C4-hydroxyl group on the

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
53
O
O
O
O
C22H45
C22H45
OBn
OBn
BnO
OH
BnO
BnO
HO
HO
O
OTBDMS
O
O
OTBDMS
O
O
BnO
NH2 OTBDMS
NH OTBDMS
NH OTBDMS
a
b
BnO
BnO
HO
O
O
O
C14H29
C14H29
C14H29
OTBDMS
OTBDMS
OTBDMS
71
72
60
C22H45
HO
O
OH
OH
OH
HO
HO
O
c
NH OH
HO
O
C14H29
OH
73
(RCAI-160)
Scheme 13. Reagents and conditions: (a) 70, EDAC, DMAP, THF–CH2Cl2 (1:1), rt, 16 h, 38%; (b) H2, Pd(OH)2/C, THF, 16 h, 54%; (c) aq 46% HF, CH2Cl2–MeCN (3:2), H2O, rt, 16 h,
48%.
γ
IFN
apg/mL
a
KRN7000
pg/mL
γ
IFN
30000
25000
20000
15000
10000
5000
0
14 (RCAI-104)
30 (RCAI-120)
43 (RCAI-129)
47 (RCAI-130)
30000
25000
20000
15000
10000
5000
0
KRN7000
55 (RCAI-142)
59 (RCAI-147)
63 (RCAI-151)
73 (RCAI-160)
hr
hr
b
IL-4
pg/mL
1800
1600
1400
1200
1000
800
bpg/mL
1000
IL-4
KRN7000
14 (RCAI-104)
30 (RCAI-120)
43 (RCAI-129)
47 (RCAI-130)
KRN7000
800
600
400
200
0
55 (RCAI-142)
59 (RCAI-147)
63 (RCAI-151)
73 (RCAI-160)
600
400
200
0
hr
0
12
24
c
(panel a) and IL-4 (panel b)15
Figure 2. (Panels a and b) The concentrations of IFN
in sera of mice (2
hr
c
(panel a) and IL-4 (panel b)15
l
g/mouse) upon administration of the -galactosylceramide
a
variants, namely four compounds 14, 30, 43, and 47 (the structures shown in
Table 1).
Figure 3. (Panels a and b) The concentrations of IFN
in sera of mice (2
l
g/mouse) upon administration of the -galactosylceramide
a
variants, namely four compounds 55, 59, 63, and 73 (the structures shown in
Table 1).
main-chain of phytosphingosine. Also the compound 14 is
retaining a hexacosanamide such as KRN7000 and 43 is having a
(2R)-2-hydroxyhexacosanamide in the amide side-chain. The
On the other hand, the IL-4 secretion of both compounds 14 and
43 was almost the same level with KRN7000. Therefore, it can be
said that the cytokine secretion ability of these two compounds
amount of IFN
c secretion mediated by compounds 14 and 43
was approximately 1.7 and 1.4 times of KRN7000, respectively.

54
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
Table 1
Structures of newly synthesized eight compounds
OH
HO
OH
OH
OH
HO
OH
C24H49
HN OH
OH
C24H49
HN OH OH
HO
HO
HO
HO
C13H27
O
O
O
O
C25H51
O
O
O
O
C25H51
HN OH
HO
C13H27
HO OH
HO
HN OH OH
HO
HO
HO
HO
O
O
O
O
C12H25
C12H25
OH OH
OH OH
HO OH
47 (RCAI-130)
30 (RCAI-120)
43 (RCAI-129)
14 (RCAI-104)
C22H45
HO
OH
HO
HO
O
HO
C23H47
OH
OH
HO
OH
HO
C23H47
OH
C23H47
OH
OH
HO
HO
HO
HO
O
O
O
OH
O
O
O
O
OH
HO
HO
C13H27
HO OH
55 (RCAI-142)
NH OH OH
NH OH
HN OH
NH OH
HO
HO
HO
HO
O
O
O
O
C12H25
C14H29
C14H29
OH OH
OH
OH
63 (RCAI-151)
59 (RCAI-147)
73 (RCAI-160)
The extra hydroxy groups added to KRN7000 are painted in red.
Clinical score
DC (n=9)
DC + KRN7000 (n=8)
DC + 59 (RCAI-147) (n=8)
DC + 63 (RCAI-151) (n=8)
DC + 73 (RCAI-160) (n=9)
Days after immunization
Figure 4. Preventive effects of the KRN7000 analogues against EAE.
is biased toward the Th1 response comparing to that of KRN7000.
However, the compound 55, which has a (2R,3R)-2,3-dihydroxy-
production ability of compound 59 decreased to ꢂ5% of
KRN7000, and yet the IL-4 production amount remained more than
70% of KRN7000. Therefore, compound 59 biased to Th2 response
(almost 14 times) compared to that of KRN7000.
Compound 63 (RCAI-151) has a phytosphingosine as the main-
chain, and also has a (2R,3R)-2,3-(dihydroxy)hexacosanamide as
hexacosanamide in the amide side-chain, decreased both IFN
c
and IL-4 productions to ꢂ40% and ꢂ85% of that of KRN7000,
respectively, even though it has the same (5S)-hydroxyl group on
the phytosphingosine main-chain as compounds 14 and 43. There-
fore, compound 55 showed a weak tendency to bias to Th2 re-
sponse compared to that of KRN7000.
Compounds 30 and 47 have two successive (5S,6S)-5,6-dihydr-
oxyl groups next to C4-hydroxyl group on the phytosphingosine
main-chain. The amide side-chain of 30 is a hexacosanamide, and
that of 47 is a (2R)-2-hydroxyhexacosanamide. Each amount of
the amide side-chain. The IFNc production ability of compound
63 decreased to ꢂ41% of KRN7000, and its IL-4 production amount
increased to ꢂ138% of that of KRN7000. Therefore, compound 63
biased to Th2 response (almost 3.4 times) compared to that of
KRN7000.
Compound 73 (RCAI-160) has a phytosphingosine moiety as the
main-chain, and has a (2R,3R,4R)-2,3,4-trihydroxyhexacosanamide
both IFN
most the same level as KRN7000, and compound 30 decreased both
IFN
and IL-4 productions to ꢂ70% and ꢂ56% of that of KRN7000,
respectively. Thus, the compound 47 has almost the same profile to
KRN7000 about both IFN and IL-4 secretions, and compound 30
c and IL-4 secretions mediated by compound 47 was al-
as the amide side-chain. The IFNc production ability of compound
c
73 decreased to ꢂ7% of that of KRN7000, and its IL-4 production
amount still remained ꢂ55% of that of KRN7000. Therefore, com-
pound 73 biased to Th2 response (almost 7.8 times) comparing
to that of KRN7000.
c
shows a little bit bias to Th1 response, but it will be close to the
same with that of KRN7000.
The EAE test of compounds 59, 63, and 73 was performed as
shown in Figure 4. Compounds 59 (RCAI-147) and 73 (RCAI-160)
were effective for EAE test. This may be because the IFNc produc-
Compound 59 (RCAI-147) has two successive (5S,6S)-5,6-dihy-
droxy groups next to C4-hydroxyl group on the phytosphingosine
main-chain such as compounds 30 and 47, and also (2R,3R)-2,3-
tion by 59 (RCAI-147) and 73 (RCAI-160) was close to zero, and the
dihydroxyhexacosanamide on the amide side-chain. The IFN
c
IL-4 production was still remaining in high level (approximately

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
55
ꢂ70% and ꢂ55% of KRN7000, respectively). Although, compound
63 (RCAI-151) produced IL-4 more than KRN7000, it could not
suppress the development of EAE. It may be attributable to mask-
H). ESI-MS: m/z 473.19 [M+Na]+. HR ESI-MS: calcd for C27H30O6Na:
473.1940; observed: 473.1926.
4.1.2. 2,3,4-Tri-O-benzyl-6-O-tert-butyldimethylsilyl-D-
galactopyranose (3)
ing of the therapeutic effect of IL-4 by the IFN
neously from iNKT cells.
c derived simulta-
To a solution of 2 (10.5 g, 23.3 mmol) in CH2Cl2 (250 mL) were
added imidazole (1.67 g, 24.5 mmol, 1.05 equiv) and TBDMS-Cl
(3.65 g, 24.2 mmol, 1.04 equiv). After stirring for 1.5 h at room
temperature, the reaction mixture 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 hexane–EtOAc (9:1, then 4:1) gave 3
(10.3 g, 78%) as an oil and tert-butyldimethylsilyl 2,3,4-tri-O-ben-
3. Conclusions
The cytokine secretion ability of 14 and 43 was biased toward
the Th1 response compared to that of KRN7000. Compounds 30,
47, and 55 were much the same with KRN7000. On the contrary,
compounds 59, 63, and 73 behaved obviously as Th2 type antigens.
Most of all, both compounds 59 and 73 were biased largely to Th2
response, and showed good suppressive effects against EAE symp-
toms. From these results, it can be said that the existence of 2–3
consecutive hydroxyl groups on the amide side chain and 2–4 con-
secutive hydroxyl groups on the octadecyl main chain should con-
tribute to the secretion of Th2 cytokines from iNKT cells. And also
these results might show that the balance of hydroxyl groups both
on the amide side chain and on the octadecyl main chain should be
essential for Th2 response. In addition, not only making the cyto-
zyl-6-O-tert-butyldimethylsilyl-b-
3%). Physical data of 3; IR max(KBr): 3411 (br), 2952, 2928, 2883,
2856, 1496, 1470, 1454 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.03
D-galactopyranoside
(0.47 g,
m
.
(3H, s), 0.04 (3H, s), 0.88 (9H, s), 2.93 (0.5H, d, J 2.0 Hz, OH), 3.06
(0.5H, d, J 7.1 Hz, OH), 3.43–3.76 (3H, m), 3.88–4.07 (3H, m),
4.61–4.97 (6.5H, m), 5.26 (0.5H, dd, J 2.0, 3.4 Hz), 7.26–7.39
(15H, m). ESI-MS: m/z 587 [M+Na]+. HR ESI-MS: calcd for
C
33H44O6SiNa: 587.2799; observed: 587.2796. Physical data of
tert-butyldimethylsilyl 2,3,4-tri-O-benzyl-6-O-tert-butyldimethyl-
silyl-b- -galactopyranoside; IR max(KBr) 2954, 2929, 2857, 1496,
1471, 1254 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.02 (6H, s), 0.12
kine secretion bias to Th2 but also decreasing the INFc production
to almost zero may be important for the preventive effects against
EAE.
D
m
.
(3H, s), 0.13 (3H, s), 0.87 (9H, s), 0.92 (9H, s), 3.34 (1H, t, J
6.4 Hz), 3.48 (3H, dd, J 3.0, 9.9 Hz), 3.58 (1H, dd, J 6.9, 10.1 Hz),
3.65 (1H, dd, J 5.1, 9.8 Hz), 3.75 (1H, dd, J 7.4, 9.9 Hz), 3.81 (1H,
d, J 2.6 Hz), 4.58 (1H, d, J 7.3 Hz), 4.63, 4.95 (2H, AB-q, J 11.6 Hz),
4.69, 4.77 (2H, AB-q, J 12.0 Hz), 4.78, 4.93 (2H, AB-q, J 10.9 Hz),
7.26–7.37 (15H, m). ESI-MS: m/z 701 [M+Na]+. HR ESI-MS: calcd
for C39H58O6Si2Na: 701.3664; observed: 701.3664.
4. Experimental
4.1. General
IR spectra were measured with a Jasco FT/IR-460 plus spectrom-
eter. 1H NMR spectra (TMS as an internal standard) and 13C NMR
spectra were recorded with a Varian VNMRS-500 spectrometer
using tetramethylsilane (TMS) as an internal standard. Mass spec-
tra 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 apparatus. Optical
rotation values were measured with a Jasco P-1010 polarimeter.
Column chromatography was carried out using silica gel 60 N
(70–230 mesh ASTM, using ca 10–50 times the weight of substrate
if there is no indication) under a slightly elevated pressure for elu-
tion. Preparative TLC was carried out on a PLC plate (Merck, Silica
gel 60 F254, 0.5 mm).
4.1.3. (2R,3S,4R,5S,6EZ)-3,4,5-Tris(benzyloxy)-1-[(tert-
butyldimethylsilyl)oxy]octadec-6-en-2-ol (4)
To a solution of Ph3P(Br)C12H25 (25.3 g, 49.5 mmol) in dry THF
(300 mL) was added n-BuLi (1.59 M in hexane, 32 mL, 50.9 mmol)
at ꢁ10 °C under an atmosphere of argon. After stirring for 30 min
at ꢁ10 °C, a solution of 3 (9.31 g, 16.48 mmol) in THF (50 mL)
was added dropwise at ꢁ10 °C. The mixture was stirred for
15 min at ꢁ10 °C at room temperature for 16 h, and then concen-
trated in vacuo to one-fifth of the volume, and quenched with
MeOH (30 mL). The mixture was diluted with hexane, and the
whole was washed with water, and aq 3.5% H2O2, and again with
water, 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 hexane–EtOAc (10:1) gave 4
4.1.1. 2,3,4-Tri-O-benzyl-
D-galactopyranose (2)
(1) To a solution of methyl 2,3,4,6-tetra-O-benzyl-
a-D-galacto-
pyranoside 1 (20.0 g, 36.1 mmol) in Ac2O (600 mL) was added con-
cd H2SO4 (0.6 mL). The solution was stirred for 1 h at room
temperature, and diluted with EtOAc (1.2 L). The solution was
washed with aq satd NaHCO3 (200 mL ꢃ 2) and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, to which toluene (100 mL) was added and evaporated
under reduced pressure. This procedure was repeated once more,
and dried in vacuo to give crude 1,6-di-O-acetyl-2,3,4-tri-O-ben-
(5.36 g, 45%) as an oily mixture of E- and Z-isomers. IR
mmax(KBr)
3489, 2926, 2855, 1455, 1253 cmꢁ1 1H NMR (500 MHz, CDCl3): d
.
0.03 (6H, s), 0.86–0.89 (12H, m), 1.26 (16H, br s), 1.31–1.38 (2H,
m), 2.00–2.08 (2H, m), 3.09 (1H, d, J 4.9 Hz, OH), 3.59–3.62 (2H,
m), 3.78 (1H, m), 3.87 (1H, m), 3.90 (1H, m), 4.31, 4.63 (2H, AB-q,
J 12.0 Hz), 4.44, 4.50 (2H, AB-q, J 11.4 Hz), 4.46 (1H, m), 4.77 (2H,
s), 5.43 (1H, m), 5.64 (1H, m), 7.22–7.37 (15H, m). ESI-MS: m/z
739.5 [M+Na]+. HR ESI-MS: calcd for C45H68O5SiNa: 739.4728; ob-
served: 739.4727.
zyl-D-galactopyranose (23.4 g).
(2) The above obtained diacetate was dissolved in MeOH (1.2 L)
containing KOH (1.4 g). After stirring for 1 h at room temperature,
the reaction mixture was concentrated in vacuo to give a residue,
which was chromatographed on a silica gel column. Elution with
hexane–EtOAc (1:1, and then EtOAc) gave 2 (15.5 g, 95%) as a 1:1
4.1.4. (2R,3S,4R,5S)-3,4,5-Tris(benzyloxy)-1-[(tert-
butyldimethylsilyl)oxy]octadecan-2-ol (5)
A solution of 4 (1.00 g, 1.39 mmol) in hexane (100 mL) was stir-
red for 50 min under an atmosphere of H2 using 10% Pd/C (500 mg)
at room temperature. The reaction mixture was filtered, and the fil-
trate was concentrated to give a residue, which was chromato-
graphed on a silica gel column. Elution with hexane–EtOAc (9:1)
anomeric mixture as a viscous oil. IR
mmax(KBr) 3410 (br), 1497,
1454 cmꢁ1 1H NMR (500 MHz, CDCl3): d 3.05 (1H, br s, OH), 3.36
.
(1H, br s, OH), 3.45–3.52 (1.5H, m), 3.57 (0.5H, dd, J 2.8, 9.4 Hz),
3.71–3.81 (2H, m), 3.90–3.92 (1H, m), 3.99 (0.5H, t, J 5.7 Hz), 4.05
(0.5H, m), 4.63–4.98 (6.5H, m), 5.31 (0.5H, d, J 3.4 Hz, anomeric
gave
5
(710 mg, 71%) as an oil. IR
.
mmax(KBr) 3495, 2927,
2854 cmꢁ1
1H NMR (500 MHz, CDCl3): d 0.04 (3H, s), 0.07 (3H, s),

56
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
0.86–0.90 (12H, m), 1.26 (22H, br s), 1.52–1.58 (2H, m), 3.36 (1H, d, J
4.4 Hz, OH), 3.58–3.66 (3H, m), 3.82–3.86 (2H, m), 3.90 (1H, m),
4.56, 4.58 (2H, AB-q, J 11.7 Hz), 4.61 (2H, s), 4.73, 4.81 (2H, AB-q, J
11.2 Hz), 7.22–7.35 (15H, m). ESI-MS: m/z 741.49 [M+Na]+. HR
ESI-MS: calcd for C45H70O5SiNa: 741.4885; observed: 741.4887.
8.26 mmol). After stirring for 40 min at ꢁ20 °C, the reaction mix-
ture was quenched with aq satd NaHCO3 (35 mL), concentrated
in vacuo, and diluted with EtOAc, which was washed with aq
10% Na2S2O3 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 hexane–EtOAc (2:1,
4.1.5. (2S,3S,4R,5S)-2-Azido-1-(tert-butyldimethylsilyl)oxy-
3,4,5-tris(benzyloxy)octadecane (6)
then 3:2, finally 2:3) gave 9 (2.24 g, 65%) as a gum. IR
mmax(KBr)
3539, 3419 cmꢁ1 1H NMR (500 MHz, CDCl3): d 1.01 (4.5H, s),
.
To a solution of 5 (72 mg, 0.10 mmol) in dry THF (2 mL) were
added Ph3P (118 mg, 0.45 mmol) and diethyl azodicarboxylate
(2.2 M in toluene, 0.20 mL, 0.44 mmol) under an atmosphere of ar-
gon. After stirring for 10 min at room temperature, (PhO)2P(O)N3
(124 mg, 0.45 mmol) was added at 0 °C. The mixture was stirred
vigorously for 30 min at 0 °C, then at room temperature for 3 h,
and concentrated in vacuo to give a mixture, which was chromato-
graphed on a silica gel column. Elution with hexane–EtOAc (19:1)
1.06 (4.5H, s), 1.076 (4.5H, s), 1.084 (4.5H, s), 2.83–2.85 (1H, m,
OH), 3.35 (0.5H, s), 3.45 (0.5H, dd, J 3.0, 7.9 Hz), 3.71 (0.5H, dd, J
7.8, 9.3 Hz), 3.77 (0.5H, dd, J 2.8, 9.7 Hz), 3.85 (0.5H, s), 3.98
(0.5H, dd, J 3.7, 10.0 Hz), 4.13–4.23 (2H, m), 4.47 (0.5H, d, J
2.9 Hz), 4.52 (0.5H, d, J 3.2 Hz), 4.64–4.78 (3H, m), 4.88–4.91
(1.5H, m), 5.20 (0.5H, d, J 3.9 Hz, on addition of D2O), 7.27–7.43
(10H, m). ESI-MS: m/z 523.25 [M+Na]+. HR ESI-MS: calcd for
C28H40O6SiNa: 523.2486; observed: 523.2486.
gave 6 (58 mg, 78%) as a viscous oil. IR
mmax(KBr) 2926, 2854,
2097 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.04 (3H, s), 0.05 (3H,
.
4.1.8. (2S,3S,4R,5S)-2-Azido-3,4,5-tris(benzyloxy)octadecyl 2,3-
s), 0.88 (3H, t, J 7.1 Hz), 0.90 (9H, s), 1.26 (20H, br s), 1.30–1.44
(2H, br), 1.52–1.61 (2H, br), 3.65–3.75 (3H, m), 3.79–3.87 (2H,
m), 4.01 (1H, dd, J 2.4, 10.3 Hz), 4.53–4.58 (3H, m), 4.65 (1H, d, J
11.2 Hz), 4.71, 4.75 (2H, AB-q, J 11.4 Hz), 7.27–7.36 (15H, m).
ESI-MS: m/z 766.49 [M+Na]+. HR ESI-MS: calcd for C45H69O4SiNa:
766.4950; observed: 766.4950.
di-O-benzyl-4,6-O-(di-tert-butyl)silylene-a-D-galactopyranoside
(10)
To a solution of 9 (462 mg, 0.923 mmol) in dry CH2Cl2 (10 mL)
were added Cl3CCN (1.33 g, 9.23 mmol) and Cs2CO3 (350 mg),
and the mixture was stirred for 16 h at room temperature. The
reaction mixture was diluted with CH2Cl2. The solution was
washed with water and brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to give crude imidate
4.1.6. (2S,3S,4R,5S)-2-Azido-3,4,5-tris(benzyloxy)octadecan-1-ol
(7)
(590 mg). To the solution of this imidate (590 mg) and
7
A solution of 6 (580 mg, 0.779 mmol) in THF (2 mL) containing
n-Bu4NF (1 M solution in THF, 3.3 mL) was stirred for 30 min at
room temperature, and the solution was concentrated in vacuo
to give a mixture, which was diluted with EtOAc. The solution
was washed with H2O 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-
ane–EtOAc (9:1, then 4:1) gave 7 (456 mg, 93%) as an oil. IR
(348 mg, 0.55 mmol) in CH2Cl2 (8 mL) was added molecular sieves
(MS) 4 Å (890 mg). The mixture was stirred for 30 min at room
temperature, and cooled to 0 °C. To this mixture was added AgOTf
(83 mg, 0.323 mmol), and stirred for 1 h at 0 °C, and then 16 h at
room temperature. The mixture was filtered, and the filter cake
was washed with CH2Cl2. The combined filtrate was washed with
aq satd NaHCO3 and brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to give a mixture, which was chro-
matographed on a silica gel column. Elution with hexane–EtOAc
m
max(KBr) 3721 (br), 2925, 2854, 2097, 1454 cmꢁ1 1H NMR
.
(500 MHz, CDCl3): d 0.88 (3H, t, J 7.0 Hz), 1.26 (20H, s), 1.39 (2H,
br s), 1.53 (2H, m), 2.59 (1H, t, J 6.4 Hz, OH), 3.61 (1H, m),
3.75–3.84 (4H, m), 3.95 (1H, m), 4.56, 4.64 (2H, AB-q, J 11.3 Hz),
4.57, 4.61 (2H, AB-q, J 11.3 Hz), 4.75 (2H, s), 7.27–7.35 (15H, m).
ESI-MS: m/z 624.40, 652.40 [M+Na]+ (on addition of HCOONa).
(9:1) gave 10 (615 mg, quantitative) as a gum. IR
mmax(KBr) 2927,
2856, 2099 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.88 (3H, t, J
.
6.8 Hz) 0.98 (9H, s), 1.04 (9H, s), 1.20–1.31 (20H, m), 1.31–1.43
(2H, m), 1.50–1.60 (2H, m), 3.51 (1H, br s), 3.62 (1H, m), 3.69–
3.73 (2H, m), 3.79–3.83 (2H, m), 3.93–4.03 (5H, m), 4.42 (1H, d, J
3.0 Hz), 4.42–4.85 (12H, m containing 1H, s, at 4.58 ppm, and 1H,
d, J 3.4 Hz at 4.79 ppm as an anomeric proton), 7.22–7.42 (25H,
m). ESI-MS: m/z 1134.64 [M+Na]+. HR ESI-MS: calcd for
HR ESI-MS: calcd for
652.4083.
C39H55N3O4Na: 652.4090; observed:
4.1.7. 2,3-Di-O-Benzyl-4,6-O-di-tert-butylsilylene-
D
-
C67H93N3O9SiNa: 1134.6579; observed: 1134.6588.
galactopyranose (9)
(1) To a solution of 86 (2.93 g, 6.86 mmol) in DMF (8 mL) were
added BnBr (4.69 g, 27.4 mmol) and NaH (60% oil dispersion,
1.65 g, 41.3 mmol) at 0 °C under an atmosphere of argon. After stir-
ring for 15 min at 0 °C, the mixture was stirred for 15 min at room
temperature, diluted with EtOAc, and quenched with ice water.
The organic layer was washed with 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
hexane–EtOAc (19:1, then 9:1) gave a di-benzyl ether (4.17 g,
quantitatively) as a viscous oil. 1H NMR (500 MHz, CDCl3): d 1.08
(9H, s), 1.13 (9H, s), 2.32 (3H, s), 3.25 (1H, s), 3.46 (1H, dd, J 2.9,
9.0 Hz), 3.83 (1H, t, J 9.5 Hz), 4.16 (1H, dd, J 2.3, 12.4 Hz), 4.20
(1H, dd, J 1.7, 12.4 Hz), 4.49 (1H, d, J 2.4 Hz), 4.58, (1H, d, J
3.9 Hz), 4.69, 4.77 (2H, AB-q, J 12.0 Hz), 4.89, 4.91 (2H, AB-q, J
10.4 Hz), 7.07 (2H, d, J 8.0 Hz), 7.27–7.36 (6H, m), 7.40–7.45 (6H,
m). ESI-MS: m/z 629.27 [M+Na]+. HR ESI-MS: calcd for
4.1.9. (2S,3S,4R,5S)-2-Amino-3,4,5-tris(benzyloxy)octadecyl 2,3-
di-O-benzyl-4,6-O-(di-tert-butyl)silylene-a-D-galactopyranoside
(11)
To a solution of 10 (318 mg, 0.29 mmol) in dry THF (5.6 mL) was
added Me3P (1 M solution in THF, 1.5 mL). After stirring for 2 h at
room temperature, aq NaOH (1 M solution in H2O, 5.45 mL) was
added to this solution. The mixture was stirred for 2 h at room
temperature, diluted with water, and extracted with CHCl3, which
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 hex-
ane–EtOAc (4:1, then 1:1) gave 11 (240 mg, 77%) as a gum. IR
m
max(KBr) 2926, 2856 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.88
.
(3H, t, J 6.8 Hz) 0.99 (9H, s), 1.04 (9H, s), 1.20–1.30 (22H, m),
1.50–1.70 (4H, br s, containing NH2), 3.53 (1H, br s), 3.76 (1H,
m), 3.81 (1H, dd, J 3.2, 10.1 Hz), 3.97–4.02 (2H, m), 4.08 (1H, m),
4.47 (1H, m), 4.52–4.75 (9H, m), 4.78 (1H, d, J 3.0 Hz), 4.83 (1H,
d, J 12.0 Hz, benzylic H), 7.22–7.33 (23H, m), 7.42 (2H, d, J
7.0 Hz). ESI-MS: m/z 1086.67 [M+H]+. HR ESI-MS: calcd for
C35H46O5SSiNa: 629.2727; observed: 629.2726.
(2) The above obtained compound (4.17 g, 6.86 mmol) was dis-
solved in acetone (140 mL), and the solution was cooled to ꢁ20 °C.
To this solution was added N-bromosuccinimide (1.47 g,
C67H96NO9Si: 1086.6854; observed: 1086.6843.

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
57
4.1.10. (2S,3S,4R,5S)-3,4,5-Tris(benzyloxy)-2-
73.78, 73.97, 101.31, 173.32. ESI-MS: m/z 896.72 [M+Na]+. HR
(hexacosanoylamino)octadecyl 2,3-di-O-benzyl-4,6-O-di-tert-
ESI-MS: calcd for C50H99NO10Na: 896.7161; observed: 896.7157.
butylsilylene-a-D-galactopyranoside (12)
To a solution of 11 (170 mg, 0.15 mmol) in THF–CH2Cl2 (1:1,
14 mL) were added DMAP (150 mg, 1.22 mmol), n-hexacosanoic
acid (182 mg, 0.46 mmol), and EDAC (176 mg, 0.919 mmol). The
mixture was stirred for 1.5 h at room temperature, and concen-
trated in vacuo to give a mixture, which was diluted with CH2Cl2.
The solution was washed with H2O and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a mix-
ture, which was chromatographed on a silica gel column. Elution
with hexane–EtOAc (9:1) gave 12 (161 mg, 70%) as a gum. IR
4.1.13. (3R,4R,5S,6S,7R)-7-[(tert-Butyldimethylsilyloxy)methyl]-
5,6-O-isopropylidene-3,4,5,6-tetrahydroxyoxepan-2-one (16)
To a solution of 15 (3.00 g, 9.13 mmol), 4-methylmorpholine
N-oxide (1.28 g, 10.96 mmol), and p-TsOHꢀH2O (2.07 g,
10.90 mmol) in acetone–H2O (4:1, 150 mL) was added OsO4 (1%
solution in t-BuOH, 30 mL) at 0 °C. The solution was stirred for
15 min at 0 °C, and for 16 h at room temperature. The reaction mix-
ture was concentrated in vacuo to half volume at 25 °C, and diluted
with EtOAc (500 mL), which was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column. Elu-
tion with hexane–EtOAc (4:1, then 2:1) gave 16 (2.10 g, 63%); mp
107–108 °C (as a floc from hexane–EtOAc (5:1)). This compound
m
max(KBr) 2925, 2853, 1674 cmꢁ1 1H NMR (500 MHz, CDCl3): d
.
0.88 (3H, t, J 7.0 Hz), 0.99 (9H, s), 1.02 (9H, s), 1.25 (6H, br s),
1.37–1.44 (2H, m), 1.53–1.66 (2H, m), 1.73–1.78 (2H, m),
3.50–3.55 (2H, m), 3.69 (1H, dd, J 4.0, 5.9 Hz), 3.75 (1H, dd, J 2.7,
10.1 Hz), 3.79 (1H, dd, J 7.6, 11.0 Hz), 3.87 (1H, t, J 3.8 Hz), 3.96
(1H, dd, J 3.7, 10.1 Hz), 4.01–4.08 (3H, m), 4.33 (1H, m), 4.46 (1H,
d, J 2.3 Hz), 4.49–4.74 (9H, m), 4.79 (1H, d, J 3.6 Hz), 4.82 (1H, d,
J 11.5 Hz), 6.12 (1H, d, J 8.4 Hz, NH), 7.22–7.34 (23H, m), 7.40
(2H, m). ESI-MS: m/z 1487.05 [M+Na]+. HR ESI-MS: calcd for
decomposes gradually at room temperature. IR
mmax(KBr) 3390
(br), 3000–2858, 1725, 1472 cmꢁ1 1H NMR (500 MHz, CDCl3): d
.
0.089 (3H, s), 0.092 (3H, s), 0.90 (9H, s), 1.38 (3H, s), 1.50 (3H, s),
2.72 (1H, d, J 3.9 Hz, OH), 3.51 (1H, s, OH), 3.81 (1H, dd, J 9.3,
9.6 Hz), 3.94 (1H, dd, J 5.6, 9.5 Hz), 4.24 (1H, m, changed to dd, J
1.5, 3.9 Hz, on addition of D2O), 4.44 (1H, dd, J 4.3, 7.6 Hz), 4.52
(1H, br s, changed to doublet, J 1.5 Hz, on addition of D2O), 4.70
(1H, d, J 7.6 Hz), 4.85 (1H, dd, J 5.6, 9.3 Hz). FABMS: m/z 363.2
[M+H]+. HRFABMS: calcd for C16H31O7Si: 363.1839; observed:
363.1853.
C93H145NO10SiNa: 1487.0535; observed: 1487.0523.
4.1.11. (2S,3S,4R,5S)-3,4,5-Tris(benzyloxy)-2-
(hexacosanoylamino)octadecyl 2,3-di-O-benzyl-
galactopyranoside (13)
a-D-
To a solution of 12 (55 mg, 0.04 mmol) and pyridine (64 mg,
0.79 mmol) in dry THF (4 mL) was added HFꢀpyridine (HF: ꢂ70%;
pyridine: ꢂ30%, 30 mg, ca. 1.05 mmol) under an atmosphere of ar-
gon at room temperature. After stirring for 50 min, the reaction
mixture was diluted with CHCl3, and washed with aq satd NaHCO3
and brine, 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 hexane–EtOAc (2:1, then 1:1) gave
4.1.14. (3R,4R,5S,6S,7R)-7-[(tert-Butyldimethylsilyloxy)methyl]-
3,4-bis(tert-butyldimethylsilyloxy)-5,6-O-isopropylidene-5,6-
dihydroxyoxepan-2-one (17)
To a solution of 16 (580 mg, 1.60 mmol) in dry CH2Cl2 (30 mL)
containing 2,6-lutidine (1.72 g, 16.0 mmol) was added TBDMSOTf
(2.54 g, 9.60 mmol). After stirring for 16 h at room temperature,
the reaction mixture was diluted with CH2Cl2, and washed with
H2O and brine, dried over MgSO4, and filtered. The filtrate was con-
centrated in vacuo to give a residue, which was chromatographed
on a silica gel column. Elution with hexane–EtOAc (9:1) gave 17
13 (46 mg, 92%) as a gum. IR
mmax(KBr) 3744–3535 (br), 2923,
2852, 1650 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.879 (3H, t, J
.
7.0 Hz), 0.882 (3H, t, J 6.9 Hz), 1.25 (66H, br s), 1.44 (2H, quintet,
J 7.2 Hz), 1.52–1.70 (2H, m), 1.80–1.84 (2H, m), 2.50 (1H, s, OH),
3.60 (1H, m), 3.64–3.69 (2H, m), 3.72–3.78 (4H, m), 3.81 (1H, dd,
J 3.7, 9.8 Hz), 3.86 (1H, dd, J 8.6, 11.6 Hz), 3.96 (1H, d, J 2.9 Hz),
4.13 (1H, dd, J 4.4, 11.5 Hz), 4.42 (1H, d, J 11.7 Hz), 4.45 (1H, m),
4.54–4.75 (9H, m), 4.84 (1H, d, J 3.7 Hz, anomeric H), 5.94 (1H, d,
J 8.4 Hz, NH), 7.24–7.35 (25H, m). ESI-MS: m/z 1346.95 [M+Na]+.
(908 mg, 96%) as an amorphous. IR
mmax(KBr) 2990–2858, 1732,
1471, 1464 cmꢁ1 1H NMR (600 MHz, CDCl3): d 0.07 (6H, s), 0.10
.
(3H, s), 0.11(3H, s), 0.12 (3H, s), 0.14 (3H, s), 0.88 (9H,s), 0.89
(9H, s), 0.91 (9H, s), 1.36 (3H, s), 1.48 (3H, s), 3.81 (1H, dd, J 8.2,
9.6 Hz), 3.87 (1H, dd, J 6.1, 9.6 Hz), 3.89 (1H, dd, J 1.0, 6.4 Hz),
4.22 (1H, dd, J 6.4, 6.9 Hz), 4.50 (1H, d, J 1.0 Hz, C3-H), 4.50 (1H,
d, J 6.9 Hz, C6-H), 5.13 (1H, dd, J 6.1, 8.2 Hz). 13C NMR (150 MHz,
CDCl3): d ꢁ5.50, ꢁ5.43, ꢁ5.40, ꢁ5.08, ꢁ4.81, ꢁ4.51, 18.02, 18.11,
18.20, 25.31, 25.70, 25.78, 27.62, 62.51, 73.51, 74.51, 74.97,
77.64, 78.24, 108.59, 170.07. ESI-MS: m/z 613.34 [M+Na]+. HR
ESI-MS: calcd for C28H58O7Si3Na: 613.3383; observed: 613.3381.
HR ESI-MS: calcd for
1346.9498.
C85H129NO10Na: 1346.9514; observed:
4.1.12. (2S,3S,4R,5S)-2-Hexacosanoylamino-3,4,5-
trihydroxyoctadecyl -galactopyranoside (14) (RCAI-104)
a-D
To a solution of 13 (32 mg, 0.024 mmol) in THF (25 mL) was
added 20% Pd(OH)2/C (Degussa type, wet, ꢂ50%, 32 mg). After stir-
ring for 24 h under an atmosphere of H2 at room temperature, the
catalyst was removed by filtration, and washed with THF, and then
CHCl3–MeOH (5:1). The combined filtrate was concentrated in va-
cuo to give a residue, which was chromatographed on a silica gel
(1 g) column. Elution with CHCl3–MeOH (19:1, then 9:1) gave 14
4.1.15. (3R,4R,5S,6S,7R)-7-[(tert-Butyldimethylsilyloxy)methyl]-
3,4-bis(tert-butyldimethylsilyloxy)-5,
6-O-(isopropylidene)oxepan-2,5,6-triol (18)
To a solution of 17 (5.97 g, 10.1 mmol) in toluene (180 mL) was
added DIBAH (1.0 M in toluene, 10.8 mL, 10.8 mmol) under an
atmosphere of argon at ꢁ78 °C. The solution was stirred for
45 min, and the reaction mixture was quenched with MeOH
(15 mL). After stirring for 30 min at ꢁ78 °C, the reaction mixture
was allowed to warm up to room temperature, and diluted with
EtOAc, and the solution was washed with H2O. The whole was fil-
tered on Celite. Water, separated from the organic layer, was re-
extracted with EtOAc. The combined organic layers were washed
with brine, dried over MgSO4, and filtered. The filtrate was concen-
trated in vacuo to give a residue, which was chromatographed on a
silica gel column. Elution with hexane–EtOAc (19:1, then 14:1)
(10 mg, 47%) as a white powder. ½a D29
ꢄ
+39.9 [c 1.2, CHCl3–CH3OH
mmax(KBr) 3372 (br), 2920, 2851, 1645, 1541,
(1:1)]. IR
1468 cmꢁ1
.
1H NMR [500 MHz, CDCl3–CD3OD (9:1)]: d 0.88 (6H,
t, J 7.0 Hz), 1.25 (64H, br s), 1.40–1.63 (4H, m), 2.20 (2H, t, J
7.6 Hz), 3.43 (1H, d, J 7.9 Hz), 3.66 (1H, dd, J 4.4, 10.8 Hz), 3.72
(1H, dd, J 3.3, 9.9 Hz), 3.74–3.84 (7H, m), 3.91 (1H, dd, J 5.4,
11.0 Hz), 3.96 (1H, d, J 3.1 Hz), 4.22 (1H, m), 4.93 (1H, d, J 3.9 Hz,
anomeric H), 7.15 (1H, d, J 8.3 Hz). 13C NMR (126 MHz, pyridine-
d5): d 14.28, 22.93, 26.36, 26.88, 29.60, 29.61, 29.75, 29.79, 29.85,
29.91, 29.92, 30.00, 30.02, 30.04, 30.11, 30.23, 32.12, 35.01,
36.75, 51.74, 62.68, 68.22, 70.31, 71.00, 71.13, 71.64, 73.06,
gave 18 (5.77 g, 96%) as an oily 2:1 anomeric mixture. IR
mmax(KBr)
3440, 2954, 2930, 2887, 2858, 1748 (w), 1472, 1464, 1389, 1379,

58
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
1362, 1255 cmꢁ1
.
1H NMR (500 MHz, CDCl3): d 0.05–0.06 (6H, m),
with EtOAc, which was 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
hexane–EtOAc (9:1) gave 21 (68 mg, 62%) as an oily 12:1 mixture
of E- and Z-isomers. Partly this mixture was separated on a silica
gel TLC plate by development with hexane–EtOAc (4:1). Physical
data of the major isomer (larger Rf value than that of minor isomer).
0.10–0.14 (12H, m), 0.87–0.93 (27H, m), 1.33 (2/3H, s), 1.34 (1/3H,
s), 1.56 (3H, s), 2.63 (2/3H, d, J 4.2 Hz, OH), 3.68–3.74 (3H, m),
3.92–3.97 (1H, m), 4.22 (2/3H, m), 4.26–4.37 (2H, m), 4.55 (1/3H,
m), 4.88 (2/3H, dd, J 4.4, 6.8 Hz, changed to doublet, J 6.8 Hz, on
addition of D2O), 5.06 (1/3H, m), 5.53 (1/3H, m, OH). ESI-MS: m/z
615.35 [M+Na]+. HR ESI-MS: calcd for C28H60O7Si3Na: 615.3539;
observed: 615.3540.
IR m .
max(KBr) 3540, 2927, 2856, 1466, 1342, 1253 cmꢁ1 1H NMR
(500 MHz, CDCl3): d 0.05 (3H, s), 0.06 (3H, s), 0.07 (3H, s), 0.12 (3H,
s), 0.88 (18H, s, and 3H, t, J 7.1 Hz), 1.25–1.40 (19H, m, containing
3H singlet at 1.33 ppm), 1.49 (3H, s), 2.02 (1H, m), 2.10 (1H, m),
2.87 (1H, dd, dd, J 4.7, 7.9 Hz), 3.16 (3H, s), 3.94–3.99 (2H, m), 4.08
(1H, dd J 6.1, 9.6 Hz), 4.17 (1H, dd J 4.8, 9.5 Hz), 4.25 (1H, dd J 1.3,
6.3 Hz), 4.32 (1H, dd J 4.6, 9.0 Hz), 5.00 (1H, t J 4.4 Hz), 5.40 (1H,
m), 5.48 (1H, m). ESI-MS: m/z 717.4 [M+Na]+. HR ESI-MS: calcd for
4.1.16. (2R,3S,4S,5R,6S,7EZ)-1,5,6-Tris(tert-
butyldimethylsilyloxy)-3,4-O-(isopropylidene)octadec-7-en-
2,3,4-triol (19)
To a solution of Ph3P(Br)C11H23 (9.46 g, 19.0 mmol) in dry THF
(47 mL) was added a solution of 18 (2.82 g, 4.76 mmol) in dry THF
(23 mL) under an atmosphere of argon at ꢁ30 °C. To this solution
was added LiN(TMS)2 (1 M in THF, 28 mL) with stirring. After stirring
for 5 min at ꢁ30 °C, the temperature was gradually elevated from
ꢁ30 °C to ꢁ10 °C over 20 min. After stirring for 15 min at ꢁ10 °C,
the solution was stirred for 15 min at ꢁ10 °C, and 3 h at room tem-
perature. The reaction mixture was quenched with MeOH (4 mL) at
0 °C, and diluted with EtOAc, 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 hexane–EtOAc (40:1) gave 19 (1.88 g,
54%) as an oily mixture of E- and Z-isomers, and elution with hex-
C34H70O8Si2SNa: 717.4222; observed: 717.4224.
4.1.19. (2R,3R,4S,5R,6S)-5,6-Bis(tert-butyldimethylsilyloxy)-3,4-
O-isopropylidene-2-(methanesulfonyloxy)octadecane-1,3,4-
triol (22)
A solution of 21 (68 mg, 0.10 mmol) in EtOAc (7 mL) was stirred
for 16 h at room temperature under hydrogen using 10% Pd/C
(50 mg), and filtered. The filtrate was concentrated in vacuo to give
22 (68 mg, quantitatively) as a viscous oil, which was employed for
the next reaction without purification. IR
m
max(KBr) 3526, 2928,
ane–EtOAc (30:1) gave recovered 18 (380 mg, 14%). IR
mmax(KBr)
2856, 1517 (w), 1471, 1465, 1361, 1255, 1219, 1175 cmꢁ1
.
1H
3484, 2927, 2856 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.03 (3H, s),
.
NMR (500 MHz, CDCl3): d 0.10 (6H, s), 0.12 (6H, s), 0.87–0.90
(21H, m, containing two 9H singlets at 0.88 and 0.90 ppm), 1.26
(18H, br s), 1.33 (3H, s), 1.38–1.49 (2H, m), 1.51 (3H, s),
1.73–1.77 (2H, m), 3.03 (1H, dd, J 3.0, 9.4 Hz, OH), 3.16 (3H, s),
3.65 (1H, m), 3.94 (1H, m), 4.00–4.07 (2H, m), 4.22 (1H, m), 4.30
(1H, d, J 6.4 Hz), 4.90 (1H, m). ESI-MS: m/z 719.4 [M+Na]+. HR
ESI-MS: calcd for C34H72O8Si2SNa: 719.4379; observed: 719.4380.
0.05 (3H, s), 0.07 (6H, s), 0.08 (3H, s), 0.09 (3H, s), 0.87–0.91 (30H,
m, containing 9H singlet and 18H singlet at 0.87 and 0.90 ppm,
respectively), 1.26–1.40 (19H, m, containing 3H singlet at
1.32 ppm), 1.48 (3H, s), 1.98 (1H, m), 2.06 (1H, m), 2.23 (1H, d, J
6.1 Hz, OH), 3.58 (1H, d, J 1.7 Hz), 3.59 (1H, s), 3.71 (1H, q, J
6.1 Hz), 3.99 (1H, dd, J 6.5, 9.7 Hz), 4.14 (1H, d, J 6.4 Hz), 4.23 (1H,
dd, J 2.7, 9.8 Hz), 4.27 (1H, dd, J 2.6, 8.4 Hz), 5.44–5.53 (2H, m).
ESI-MS: m/z 753.5 [M+Na]+. HR ESI-MS: calcd for C39H82O6Si3Na:
753.5311; observed: 753.5314.
4.1.20. (2S,3S,4S,5R,6S)-2-Azido-5,6-bis(tert-
butyldimethylsilyloxy)-3,4-O-(isopropylidene)octadecane-
1,3,4-triol (23)
4.1.17. (2R,3R,4S,5R,6S,7EZ)-1,5,6-Tris(tert-
A solution of 22 (20 mg, 0.03 mmol) and NaN3 (20 mg,
butyldimethylsilyloxy)-3,4-O-isopropylidene-2-
(methanesulfonyloxy)octadec-7-ene-3,4-diol (20)
0.31 mmol) in dry DMF (2 mL) was stirred for 14 h at 100 °C, and
concentrated in vacuo. The residual 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 mix-
ture, which was chromatographed on a silica gel column. Elution
with hexane–EtOAc (9:1) gave 23 (14 mg, 76%) as a viscous oil.
To a solution of 19 (840 mg, 1.15 mmol) in dry CH2Cl2 (40 mL)
and pyridine (4 mL) was added methanesulfonic anhydride
(840 mg, 4.82 mmol). The mixture was stirred for 45 min at room
temperature, and diluted with CH2Cl2, which was washed with
aq satd NaHCO3 (twice) 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 hex-
ane–EtOAc (30:1) gave 20 (719 mg, 77%) as an oily mixture of E-
IR
m .
max(KBr) 3470, 2927, 2856, 2100, 1518 cmꢁ1 1H NMR
(500 MHz, CDCl3): d 0.09 (3H, s), 0.11 (6H, s), 0.14 (3H, s),
0.87–0.92 (21H, m, containing two 9H singlets at 0.90 and
0.92 ppm), 1.26 (18H, br s), 1.32 (3H, s), 1.41 (2H, m), 1.47 (3H,
s), 1.52–1.65 (2H, m), 2.03 (1H, t, J 6.1 Hz, OH), 3.66 (3H, m),
3.71 (1H, quintet, J 3.4 Hz), 3.92 (1H, m), 3.98–4.01 (2H, m),
4.08–4.12 (2H, m). ESI-MS: m/z 666.5 [M+Na]+. HR ESI-MS: calcd
for C33H69N3O5Si2Na: 666.4668; observed: 666.4667.
and Z-isomers. IR
mmax(KBr) 2929, 2857, 1471, 1345, 1254,
1178 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.04 (3H, s), 0.088 (6H,
.
s), 0.093 (3H, s), 0.10 (3H, s), 0.11 (3H, s), 0.88 (9H, s), 0.89 (3H,
m), 0.90 (9H, s), 0.91 (9H, s), 1.25–1.40 (19H, m, containing 3H sin-
glet at 1.30 ppm), 1.45 (3H, s), 1.99 (1H, m), 3.08 (3H, s), 3.84 (1H,
dd, J 7.5, 10.0 Hz), 3.97–4.02 (2H, m), 4.21 (1H, dd, J 2.2, 9.7 Hz),
4.25 (1H, d, J 6.1 Hz), 4.31 (1H, dd, J 2.2, 8.5 Hz), 4.77 (1H, t, J
6.5 Hz), 5.44–5.53 (2H, m). ESI-MS: m/z 831.5 [M+Na]+. HR ESI-
MS: calcd for C40H84O8Si3SNa: 831.5087; observed: 831.5088.
4.1.21. (2S,3S,4S,5R,6S)-2-Azido-5,6-bis(tert-
butyldimethylsilyloxy)-3,4-O-isopropylidene-3,4-
dihydroxyoctadecyl 2,3-bis-O-(4-methoxybenzyl)-4,6-O-(di-
tert-butyl)silylene-a-D-galactopyranoside (25)
To a solution of 24 (225 mg, 0.40 mmol) in dry CH2Cl2 (4 mL)
were added Cl3CCN (580 mg, 4.02 mmol) and Cs2CO3 (200 mg,
0.61 mmol), and the mixture was stirred for 16 h at room temper-
ature. The reaction mixture was diluted with CH2Cl2. The solution
was washed with water and brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to give crude imidate
(280 mg). This compound was unstable for silica gel column
chromatography. Therefore, this imidate was used for the next
reaction without purification. Physical data of this crude imidate:
4.1.18. (2R,3R,4S,5R,6S,7EZ)-5,6-Bis(tert-butyldimethylsilyloxy)-
3,4-O-isopropylidene-2-(methanesulfonyloxy)octadec-7-en-
1,3,4-triol (21)
To a solution of 20 (128 mg, 0.16 mmol) and pyridine (1.0 mL) in
dry THF (1.72 mL) was added HFꢀpyridine (HF: ꢂ70%; pyridine:
ꢂ30%, 0.34 mL) under an atmosphere of argon at 0 °C. After stirring
for 5 min, the mixture was stirred for 5 h at room temperature, the
reaction mixture was quenched with aq satd NaHCO3, and diluted

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
59
IR
m
max(KBr) 3343 (w), 2935, 2859, 1728, 1672, 1514 cmꢁ1. 1H NMR
NMR (500 MHz, CDCl3): d 0.01 (3H, s), 0.08 (3H, s), 0.10 (3H, s),
0.11 (3H, s), 0.86–0.89 (24H, m, containing two 9H singlets at
0.88 and 0.89 ppm), 0.99 (9H, s), 1.05 (9H, s), 1.22–1.36 (64H, m,
containing 3H singlet at 1.35 ppm), 1.45 (3H, s), 1.53–1.58 (4H,
m), 2.00 (1H, m), 2.08 (1H, m), 3.55 (1H, s), 3.59 (1H, dd, J 4.4,
10.5 Hz), 3.66 (1H, m), 3.72 (1H, dd, J 2.7, 10.0 Hz), 3.77–3.81
(7H, m, containing two 3H singlets at 3.79 and 3.81 ppm),
3.88–4.00 (4H, m), 4.08–4.18 (2H, m), 4.32 (1H, dd, J 5.4, 7.8 Hz),
4.47 (1H, d, J 2.4 Hz, galactose C4-H), 4.58–4.71 (4H, benzylic
4H), 4.83 (1H, J 3.7 Hz, anomeric H), 6.24 (1H, d, J 6.1 Hz, NH),
6.81 (2H, d, J 8.7 Hz), 6.87 (2H, d, J 8.7 Hz), 7.28 (2H, d, J 8.6 Hz),
7.33 (2H, d, J 8.6 Hz). ESI-MS: m/z 1561.13 [M+Na]+. HR ESI-MS:
calcd for C89H163NO13Si3Na: 1561.1324; observed: 1561.1324.
(500 MHz, CDCl3): d 1.00–1.07 (18H, m), 3.79–4.19 (12H, m, con-
taining two methoxy 6H), 4.54–4.71 (5H, m, containing benzylic
4H), 6.39 (1H, d, J 3.6 Hz, anomeric H. Therefore, the major com-
pound of this imidate is
a-anomer.), 6.83–6.87 (4H, m),
7.24–7.33 (4H, m), 8.53 (1H, s, C@NH). ESI-MS: m/z 726.18
[M+Na]+. HR ESI-MS: calcd for C32H44NO8SiCl3Na: 726.1794;
observed: 726.1791.
To the solution of this imidate and 23 (127 mg, 0.20 mmol) in
CH2Cl2 (6 mL) was added MS 4 Å (640 mg). The mixture was stirred
for 30 min at room temperature, and cooled to 0 °C. To this mixture
was added AgOTf (60 mg, 0.23 mmol), and stirred for 1 h at 0 °C,
and then 1.5 h at room temperature. The mixture was filtered,
and the filter cake was washed with CH2Cl2. The combined filtrate
was washed with aq satd NaHCO3 and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a mix-
ture, which was chromatographed on a silica gel column. Elution
with hexane–EtOAc (19:1, then 9:1) gave 25 (184 mg, 78%) as a
4.1.24. (2S,3S,4S,5R,6S)-5,6-Bis(tert-butyldimethylsilyloxy)-2-
hexacosanoylamino-3,4-O-isopropylidene-3,4-
dihydroxyoctadecyl 4,6-O-(di-tert-butyl)silylene-a-D-
galactopyranoside (28)
gum. IR
m
max(KBr) 2928, 2857, 2099, 1513 cmꢁ1
.
1H NMR
To a solution of 27 (154 mg, 0.10 mmol) in CH2Cl2–H2O (10:1,
22 mL) was added DDQ (154 mg, 0.68 mmol), and the solution
was stirred for 2 h at room temperature, then diluted with CH2Cl2.
The solution was washed with satd aq NaHCO3 (two 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 hexane–EtOAc (6:1, then 3:1) gave 28
(500 MHz, CDCl3): d 0.08 (6H, s), 0.09 (3H, s), 0.13 (3H, s), 0.86–
0.89 (12H, m, containing 9H singlet at 0.88 ppm), 0.91 (9H, s),
0.99 (9H, s), 1.05 (9H, s), 1.25 (18H, br s), 1.29 (3H, s), 1.38–1.44
(5H, m, containing 3H singlet at 1.43 ppm), 1.54–1.59 (2H, m),
3.61 (1H, s), 3.65 (1H, m), 3.68–3.70 (2H, m), 3.78–3.81 (7H, m, con-
taining 6H singlet at 3.80 ppm), 3.94–3.98 (2H, m), 4.04–4.06 (2H,
m), 4.09–4.13 (2H, m), 4.18 (1H, dd, J 2.0, 12.5 Hz), 4.43 (1H, d, J
3.7 Hz, galactose C4-H), 4.61, 4.74 (2H, AB-q, J 11.6 Hz), 4.63, 4.66
(2H, AB-q, J 11.7 Hz), 4.75 (1H, d, J 3.4 Hz, anomeric H), 6.83–6.86
(4H, m), 7.30–7.33 (4H, m). ESI-MS: m/z 1208.7 [M+Na]+. HR ESI-
MS: calcd for C63H111N3O12Si3Na: 1208.7368; observed: 1208.7362.
(111 mg, 85%) as a gum. IR
mmax(KBr): 3429, 3326, 2925, 2855,
1656, 1467 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.06 (3H, s), 0.09
.
(3H, s), 0.15 (3H, s), 0.16 (3H, s), 0.875 (9H, s), 0.882 (6H, t, J
6.8 Hz), 0.94 (9H, s), 1.02 (9H, s), 1.05 (9H, s), 1.25 (64H, br s),
1.32 (3H, s), 1.44 (3H, s), 1.53–1.66 (2H, m), 2.06–2.20 (2H, m),
2.52 (1H, d, J 10.0 Hz, OH), 2.58 (1H, d, J 10.3 Hz, OH), 3.60–3.65
(2H, m), 3.69 (1H, s), 3.77 (1H, m), 3.81 (1H, dd, J 6.3, 9.4 Hz),
4.03 (1H, dd, J 5.1, 9.3 Hz), 4.07 (1H, m), 4.14–4.16 (2H, m),
4.20–4.25 (2H, m), 4.39 (1H, dd, J 3.4 Hz, galactose C4-H), 4.87
(1H, d, J 3.7 Hz, anomeric H), 6.17 (1H, d, J 6.3 Hz, NH). ESI-MS:
m/z 1321.02 [M+Na]+. HR ESI-MS: calcd for C73H147NO11Si3Na:
1321.0174; observed: 1321.0177.
4.1.22. (2S,3S,4S,5R,6S)-2-Amino-5,6-bis(tert-
butyldimethylsilyloxy)-3,4-O-isopropylidene-3,4-
dihydroxyoctadecyl 2,3-bis-O-(4-methoxybenzyl)-4,6-O-(di-tert-
butyl)silylene-a-D-galactopyranoside (26)
To a solution of 25 (180 mg, 0.15 mmol) in dry THF (3 mL) was
added Me3P (1 M solution in THF, 0.80 mL). After stirring for 2 h at
room temperature, NaOH (1 M solution in H2O, 2.9 mL) was added
to this solution. The mixture was stirred for 2 h at room tempera-
ture, diluted with water, and extracted with CHCl3, which was
washed with H2O and brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to give a mixture, which was chro-
matographed on a silica gel column. Elution with hexane–EtOAc
4.1.25. (2S,3S,4S,5R,6S)-5,6-Bis(tert-butyldimethylsilyloxy)-2-
hexacosanoylamino-3,4-O-isopropylidene-3,4-
dihydroxyoctadecyl a-D-galactopyranoside (29)
To a solution of 28 (61 mg, 0.05 mmol) and pyridine (40 mg,
0.77 mmol) in dry THF (5 mL) was added HFꢀpyridine (HF: ꢂ70%;
pyridine: ꢂ30%, 30 mg, ca. 1.05 mmol) under an atmosphere of ar-
gon at room temperature. After stirring for 1 h, the reaction mix-
ture was diluted with CHCl3, and washed with aq satd NaHCO3
and brine, 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 hexane–EtOAc (1:4) gave 29
(9:1, then 2:1) gave 26 (136 mg, 77%) as a gum. IR
m
max(KBr)
2928, 2856, 1613, 1513, 1472, 1464, 1250 cmꢁ1
.
1H NMR
(500 MHz, CDCl3): d 0.03 (3H, s), 0.056 (3H, s), 0.061 (3H, s), 0.10
(3H, s), 0.86–0.89 (21H, m, containing two 9H singlets at 0.88
and 0.89 ppm), 1.00 (9H, s), 1.06 (9H, s), 1.25–1.29 (21H, br s, con-
taining 3H singlet at 1.29 ppm), 1.39–1.42 (5H, m, containing 3H
singlet at 1.42 ppm), 1.54–1.57 (4H, m, containing NH2), 3.08
(1H, m), 3.28 (1H, dd, J 7.8, 10.0 Hz), 3.59 (1H, s), 3.67 (1H, m),
3.77 (1H, dd, J 2.8, 9.9 Hz), 3.79 (3H, s), 3.80 (3H, s), 3.89 (1H, m),
3.94–3.98 (3H, m), 4.03 (1H, m), 4.11 (1H, dd, J 1.6, 12.4 Hz), 4.18
(1H, dd, J 2.0, 12.4 Hz), 4.48 (1H, d, J 2.5 Hz, galactose C4-H), 4.59,
4.74 (2H, AB-q, J 11.4 Hz), 4.63, 4.66 (2H, AB-q, J 11.4 Hz), 4.76
(1H, d, J 3.4 Hz, anomeric H), 6.83–6.87 (4H, m), 7.26–7.34 (4H,
m). ESI-MS: m/z 1160.8 [M+H]+. HR ESI-MS: calcd for C63H114NO12-
Si3: 1160.7643; observed: 1160.7644.
(46 mg, 85%) as a gum. IR
mmax(KBr): 3411 (br), 2925, 2854,
1655 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.07 (3H, s), 0.10 (3H,
.
s), 0.15 (6H, s), 0.88 (9H, s, and 3H, t, J 6.6 Hz), 0.94 (9H, s), 1.25
(62H, br s), 1.32 (3H, s), 1.34–1.38 (2H, m), 1.44 (3H, s),
1.56–1.65 (4H, m), 2.15 (1H, t, J 7.0 Hz), 2.41 (1H, br s, OH), 2.65
(1H, br s, OH), 2.75 (1H, br s, OH), 2.87 (1H, s, OH), 3.47 (1H, m),
3.63 (1H, m), 3.71–3.79 (3H, m), 3.93 (1H, m), 4.04 (1H, m), 4.08
(1H, s), 4.12 (2H, s), 4.27 (1H, d, J 10.6 Hz), 4.88 (1H, d, J 3.4 Hz,
anomeric H), 6.20 (1H, d, J 6.2 Hz, NH). ESI-MS: m/z 1180.91
[M+Na]+. HR ESI-MS: calcd for C65H131NO11SiNa: 1180.9153; ob-
served: 1180.9149.
4.1.23. (2S,3S,4S,5R,6S)-5,6-Bis(tert-butyldimethylsilyloxy)-2-
hexacosanoylamino-3,4-O-isopropylidene-3,4-
dihydroxyoctadecyl 2,3-bis-O-(4-methoxybenzyl)-4,6-O-(di-tert-
4.1.26. (2S,3S,4S,5S,6S)-2-Hexacosanoylamino-3,4,5,6-
butyl)silylene-
a-
D-galactopyranoside (27)
tetrahydroxyoctadecyl a-D-galactopyranoside (30) (RCAI-120)
Compound 26 (135 mg, 0.12 mmol) was treated as described for
the formation of 12 from 11 to give 27 (154 mg, 86%) as a gum. IR
m
To a solution of 29 (45 mg, 0.04 mmol) in CH2Cl2–CH3CN (1:1,
20 mL) and H2O (384 mg) was added aq 46% HF (294 mg,
7.71 mmol). The solution was stirred for 16 h at room temperature
max(KBr) 3349 (w), 2925, 2854, 1678, 1613, 1514, 1465 cmꢁ1 1H
.

60
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
to yield insoluble powder, and the whole was filtered. The filter
cake was washed with aq satd NaHCO3 and water, and a small
amount of CHCl3–CH3CN (1:1), consecutively. The residual powder
was dried under reduced pressure, and chromatographed on a sil-
ica gel (1 g) column. [It was loaded on the column with hot CHCl3
or hot CHCl3–MeOH (19:1).] Elution with CHCl3–MeOH (9:1, then
to give a mixture, which was chromatographed on a silica gel col-
umn. Elution with hexane–EtOAc (16:1, then 9:1) gave 34 (307 mg,
61%) as an oily mixture of E- and Z-isomers. IR
mmax(KBr): 3519,
2917, 2850, 1735, 1468 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.88
.
(3H, t, J 7.0 Hz), 1.21–1.31 (36H, m), 1.70 (1H, m), 1.85 (1H, m),
1.95 (2H, q like, J 6.6–7.4 Hz), 2.04–2.17 (2H, m), 2.69 (1H, d, J
5.9 Hz, OH), 4.23 (1H, m), 5.21 (2H, s), 5.33–5.46 (2H, m),
7.35–7.40 (5H, m). ESI-MS: m/z 523.41 [M+Na]+. HR ESI-MS: calcd
for C33H56O3: 523.4127; observed: 523.4123.
7:1) gave 30 (32 mg, 92%) as a powder. ½a D26
ꢄ
+35.1 (c 0.5, pyridine).
m .
IR
max(KBr): 3293, 2919, 2850, 1649 cmꢁ1 1H NMR [500 MHz,
CDCl3–CD3OD (10:1)]: d 0.88 (6H, t, J 6.8 Hz), 1.26 (62H, br s),
1.31–1.63 (4H, m), 1.67–1.74 (2H, m), 2.20 (1H, t, J 7.6 Hz),
3.58–3.64 (3H, m), 3.71–3.76 (2H, m), 3.77–3.83 (4H, m),
3.90–3.96 (3H, m), 4.26 (1H, m), 4.92 (1H, d, J 3.7 Hz, anomeric
H). 13C NMR (126 MHz, pyridine-d5): d 14.26, 22.92, 26.30, 29.59,
29.74, 29.78, 29.83, 29.90, 29.96, 29.99, 30.03, 30.12, 30.32,
32.10, 35.13, 36.76, 52.02, 62.64, 68.42, 70.35, 70.96, 71.44,
71.59, 72.91, 73.57, 73.77, 101.40, 173.50. ESI-MS: m/z 912.71
[M+Na]+. HR ESI-MS: calcd for C50H99NO11Na: 912.7110; observed:
912.7116.
4.1.30. (2R,5EZ)-Benzyl 2-(tert-butyldimethylsilyloxy)hexacos-
5-enoate (35)
To a solution of 34 (305 mg, 0.607 mmol) in dry CH2Cl2 (10 mL)
containing 2,6-lutidine (100 mg, 0.93 mmol) was added TBDMSOTf
(190 mg, 0.72 mmol). After stirring for 1.5 h at room temperature,
the reaction mixture was diluted with CH2Cl2. To this solution was
added an aq 3% solution of H2O2 (5 mL), the mixture was stirred for
5 min at room temperature, and washed with H2O 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 hexane–EtOAc (39:1) gave 35 (297 mg, 79%) as an oily
mixture of E- and Z-isomers. The Rf values of these isomers were
different, but their 1H NMR spectra were almost identical. IR
4.1.27. (2R)-Benzyl 5-oxo-2-tetrahydrofurancarboxylate (32)
To a solution of (R)-5-oxo-2-tetrahydrofurancarboxylic acid (31,
97% ee) (1.35 g, 10.4 mmol) and BnOH (2.25 g, 20.8 mmol) in
CH2Cl2 (30 mL) was added EDAC (6.00 g, 31.2 mmol). After stirring
for 1.5 h at room temperature, the reaction mixture was diluted
with CH2Cl2 (100 mL), 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
hexane–EtOAc (2:1, then 1:2) gave 32 (1.60 g, 70%) as an oil. IR
m
max(KBr): 2925, 2853, 1757, 1458 cmꢁ1 1H NMR (500 MHz,
.
CDCl3): d 0.03 (3H, s), 0.04 (3H, s), 0.86–0.89 (12H, m), 1.25
(36H, m), 1.73–1.79 (2H, m), 1.93–2.00 (2H, m), 2.03–2.18 (2H,
m), 4.24 (1H, dd, J 5.3, 6.8 Hz), 5.13, 5.17 (2H, AB-q, J 12.3 Hz),
5.29–5.42 (2H, m), 7.32–7.36 (5H, m). ESI-MS: m/z 637.50
[M+Na]+. HR ESI-MS: calcd for C39H70O3SiNa: 637.4992; observed:
637.4989.
m
max(KBr): 1771, 1746, 1467 cmꢁ1 1H NMR (500 MHz, CDCl3): d
.
2.30 (1H, m), 2.47–2.65 (2H, m), 4.98 (1H, dd, J 4.3, 8.2 Hz), 5.22,
5.24 (2H, AB-q, J 12.1 Hz), 7.34–7.41 (5H, m). ESI-MS: m/z 220.07
[MÅ]+. HR ESI-MS: calcd for C12H12O4: 220.0736; observed:
220.0726.
4.1.31. (2R)-2-(tert-Butyldimethylsilyloxy)hexacosanoic acid
(36)
The suspension of 35 (66 mg, 0.107 mmol) in THF (6.6 mL) con-
taining 20% Pd(OH)2 on carbon (66 mg) was stirred under an atmo-
sphere of H2 for 5–16 h. The resulting solution was filtered, and the
catalyst was washed with THF. The combined THF solution was
concentrated in vacuo to give 36 (57 mg, quantitatively) as a crude
crystalline solid, which was employed for the next reaction with-
out further purification. (This product was unstable for silica gel
4.1.28. (2R)-Benzyl 5-(hydroxyl)tetrahydrofuran-2-carboxylate
(33)
A solution of disiamylborane was prepared by mixing BH3 (1 M
solution in THF, 14 mL) and 2-methyl-2-butene (2 M solution in
THF, 14 mL) at 0 °C under Ar, and by stirring for 75 min at 0 °C.
To this disiamylborane solution was introduced a solution of 32
(1.60 g, 7.26 mmol) in THF (2 mL). The combined solution was
warmed to room temperature over a period of 2.5 h, and then stir-
red for another 15 h, and quenched with aq satd NH4Cl, followed
by dilution of EtOAc. The mixture was stirred for 10 min, and the
organic phase was separated, washed with aq satd NH4Cl 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 hexane–EtOAc (3:2) gave 33 (0.83 g, 51%)
chromatography.) IR
mmax(KBr): 3300–3000 (br), 2919, 2850,
1727 (br) cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.13 (3H, s), 0.14
.
(3H, s), 0.88 (3H, t, J 6.9 Hz), 0.94 (9H, s), 1.25 (44H, br s),
1.71–1.84 (2H, m), 4.30 (1H, t, J 5.0 Hz). FABMS (negative-ion):
m/z 507.4, 525.5 [MꢁH]ꢁ. HRFABMS: calcd for C32H65O3Si:
525.4703; observed: 525.4679.
4.1.32. Authentic sample of N-[(R)-1-(1-naphthyl)ethyl]-(R)-2-
(tert-butyldimethylsilyloxy)hexacosanamide (38) or N-[(S)-1-
(1-naphthyl)ethyl]-(R)-2-(tert-
as an oily 2:3 mixture of anomers. IR m .
max(KBr): 3650, 1740 cmꢁ1
1H NMR (500 MHz, CDCl3): d 1.97–2.05 (2H, m), 2.17–2.47 (2H, m),
2.92 (0.4H, s, OH), 3.55 (0.6H, s, OH), 4.63 (0.6H, dd, J 6.6, 8.3 Hz),
4.76 (0.4H, dd, J 4.1, 8.8 Hz), 5.17–5.23 (2H, m), 5.60 (0.6H, d, J
3.2 Hz), 5.74 (0.4H, d, J 3.9 Hz), 7.36–7.37 (5H, m). ESI-MS: m/z
204 [MꢁH2O]+, 222 [M]+. HR ESI-MS: calcd for C12H14O4:
222.0893; observed: 222.0901.
butyldimethylsilyloxy)hexacosanamide (40)
To a solution of 36 (4.9 mg, 0.01 mmol) and (R)-1-(1-naph-
thyl)ethylamine (37, 3.2 mg, 0.02 mmol) or (S)-1-(1-naphthyl)eth-
ylamine (39, 3.2 mg, 0.02 mmol) in CH2Cl2 (0.4 mL) was added
EDAC (7.0 mg, 0.04 mmol). The mixture was stirred for 2 h at room
temperature, and the whole was loaded on a preparative TLC plate.
Development with hexane–EtOAc (6:1) gave 38 [(R,R)-isomer,
4 mg] or 40 [(S,R)-isomer, 4 mg]. Physical data of 38: Rf = 0.446
(hexane:EtOAc = 6:1); 1H NMR (500 MHz, CDCl3): d ꢁ0.37 (3H, s),
ꢁ0.06 (3H, s), 0.68 (9H, s), 0.88 (3H, t, J 7.0 Hz), 1.25 (44H, br s),
1.68 (3H, d, J 6.8 Hz), 1.70–1.76 (2H, m), 4.11 (1H, dd, J 4.4,
5.7 Hz), 5.93 (1H, m), 6.84 (1H, d, J 8,8 Hz, NH), 7.44–7.53 (4H,
m), 7.79 (1H, d, J 7.6 Hz), 7.85 (1H, d, J 7.8 Hz), 8.12 (1H, d, J
8.3 Hz). ESI-MS: m/z 702.56 [M+Na]+. HR ESI-MS: calcd for
4.1.29. (2R,5EZ)-Benzyl 2-hydroxyhexacos-5-enoate (34)
To a suspension of Ph3P(Br)C21H43 (1.59 g, 2.50 mmol) in dry
THF (5 mL) was added LiN(TMS)2 (1 M in THF, 4 mL) with stirring
under an atmosphere of Ar. After stirring for 1 h at 0 °C, the mix-
ture became a red-colored solution. To this solution was added a
solution of 33 (222 mg, 1.00 mmol) in THF (3 mL). The solution
was stirred for 1 h at 0 °C, and for 2 h at room temperature. The
reaction mixture was quenched with aq satd NH4Cl, and extracted
with EtOAc–hexane (1:1), which was washed with brine, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo
C
44H77NO2SiNa, 702.5621; observed, 702.5635. Physical data of
40: Rf= 0.429 (hexane:EtOAc = 6:1); 1H NMR (500 MHz, CDCl3): d

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
61
0.08 (3H, s), 0.11 (3H, s), 0.88 (3H, t, J 7.0 Hz), 0.90 (9H, s),
1.12–1.32 (44H, m), 1.62–1.67 (5H, m, containing 3H, d, J 6.9 Hz
at 1.65 ppm), 4.18 (1H, t, J 5.3 Hz), 5.93 (1H, m), 6.89 (1H, d, J
8.8 Hz, NH), 7.44–7.54 (4H, m), 7.79 (1H, m), 7.86 (1H, m), 8.10
(1H, d, J 8.5 Hz). ESI-MS: m/z 702.56 [M+Na]+. HR ESI-MS: calcd
for C44H77NO2SiNa, 702.5621; observed, 702.5625.
HPLC conditions: [Column: PEGASIL silica 60-5, 4.6ø ꢃ 250 mm;
Eluent: hexane–EtOAc (9:1); Flow rate: 1 mL/min; Detection:
254 nm; Column temperature: 10 °C.]
HPLC retention time of authentic 38 [(R,R)-isomer] was
5.41 min, and that of authentic 40 [(S,R)-isomer] was 6.56 min.
Preparation of HPLC analytical sample: To a solution of 36
(4.9 mg, 0.01 mmol) and (S)-1-(1-naphthyl)ethylamine (39,
3.2 mg, 0.02 mmol) in CH2Cl2 (0.4 mL) was added EDAC (7.0 mg,
0.04 mmol). The mixture was stirred for 2 h at room temperature,
and evaporated in vacuo to give a residue. To this residue were
added EtOAc (0.3 mL) and H2O (0.3 mL). This mixture was stirred
for 15 min at room temperature, and allowed to stand to separate
into two layers. The upper EtOAc layer was concentrated in vacuo
to obtain a HPLC analytical sample of 40.
The HPLC sample of 40 showed that the area integrated at
5.41 min was 0.861, and the area integrated at 6.56 min was
82.463. The diastereomeric excess of compound 40 is 97.9%
(81.602/83.324 = 0.97933). The enantiomeric excess of starting
31 was 97%, and that of 36 was 97.9%. Therefore, it is obvious that
no racemization occurred during the conversion from 31 to 36.
with aq satd 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 hexane–EtOAc (1:1,
then 2:3) gave 42 (23 mg, two steps 68% yield) as a gum. IR
m
max(KBr): 3800–3400 (br), 2924, 2853, 1653 cmꢁ1 1H NMR
.
(500 MHz, CDCl3): d 0.88 (6H, t, J 6.8 Hz), 1.22–1.36 (66H, m),
1.52–1.86 (4H, m), 2.49 (1H, s, OH), 2.54 (1H, br s, OH), 2.98 (1H,
d, J 4.8 Hz, OH), 3.59–3.65 (2H, m), 3.67–3.72 (2H, m), 3.73–3.81
(4H, m), 3.82–3.87 (2H, m), 3.92 (1H, br s), 4.05 (1H, dd, J 3.6,
11.1 Hz), 4.48 (1H, m), 4.49–4.76 (12H, m), 6.86 (1H, d, J 9.5 Hz,
NH), 7.24–7.37 (25H, m). ESI-MS: m/z 1362.94 [M+Na]+. HR ESI-
MS: calcd for C85H129NO11Na: 1362.9463; observed: 1362.9497.
4.1.35. (2S,3S,4R,5S)-3,4,5-Tri(hydroxy)-2-[(R)-20-
hydroxyhexacosanoylamino]octadecyl
(43) (RCAI-129)
a-D-galactopyranoside
Compound 42 (23 mg, 0.02 mmol) was treated as described for
the formation of 14 from 13 to give 43 (8 mg, 52%) as a powder.
Column chromatography was performed by elution with CHCl3–
CH3OH (9:1, then 7:1). ½a D19
ꢄ
+63.2 (c 0.65, CHCl3–CH3OH (7:1)). IR
m .
max(KBr) 3810–3291, 2919, 2850, 1646 cmꢁ1 1H NMR (500 MHz,
CDCl3–CD3OD (9:1)): d 0.88 (6H, t, J 6.9 Hz), 1.10–1.62 (69H, m),
1.77 (1H, m), 3.70–3.90 (10H, m), 3.92 (1H, d, J 2.9 Hz), 4.01(1H,
dd, J 3.3, 8.7 Hz), 4.23 (1H, dd, J 4.3, 10.1 Hz), 4.88 (1H, d, J
3.9 Hz, anomeric H), 7.56 (1H, d, J 9.1 Hz, NH, gradually disap-
peared). 13C NMR [126 MHz, CDCl3–CD3OD (9:1)]: d 14.03, 22.63,
25.44, 25.89, 29.32, 29.43, 29.67, 31.87, 33.62, 34.40, 50.18,
61.87, 67.34, 68.87, 69.53, 70.04, 70.48, 70.86, 71.85, 71.96,
72.32, 99.19, 175.65. ESI-MS: m/z 912.73 [M+Na]+. HR ESI-MS:
calcd for C50H99NO11Na: 912.7166; observed: 912.7107.
4.1.33. (2S,3S,4R,5S)-3,4,5-Tris(benzyloxy)-2-[(R)-20-(tert-
butyldimethylsilyloxy)hexacosanoylamino]octadecyl 2,3-di-O-
benzyl-4,6-O-(di-tert-butyl)silylene-a-D-galactopyranoside (41)
To a solution of 11 (30 mg, 0.03 mmol) in THF–CH2Cl2 (1:1,
2 mL) were added DMAP (27 mg, 0.22 mmol), 36 (37 mg,
0.07 mmol), and EDAC (32 mg, 0.17 mmol). The mixture was stir-
red for 3 h at room temperature, and concentrated in vacuo to give
a residue, which was diluted with CH2Cl2. The solution was washed
with H2O, 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 hexane–EtOAc (19:1, then 9:1)
4.1.36. (2S,3S,4S,5R,6S)-2-[(R)-20-(tert-
Butyldimethylsilyloxy)hexacosanoylamino]-5,6-bis(tert-
butyldimethylsilyloxy)-3,4-dihydroxy-3,4-O-
isopropylideneoctadecyl 2,3-di-O-(4-methoxybenzyl)-4,6-O-(di-
tert-butyl)silylene-a-D-galactopyranoside (44)
To a solution of 26 (103 mg, 0.09 mmol) and 36 (172 mg,
0.36 mmol) in THF–CH2Cl2 (1:1, 8 mL) were added DMAP (96 mg,
0.79 mmol) and EDAC (115 mg, 0.60 mmol) at room temperature
under Ar. After stirring for 16 h, the reaction mixture was diluted
with CH2Cl2, washed with H2O, 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 hex-
ane–EtOAc (19:1, then 9:1) gave 44 (150 mg, 100%) as a gum. IR
gave 41 (24 mg, 54%) as
a gum. IR mmax(KBr) 2925, 2854,
1675 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.01 (3H, s), 0.03 (3H,
.
s), 0.87 (9H, s), 0.88 (6H, t, J 7.0 Hz) 0.96 (9H, s), 1.02 (9H, s),
1.19–1.32 (66H, m), 1.52–1.66 (4H, m), 3.52 (1H, s), 3.65 (1H, m),
3.70–3.78 (4H, m), 3.92 (1H, dd, J 3.5, 9.9 Hz), 3.96, 4.00 (2H, AB-
q, J 12.4 Hz), 4.08 (1H, t, J 5.6 Hz), 4.16 (1H, dd, J 4.3, 11.4 Hz),
4.37 (1H, d, J 2.5 Hz), 4.45 (1H, d, J 11.3 Hz), 4.50 (1H, d, J
11.5 Hz), 4.57 (1H, d, J 11.5 Hz), 4.59–4.63 (4H, m, containing N-
CH as a multiplet), 4.67 (1H, d, J 11.5 Hz), 4.68 (1H, d, J 11.5 Hz),
4.74–4.78 (3H, m, containing 1H, d, J 4.0 Hz at 4.76 ppm as an ano-
meric H), 6.95 (1H, d, J 9.0 Hz, NH), 7.19–7.40 (25H, m). ESI-MS:
m/z 1617.14 [M+Na]+. HR ESI-MS: calcd for C99H159NO11Si2Na:
1617.1349; observed: 1617.1344.
m
max(KBr) 2925, 2854, 1685, 1678 (shoulder), 1612, 1516, 1466,
1250 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.04 (6H, s), 0.07 (3H,
.
s), 0.09 (3H, s), 0.10 (3H, s), 0.13 (3H, s), 0.88 (6H, t, J 6.8 Hz),
0.88 (9H, s), 0.89 (9H, s), 0.91 (9H, s), 0.97 (9H, s), 1.05 (9H, s),
1.21–1.34 (67H, m), 1.44 (3H, s), 1.58–1.70 (4H, m), 3.64–3.72
(3H, m), 3.77–3.84 (8H, m, containing two 3H, singlets at 3.79
and 3.80 ppm), 3.92 (1H, m), 4.20 (1H, m), 4.09–4.21 (4H, m),
4.26 (1H, m), 4.51 (1H, s), 4.52, 4.72 (2H, AB-q, J 11.3 Hz), 4.59
4.63 (2H, AB-q, J 11.1 Hz), 4.83 (1H, d, J 3.7 Hz, anomeric H),
6.78–6.86 (5H, m, containing NH), 7.23–7.31 (4H, m). ESI-MS:
m/z 1691.14 [M+Na]+. HR ESI-MS: calcd for C95H177NO14Si4Na:
1691.2144; observed: 1691.2113.
4.1.34. (2S,3S,4R,5S)-3,4,5-Tris(benzyloxy)-2-[(R)-20-
hydroxyhexacosanoylamino]octadecyl 2,3-di-O-benzyl-a-D-
galactopyranoside (42)
(1) To a solution of 41 (40 mg, 0.03 mmol) in CH2Cl2–CH3CN
(1:1, 8 mL) was added aq 46% HF (0.6 mL, 0.03 mmol). The solution
was stirred for 3 h at room temperature, and diluted with CHCl3,
which was washed with satd aq NaHCO3, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to give a mixture
(34 mg).
4.1.37. (2S,3S,4S,5R,6S)-2-[(R)-20-(tert-
Butyldimethylsilyloxy)hexacosanoylamino]-5,6-bis(tert-
butyldimethylsilyloxy)-3,4-dihydroxy-3,4-O-
isopropylideneoctadecyl 4,6-O-(di-tert-butyl)silylene-a-D-
(2) The above obtained mixture was dissolved in THF (3 mL)
containing pyridine (40 mg). To this solution was added HFꢀpyri-
dine (HF: ꢂ70%; pyridine: ꢂ30%, 33 mg, ca. 1.16 mmol) under an
atmosphere of argon at room temperature. After stirring for
1.5 h, the reaction mixture was diluted with CHCl3, and washed
galactopyranoside (45)
Compound 44 (150 mg, 0.09 mmol) was treated as described for
the formation of 28 from 27 to give 45 (77 mg, 60%) as a gum. IR
m
max(KBr) 3406, 2925, 2855, 1675, 1520, 1509, 1471 cmꢁ1 1H
.
NMR (500 MHz, CDCl3): d 0.05 (3H, s), 0.06 (3H, s), 0.10 (6H, s),

62
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
0.101 (3H, s), 0.12 (3H, s), 0.87–0.92 (24H, m), 0.95 (9H, s), 1.02
(9H, s), 1.06 (9H, s), 1.05 (9H, s), 1.23–1.32 (63H, m, containing
3H, singlet at 3.31 ppm), 1.42 (3H, s), 1.50–1.70 (4H, m), 2.50
(1H, d, J 8.8 Hz, OH), 2.83 (1H, d, J 11.3 Hz, OH), 3.27 (1H, t, J
9.7 Hz), 3.59–3.74 (4H, m), 3.90 (1H, dd, J 5.6, 8.1 Hz), 4.04–4.26
(7H, m), 4.41 (1H, m), 4.77 (1H, d, J 3.5 Hz, anomeric H), 7.20
(1H, d, J 9.5 Hz, NH). ESI-MS: m/z 1451.02 [M+Na]+. HR ESI-MS:
calcd for C79H161NO12Si4Na: 1451.0994; observed: 1451.1016.
m), 4.49 (0.8H, m), 5.08 (0.2H, m, changed to triplet, J 3.6 Hz, on
addition of D2O), 5.26 (0.8H, m, changed to dd, J 4.3, 7.0 Hz, on
addition of D2O). EIMS: m/z 159.0 [MꢁMe]Å+. HREIMS: calcd for
C7H11O4: 159.0657; observed: 159.0663.
4.1.41. (2S,3R,5EZ)-2,3-O-Isopropylidene-5-hexacosene-1,2,3-
triol (50)
To a suspension of Ph3P(Br)C21H43 (5.62 g, 8.81 mmol) in THF
(19 mL) was added LiN(TMS)2 (1 M in THF, 20.4 mmol), and the
mixture was stirred for 1 h at 0 °C under Ar. To this resulting solu-
tion was added a solution of 49 (1.03 g, 5.91 mmol) in THF (11 mL)
at 0 °C, and the mixture was stirred for 2 h at room temperature,
quenched with aq satd NH4Cl, diluted with CHCl3, which was
washed well with water, and aq 1% H2O2, and again with water,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a residue, which was chromatographed on a silica gel
column. Elution with hexane–EtOAc (19:1, then 9:1, finally 4:1)
gave 50 (1.35 g, 50%) as an oily mixture of E- and Z-isomers. IR
4.1.38. (2S,3S,4S,5R,6S)-2-[(R)-20-(tert-
Butyldimethylsilyloxy)hexacosanoylamino]-5,6-bis(tert-
butyldimethylsilyloxy)-3,4-dihydroxy-3,4-O-
isopropylideneoctadecyl a-D-galactopyranoside (46)
Compound 45 (75 mg, 0.05 mmol) was treated as described for
the formation of 29 from 28 to give 46 (50 mg, 68%) as a gum. Col-
umn chromatography was performed by elution with hexane–
EtOAc (1:1). IR
m
.
max(KBr) 3409 (br), 2925, 2854, 1670, 1523,
1515, 1465 cmꢁ1
1H NMR (500 MHz, CDCl3): d 0.08 (3H, s), 0.09
(6H, s), 0.10 (3H, s), 0.11 (3H, s), 0.12 (3H, s), 0.87–0.89 (24H, m),
0.95 (9H, s), 1.25 (64H, br s), 1.32 (3H, s), 1.41 (3H, s), 1.44–1.75
(4H, m), 2.58 (1H, m, OH), 2.65 (1H, s, OH), 2.80 (1H, s, OH), 3.09
(1H, d, J 12.0 Hz, OH), 3.25 (1H, t, J 10.3 Hz), 3.60 (1H, m), 3.70
(1H, m), 3.76–3.80 (2H, m), 3.89 (1H, m), 3.93–3.98 (2H, m),
4.04–4.13 (4H, m), 4.25–4.31 (2H, m), 4.83 (1H, d, J 3.6 Hz, anomer-
ic H), 7.30 (1H, d, J 9.5 Hz, NH). ESI-MS: m/z 1310.94 [M+Na]+. HR
m
max(KBr) 3536, 2918, 2850, 1468 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 0.88 (3H, t, J 7.0 Hz), 1.25 (36H, m), 1.38 (3H, s), 1.49 (3H, s), 1.84
(1H, dd, J 5.4, 6.6 Hz), 2.04 (1H, m), 2.28 (1H, m), 2.38 (1H, m),
3.62–3.68 (2H, m), 4.15–4.23 (2H, m), 5.38 (1H, m), 5.52 (1H, m).
ESI-MS: m/z 475.41 [M+Na] +. HR ESI-MS: calcd for C29H56O3Na:
475.4127; observed: 475.4127.
ESI-MS: calcd for
1310.9994.
C
71H145NO12Si3Na: 1310.9972; observed:
4.1.42. (2S,3R)-2,3-O-(Isopropylidene)hexacosane-1,2,3-triol
(51)
The suspension of 50 (1.74 g, 3.80 mmol) in hexane (50 mL)
containing 10% Pd/C (580 mg) was stirred under an atmosphere
of H2 for 2 h. The resulting solution was filtered, and the catalyst
was washed with THF. The combined THF solution was concen-
trated in vacuo to give a residue, which was chromatographed on
a silica gel column. Elution with hexane–EtOAc (19:1, then 4:1)
4.1.39. (2S,3S,4S,5S,6S)-2-[(R)-20-(Hydroxy)hexacosanoylamino]-
3,4,5,6-(tetrahydroxy)octadecyl
(RCAI-130)
a-D-galactopyranoside (47)
To a solution of 46 (20 mg, 0.02 mmol) in CH2Cl2–CH3CN (3:2,
10 mL) and H2O (152 mg) was added aq 46% HF (140 mg,
3.67 mmol). The solution was stirred for 16 h at room temperature
to yield insoluble powder, and the whole was filtered. The filter
cake was washed with aq satd NaHCO3 and water, and a small
amount of CHCl3–CH3CN (1:1), consecutively. The residual powder
was dried under reduced pressure, and chromatographed on a sil-
ica gel (1 g) column. [It was loaded on the column with hot CHCl3
or hot CHCl3–MeOH (19:1).] Elution with CHCl3–MeOH (9:1, then
gave 51 (1.71 g, 98%) as an oil. IR
mmax(KBr) 3458, 2924, 2848,
1468 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.88 (3H, t, J 7.0 Hz),
.
1.23–1.61 (50H, m, containing two 3H singlets at 1.37 and
1.48 ppm), 1.82 (1H, dd, J 4.9, 7.3 Hz, OH), 3.59–3.63 (2H, m),
4.13–4.17 (2H, m). ESI-MS: m/z 477.4 [M+Na] +. HR ESI-MS: calcd
for C29H58O3Na: 477.4284; observed: 477.4298.
5:1) gave 47 (9.2 mg, 65%) as a powder. ½a D27
ꢄ
+50.6 [(c 0.7,
max(KBr): 3396 (br), 2919, 2850, 1652,
4.1.43. (2R,3R)-2,3-Dihydroxy-2,3-O-
(isopropylidene)hexacosanoic acid (52)
CHCl3–CH3OH (5:1)). IR
m
1516, 1469 cmꢁ1
.
1H NMR [500 MHz, CDCl3–CD3OD (10:1)]: d
A solution of 51 (50 mg, 0.11 mmol), NaIO4 (236 mg,
0.88 (6H, t, J 7.0 Hz), 1.12–1.84 (68H, m), 3.64–3.70 (2H, m),
3.72–3.89 (8H, m), 3.90–3.98 (2H, m), 4.01 (1H, dd, J 3.6, 8.5 Hz),
4.25 (1H, m), 4.91 (1H, d, J 3.8 Hz, anomeric H). 13C NMR
(126 MHz, CDCl3–CD3OD (6:1)): d 13.91, 22.55, 25.38, 25.73,
29.23, 29.24, 29.33, 29.45, 29.52, 29.54, 29.57, 29.58, 29.61,
29.68, 31.80, 33.66, 34.29, 49.60, 61.74, 66.97, 68.70, 69.25,
69.49, 69.91, 70.81, 71.86, 71.96, 72.00, 72.54, 99.27, 175.62. ESI-
MS: m/z 928.58 [M+Na]+. HR ESI-MS: calcd for C50H99NO12Na:
928.7056; observed: 928.7080.
1.10 mmol), and RuCl3ꢀnH2O (3 mg) in CCl4–CH3CN–H2O (2:2:3,
7 mL) was stirred for 3 h at room temperature, and diluted with
CHCl3, washed with water, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to give a residue, which was chro-
matographed on a silica gel column. Elution with hexane–EtOAc
(6:1, then 1:2) gave 52 (41 mg, 80%) as a solid. IR
mmax(KBr)
3290–2560, 1728, 1469 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.88
.
(3H, t, J 7.0 Hz), 1.23–1.71 (50H, m, containing two 3H singlets at
1.40 and 1.61 ppm), 4.38 (1H, m), 4.55 (1H, d, J 7.3 Hz). ESI-MS:
m/z 467.4 [MꢁH]ꢁ. HR ESI-MS: calcd for C29H55O4: 467.4100; ob-
served: 467.4107.
4.1.40. 2-Deoxy-3,4-O-isopropylidene-
To a solution of 48 (2-deoxy- -ribose, 5.29 g, 39.44 mmol) in
DMF (40 mL) containing 2,2-dimethoxypropane (6.16 g,
L-ribopyranose (49)
L
4.1.44. (2S,3S,4R,5S)-2-[(20R,30R)-20,30-Dihydroxy-20,30-O-
(isopropylidene)hexacosanoylamino]-3,4,5-
tris(benzyloxy)octadecyl 2,3-di-O-benzyl-4,6-O-(di-tert-
59.2 mmol) was added Amberlyst 15 ion-exchange resin
(395 mg). The mixture was stirred for 18 h at room temperature,
and filtered. The filtrate was concentrated in vacuo to give a mix-
ture, which was chromatographed on a silica gel column. Elution
with hexane–EtOAc (3:2, then 2:3) gave 49 (3.41 g, 50%) as an oily
butyl)silylene-a-D-galactopyranoside (53)
To a solution of 11 (150 mg, 0.14 mmol) and 52 (130 mg,
0.28 mmol) in dry THF–CH2Cl2 (1:1, 10 mL) were added DMAP
(170 mg, 1.38 mmol) and EDAC (270 mg, 1.38 mmol) at room tem-
perature under Ar. After stirring for 16 h, the reaction mixture was
diluted with CH2Cl2, washed with H2O, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a mixture,
which was chromatographed on a silica gel column. Elution with
4:1 mixture of anomers. IR
m .
max(KBr) 3534 cmꢁ1 1H NMR
(500 MHz, CDCl3): d 1.35 (2.4H, s), 1.37 (0.6H, s), 1.51 (2.4H, s),
1.57 (0.6H, s), 1.79 (1H, m), 2.22–2.27 (1H, m), 2.69 (0.8H, d, J
3.9 Hz, OH), 3.70 (1H, dd, J 3.4, 12.2 Hz), 3.84 (0.2H, d, J 9.1 Hz,
OH), 3.95 (1H, dd, J 3.4, 12.7 Hz), 4.16–4.20 (1H, m), 4.43 (0.2H,

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
63
hexane–EtOAc (19:1, then 6:1) gave 53 (145 mg, 68%) as a gum. IR
max(KBr) 2924, 2853, 1683 cmꢁ1 1H NMR (500 MHz, CDCl3): d
containing two 3H singlets at 1.28 and 1.31 ppm), 1.42–1.62 (7H,
m, containing 3H singlet at 1.44 ppm), 3.51 (1H, m), 3.61 (1H, s),
3.72–3.78 (2H, m), 3.78–3.82 (7H, m), 3.86 (1H, m), 3.93 (1H, dd,
J 3.4, 10.0 Hz), 4.06–4.11 (2H, m), 4.12–4.16 (2H, m), 4.20 (1H,
m), 4.30 (1H, m), 4.38 (1H, d, J 7.3 Hz), 4.44 (1H, d, J 2.9 Hz),
4.57, 4.70 (2H, AB-q, J 11.4 Hz), 4.60, 4.63 (2H, AB-q, J 11.7 Hz),
4.67 (1H, d, J 3.9 Hz, anomeric H), 6.79–6.87 (5H, m, containing
NH), 7.26–7.34 (4H, m). ESI-MS: m/z 1633.15 [M+Na]+. HR ESI-
MS: calcd for C92H167NO15Si3Na: 1633.1541; observed: 1633.1559.
m
.
0.88 (6H, t, J 6.8 Hz), 0.97 (9H, s), 1.03 (9H, s), 1.18–1.30 (64H,
m), 1.31 (3H, s), 1.36–1.38 (2H, m), 1.40 (3H, s), 1.56–1.66 (2H,
m), 3.53 (1H, s), 3.65 (1H, m), 3.72 (1H, m), 3.74–3.78 (2H, m),
3.84 (1H, m), 3.92–4.09 (4H, m), 4.32 (1H, m), 4.37–4.41 (2H, m),
4.48–4.78 (11H, m), 7.00 (1H, d, J 8.5 Hz, NH), 7.21–7.41 (25H,
m). ESI-MS: m/z 1537.08 [M+H]+, 1559.07 [M+Na]+. HR ESI-MS:
calcd for C96H149NO12SiNa: 1559.0747; observed: 1559.0747.
4.1.45. (2S,3S,4R,5S)-2-[(20R,30R)-20,30-
(Dihydroxyl)hexacosanoylamino]-3,4,5-
4.1.48. (2S,3S,4S,5R,6S)-5,6-Bis(tert-butyldimethylsilyloxy)-2-
[(20R,30R)-20,30-O-(isopropylidene)hexacosanoylamino]-3,4-O-
tris(benzyloxy)octadecyl 2,3-di-O-benzyl-
a-D
-
(isopropylidene)octadecyl 4,6-O-(di-tert-butyl)silylene-a-D-
galactopyranoside (54)
galactopyranoside (57)
Compound 53 (129 mg, 0.08 mmol) was treated as described for
the formation of 42 from 41 to give 54 (78 mg, two steps 69%) as a
gum. Column chromatography was performed by elution with hex-
Compound 56 (124 mg, 0.08 mmol) was treated as described for
the formation of 28 from 27 to give 57 (99 mg, 94%) as a gum. Col-
umn chromatography was performed by elution with hexane–
ane–EtOAc (2:3, then 1:4). IR
m
max(KBr) 3645, 2924, 2853,
EtOAc (6:1, then 3:1). IR
mmax(KBr) 3405 (br), 2926, 2855, 1680,
1655 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.88 (6H, t, J 6.9 Hz),
.
1518, 1470 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.05 (3H, s), 0.07
.
1.21–1.31 (64H, m), 1.54–1.70 (4H, m), 2.23 (1H, m, OH), 2.49
(1H, s, OH), 3.38–3.48 (3H, m, containing one OH), 3.51–3.68 (6H,
m), 3.70–3.73 (2H, m), 3.74–3.79 (2H, m, containing one OH),
3.82 (1H, t, J 3.8 Hz), 3.90 (1H, m, changed to doublet, J 3.2 Hz,
on addition of D2O), 4.16 (1H, dd, J 3.7, 10.2 Hz), 4.48–4.78 (11H,
m), 6.84 (1H, d, J 9.0 Hz, NH), 7.24–7.37 (25H, m). ESI-MS: m/z
1378.86 [M+Na]+. HR ESI-MS: calcd for C85H129NO12Na:
1378.9412; observed: 1378.9444.
(3H, s), 0.12 (3H, s), 0.13 (3H, s), 0.88 (6H, m), 0.89 (9H, s), 0.90
(9H, s), 1.01 (9H, s), 1.06 (9H, s), 1.25 (64H, br s), 1.31 (3H, s),
1.37 (3H, s), 1.57 (3H, s), 2.52 (1H, d, J 9.5 Hz, OH), 3.39 (1H, t, J
9.8 Hz), 3.59 (1H, m), 3.62–3.73 (4H, m), 3.87 (1H, m), 4.06–4.18
(4H, m), 4.22–4.26 (2H, m), 4.34 (1H, m), 4.41 (1H, m), 4.46 (1H,
d, J 7.8 Hz), 4.86 (1H, d, J 3.9 Hz, anomeric H), 6.94 (1H, d, J
8.8 Hz, NH). ESI-MS: m/z 1393.05 [M+Na]+. HR ESI-MS: calcd for
C76H151NO13Si3Na: 1393.0385; observed: 1393.0405.
4.1.46. (2S,3S,4R,5S)-2-[(20R,30R)-20,30-
Dihydroxyhexacosanoylamino]-3,4,5-trihydroxyoctadecyl
galactopyranoside (55) (RCAI-142)
4.1.49. (2S,3S,4S,5R,6S)-5,6-Bis(tert-butyldimethylsilyloxy)-2-
[(20R,30R)-20,30-O-(isopropylidene)hexacosanoylamino]-3,4-O-
(isopropylidene)octadecyl a-D-galactopyranoside (58)
a-D-
To a solution of 54 (29 mg, 0.02 mmol) in THF (9 mL) was added
Pd(OH)2/C (20 wt %, Degussa type, wet, ꢂ50%, 55 mg). After stirring
for 16 h under hydrogen at room temperature, the catalyst was re-
moved by filtration, and washed with THF, and then CHCl3–MeOH
(5:1). The combined filtrate was concentrated in vacuo to give a
residue, which was chromatographed on a silica gel (1 g) column.
Elution with CHCl3–CH3OH (9:1, then 6:1) gave 55 (10 mg, 52%)
Compound 57 (96 mg, 0.07 mmol) was treated as described
for the formation of 29 from 28 to give 58 (67 mg, 78%) as a
gum. Column chromatography was performed by elution with
hexane–EtOAc (2:3, then 1:3). IR
m
max(KBr) 3418 (br), 2925,
2854, 1676, 1516, 1461, 1381 cmꢁ1
.
1H NMR (500 MHz, CDCl3):
d 0.08 (3H, s), 0.10 (3H, s), 0.12 (3H, s), 0.15 (3H, s), 0.88 (6H,
t, J 7.0 Hz), 0.89 (9H, s), 0.91 (9H, s), 1.25 (60H, br s), 1.31
(3H, s), 1.38 (3H, s), 1.42–1.73 (14H, m, containing two 3H sing-
lets at 1.43 and 1.58 ppm), 2.46 (1H, dd, J 4.2, 8.4 Hz, OH), 2.63
(1H, d, J 2.5 Hz, OH), 2.68 (1H, d, J 11.5 Hz, OH), 2.83 (1H, s, OH),
3.38 (1H, dd, J 9.3, 10.8 Hz), 3.56 (1H, m), 3.69–3.79 (3H, m),
3.85–3.98 (3H, m), 4.03–4.12 (3H, m), 4.18 (1H, m), 4.25 (1H,
m), 4.34 (1H, m), 4.45 (1H, d, J 7.6 Hz), 4.88 (1H, d, J 3.7 Hz, ano-
as a powder. ½a 2D2
ꢄ
+51.2 (c 0.71, pyridine). IR
mmax(KBr) 3383 (br),
2918, 2850, 1645 cmꢁ1
.
1H NMR (500 MHz, pyridine-d5, one drop
of 1% TMS in CDCl3): d 0.88 (6H, t, J 6.9 Hz), 1.21–1.35 (60H, m),
1.57–2.10 (8H, m), 4.32–4.81 (14H, m), 5.38 (1H, m), 5.62 (1H, br
s), 5.65 (1H, d, J 4.0 Hz, anomeric H), 6.18 (1H, br s), 6.33 (1H, br
s), 6.35 (1H, br s), 6.57 (1H, br s), 6.68 (1H, br s), 6.94 (1H, d, J
6.1 Hz), 7.77 (1H, br s), 8.68 (1H, d, J 9.0 Hz, NH). 13C NMR
(126 MHz, pyridine-d5, one drop of 1% TMS in CDCl3): d 14.38,
23.04, 26.57, 27.01, 29.71, 29.72, 30.01, 30.03, 30.07, 30.08,
30.10, 30.12, 30.14, 30.22, 30.29, 30.32, 30.37, 32.22, 32.23,
32.70, 35.13, 50.81, 62.73, 70.32, 71.08, 71.24, 71.74, 73.21,
73.28, 73.57, 74.00, 76.18, 101.11, 174.00. ESI-MS: m/z 928.63
[M+Na]+. HR ESI-MS: calcd for C50H99NO12Na: 928.7065; observed:
928.7069.
meric H), 7.03 (1H, d,
J 9.3 Hz, NH). ESI-MS: m/z 1252.93
[M+Na]+. HR ESI-MS: calcd for C68H135NO13Si2Na: 1252.9370; ob-
served: 1252.9390.
4.1.50. (2S,3S,4S,5S,6S)-2-[(20R,30R)-(20,30-
Dihydroxy)hexacosanoylamino]-3,4,5,6-tetrahydroxyoctadecyl
a-
D
-galactopyranoside (59) (RCAI-147)
Compound 58 (56 mg, 0.07 mmol) was treated as described for
the formation of 30 from 29 to give 59 (20 mg, 48%) as a powder.
4.1.47. (2S,3S,4S,5R,6S)-5,6-Bis(tert-butyldimethylsilyloxy)-2-
[(20R,30R)-20,30-O-(isopropylidene)hexacosanoylamino]-3,4-O-
(isopropylidene)octadecyl 2,3-bis-O-(4-methoxybenzyl)-4,6-O-
Column chromatography was performed by elution with CHCl3–
MeOH (9.1, then 7:1, finally 5:1). ½a D28
ꢄ
+49.6 (c 0.52, pyridine). IR
m
max(KBr) 3361 (br), 2920, 2851, 1646, 1542, 1468 cmꢁ1 1H NMR
.
(di-tert-butyl)silylene-
a
-
D
-galactopyranoside (56)
(500 MHz, pyridine-d5, one drop of 1% TMS in CDCl3): d 0.87 (6H,
t, J 7.0 Hz), 1.22–1.33 (60H, m), 1.53–1.62 (2H, m), 1.80–2.02 (5H,
m), 2.26 (1H, m), 4.31–4.40 (4H, m), 4.43–4.48 (3H, m),
4.51–4.55 (2H, m), 4.63 (1H, dd, J 3.7, 9.8 Hz), 4.70 (1H, d, J
5.1 Hz), 4.73 (1H, dd, J 6.2, 10.8 Hz), 4.83 (1H, dd, J 3.1, 8.5 Hz),
4.99 (1H, m), 5.40 (1H, m), 5.57 (1H, d, J 3.7 Hz, anomeric H),
8.71 (1H, d, J 9.5 Hz, NH). 13C NMR (126 MHz, pyridine-d5): d
14.26, 22.91, 26.42, 26.45, 29.58, 29.89, 29.95, 29.98, 30.02,
30.13, 30.16, 30.18, 30.32, 32.09, 32.69, 35.16, 50.89, 62.58,
67.89, 70.24, 70.91, 71.31, 71,59, 72.72, 72.73, 72.97, 73.49,
Compound 26 (103 mg, 0.09 mmol), 52 (125 mg, 0.27 mmol),
DMAP (130 mg, 1.06 mmol), and EDAC (205 mg, 1.06 mmol) in
THF–CH2Cl2 (1:1, 10 mL) were treated as described for the forma-
tion of 12 from 11 to give 56 (129 mg, 90%) as a gum. Column chro-
matography was performed by elution with hexane–EtOAc (19:1,
then 6:1). IR m .
max(KBr) 2925, 2855, 1684 cmꢁ1 1H NMR1H NMR
(500 MHz, CDCl3): d 0.05 (3H, s), 0.06 (3H, s), 0.09 (3H, s), 0.13
(3H, s), 0.87–0.89 (24H, m, containing two 9H, singlets at 0.88
and 0.89 ppm), 0.98 (9H, s), 1.05 (9H, s), 1.22–1.36 (71H, m,

64
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
73.80, 76.02, 101.08, 173.98. ESI-MS: m/z 944.70 [M+Na]+. HR ESI-
MS: calcd for C50H99NO13Na: 944.7014; observed: 944.7007.
4.1.54. (2S,3S,4R,5EZ)-1-[(tert-Butyldimethylsilyl)oxy]-3,4-O-
isopropylidene-5-hexacosene-2,3,4-triol (65)
To a solution of Ph3P(Br)C21H43 (638 mg, 1.00 mmol) in dry THF
(3 mL) was added a solution of n-BuLi (1.57 M in hexane, 1.20 mL,
1.88 mmol) at ꢁ10 °C under an atmosphere of argon with stirring.
After stirring for 30 min at ꢁ10 °C, to this red-colored solution was
added a solution of 64 (152 mg, 0.50 mmol) in dry THF (2 mL). This
mixture was stirred for 4 h at room temperature, quenched with
MeOH at ice cooling temperature, concentrated in vacuo, then di-
luted with EtOAc. The solution was washed with water and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a residue, which was chromatographed on a silica gel
column. Elution with hexane–EtOAc (39:1, then 19:1) gave 65
4.1.51. (2S,3S,4R)-3,4-Bis(tert-butyldimethylsilyloxy)-2-
[(20R,30R)-20,30-dihydroxy-20,30-O-
(isopropylidene)hexacosanoylamino]octadecyl 2,3,4,6-tetra-O-
benzyl-a-D-galactopyranoside (61)
Compound 60 (160 mg, 0.15 mmol) and 52 (210 mg,
0.45 mmol) were treated as described for the formation of 12 from
11 to give 61 (177 mg, 78%) as a gum. IR
mmax(KBr) 2925, 2853,
1686 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.047 (3H, s), 0.054 (3H,
.
s), 0.055 (3H, s), 0.11 (3H, s), 0.87–0.90 (24H, m), 1.20 (3H, s),
1.25 (62H, br s), 1.31–1.40 (4H, m), 1.44 (3H, s), 1.46–1.60 (2H,
m), 1.65–1.70 (2H, m), 3.47 (1H, dd, J 5.7, 9.1 Hz), 3.54 (1H, t, J
8.4 Hz), 3.69–3.72 (2H, m), 3.81–3.85 (2H, m), 3.91–3.95 (2H, m),
3.99 (1H, d, J 1.7 Hz), 4.04 (1H, d, J 3.5, 10.1 Hz), 4.18 (1H, m),
4.27 (1H, m), 4.37, 4.48 (2H, AB-q, J 11.7 Hz), 4.40 (1H, d, J
7.6 Hz), 4.55, 4.91 (2H, AB-q, J 11.3 Hz), 4.70, 4.74 (2H, AB-q, J
11.7 Hz), 4.70, 4.81 (2H, AB-q, J 11.7 Hz), 4.87 (1H, d, J 3.5 Hz, ano-
meric H), 6.80 (1H, d, J 9.5 Hz, NH), 7.24–7.36 (20H, m). ESI-MS:
m/z 1541.10 [M+Na]+. HR ESI-MS: calcd for C93H155NO11Si2Na:
1541.1036; observed: 1541.0992.
(183 mg, 63%) as an oily mixture of E- and Z-isomers. IR
mmax(KBr):
3540, 2926, 2855, 1465 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.09
.
(6H, s), 0.88 (3H, t, J 6.8 Hz), 0.91 (9H, s), 1.25–1.44 (41H, m, con-
taining 3H singlet at 1.34 ppm), 1.45 (3H, s), 2.02–2.20 (2H, m),
2.45 (1H, d, J 4.6 Hz, OH), 3.66–3.71 (2H, m), 3.81 (1H, m),
3.83–4.03 (1H, m), 4.64 (0.6H, m), 5.00 (0.4H, dd, J 7.1, 8.8 Hz),
5.51–5.85 (2H, m). ESMS: m/z 621.49 [M+Na]+. HREIMS: calcd for
C35H70O5SiNa: 621.4890; observed, 621.4877.
4.1.55. (2S,3S,4R)-1-(tert-Butyldimethylsilyloxy)-3,4-O-
(isopropylidene)hexacosane-2,3,4-triol (66)
4.1.52. (2S,3S,4R)-3,4-Bis(tert-butyldimethylsilyloxy)-2-
[(20R,30R)-20,30-dihydroxy-20,30-O-
A solution of a mixture of E- and Z-isomers (65, 3.45 g,
5.92 mmol) in EtOAc (120 mL) was stirred for 1 h at room temper-
ature under hydrogen, using 20% Pd(OH)2/C (1.3 g) as a catalyst.
The reaction mixture was filtered, and the filtrate was concen-
trated in vacuo to give 66 (3.45 g, 100%) as a wax, which was em-
ployed for the next reaction without further purification. IR
(isopropylidene)hexacosanoylamino]octadecyl
galactopyranoside (62)
a-D-
A solution of 61 (160 mg, 0.11 mmol) in THF (40 mL) containing
20% Pd(OH)2/C (300 mg) was stirred under an atmosphere of H2 for
16 h at room temperature, and filtered. The filtrate was concen-
trated to give a residue, which was chromatographed on a silica
gel column. Elution with hexane–EtOAc (1:1, then 1:4) gave 62
m
max(KBr) 3539 (br), 2923, 2853, 1518, 1464 cmꢁ1 1H NMR
.
(500 MHz, CDCl3) d 0.09 (6H, s), 0.88 (3H, t, J 6.8 Hz), 0.91 (9H, s),
1.26 (38H, br s), 1.32 (3H, s), 1.40 (3H, s), 1.50–1.80 (4H, m), 2.57
(1H, d, J 3.9 Hz), 3.64–3.69 (2H, m), 3.82 (1H, m), 3.91 (1H, dd, J
5.7, 8.8 Hz), 4.17 (1H, m). ESMS: m/z 607.51 [M+Na]+. HRESMS:
calcd for C35H72O4SiNa: 607.5098; observed, 607.5100.
(94 mg, 77%) as a gum. IR
mmax(KBr) 3415, 2925, 2854, 1667,
1523, 1466 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.07 (3H, s), 0.08
.
(3H, s), 0.12 (3H, s), 0.14 (3H, s), 0.88 (6H, t, J 7.0 Hz), 0.91 (9H,
s), 0.92 (9H, s), 1.25 (62H, br s), 1.37 (3H, s), 1.45–1.67 (11H, m,
containing 3H singlet at 1.53 ppm), 2.25 (1H, d, J 10.8 Hz, OH),
2.46 (1H, dd, J 4.8, 7.7 Hz, OH), 2.61 (1H, d, J 3.7 Hz, OH), 2.84
(1H, s, OH), 3.50 (1H, dd, J 8.3, 11.0 Hz), 3.67–3.87 (6H, m), 3.92
(1H, m), 4.09 (1H, s), 4.15 (1H, dd, J 3.2, 10.8 Hz), 4.34–4.40 (2H,
m), 4.52 (1H, d, J 7.4 Hz), 4.90 (1H, d, J 3.6 Hz, anomeric H), 6.71
(1H, d, J 9.8 Hz, NH). ESI-MS: m/z 1180.91 [M+Na]+. HR ESI-MS:
calcd for C65H131NO11Si2Na: 1180.9158; observed: 1180.9149.
4.1.56. (2S,3S,4R)-1,2-Bis(tert-butyldimethylsilyloxy)-3,4-O-
(isopropylidene)hexacosane-3,4-diol (67)
To a solution of 66 (3.45 g, 5.90 mmol) in CH2Cl2 (150 mL) were
added 2,6-lutidine (3.17 g, 29.6 mmol, 5 equiv) and TBDMSOTf
(4.69 g, 17.8 mmol, 3 equiv). After stirring for 1 h at room temper-
ature, the reaction mixture was diluted with CH2Cl2 (150 mL),
washed with aq satd 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
4.1.53. (2S,3S,4R)-3,4-Dihydroxy-2-[(20R,30R)-20,30-
(dihydroxy)hexacosanoylamino]octadecyl
galactopyranoside (63) (RCAI-151)
a-D-
hexane–EtOAc (39:1) gave 67 (3.71 g, 90%) as an oil. IR
mmax(KBr)
Compound 62 (80 mg, 0.07 mmol) was treated as described for
the formation of 30 from 29 to give 63 (40 mg, 63%) as a powder.
Column chromatography was performed by elution with CHCl3–
2925, 2855, 1464, 1254 cmꢁ1 1H NMR (500 MHz, CDCl3) d 0.06
.
(6H, s), 0.09 (3H, s), 0.12 (3H, s), 0.87 (9H, s), 0.88 (3H, t, J
7.0 Hz), 0.91 (9H, s), 1.26 (40H, br s), 1.31 (3H, s), 1.40 (3H, s),
1.52–1.54 (2H, m), 3.72 (1H, m), 3.76–3.81 (2H, m), 4.03–4.10
(2H, m). ESMS: m/z 641.58, 659.58, 721.60 [M+Na]+. HRESMS:
calcd for C41H86O4Si2Na: 721.5962; observed, 721.5974.
MeOH (9:1, then 6:1). ½a D29
ꢄ
+62.4 (c 1.04, pyridine). IR
m
max(KBr)
3364, 2918, 2850, 1646, 1636, 1541, 1469 cmꢁ1
.
1H NMR
(500 MHz, pyridine-d5, one drop of 1% TMS in CDCl3): d 0.88 (6H,
t, J 6.8 Hz), 1.20–1.34 (60H, m), 1.34–1.49 (2H, m), 1.60–1.71
(2H, m), 1.82–1.96 (3H, m), 2.00–2.08 (2H, m), 2.31 (1H, m), 4.27
(1H, m), 4.32–4.43 (4H, m), 4.47–4.57 (4H, m), 4.64–4.70 (2H,
m), 4.74 (1H, m), 5.33 (1H, m), 5.61 (1H, d, J 3.7 Hz, anomeric H),
6.11 (1H, d, J 6.6 Hz, OH), 6.32 (2H, br s, OH), 6.52 (1H, br s, OH),
6.65 (1H, br s, OH), 6.76 (1H, d, J 6.9 Hz, OH), 7.05 (1H, br s, OH),
7.78 (1H, s, OH), 8.65 (1H, d, J 9.5 Hz, NH). 13C NMR (126 MHz, pyr-
idine-d5): d 14.31, 22.96, 26.48, 26.55, 29.64, 29.65, 29.94, 29.97,
30.01, 30.03, 30.05, 30.09, 30.19, 30.22, 30.30, 30.47, 32.15,
32.16, 32.64, 34.62, 50.57, 62.67, 67.99, 70.24, 71.02, 71.65,
72.37, 73.13, 73.55, 76.21, 76.55, 101.20, 173.79. ESI-MS: m/z
912.72 [M+Na]+. HR ESI-MS: calcd for C50H99NO11Na: 912.7116;
observed: 912.7134.
4.1.57. (2S,3R,4R)-2-(tert-Butyldimethylsilyloxy)-3,4-O-
(isopropylidene)hexacosane-1,3,4-triol (68) and (2S,3S,4R)-3,4-
O-(isopropylidene)hexacosane-1,2,3,4-tetraol (69)
(1) To a solution of 67 (100 mg, 0.14 mmol) in pyridine (0.9 mL)
and dry THF (1.5 mL) was added HFꢀpyridine (0.2 mL; HF: ꢂ70%,
pyridine: ꢂ30%) at room temperature with stirring, and the mix-
ture was stirred for 16 h at room temperature. The reaction mix-
ture was quenched with aq satd NaHCO3, diluted with EtOAc,
and washed with brine. The organic solution was dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue, which was chromatographed on a silica gel column. Elu-
tion with hexane–EtOAc (19:1, 4:1, and 1:1) gave 67 (32 mg, 32%,

M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
65
recovery), 68 (39 mg, 47%) as a gum, and 69 (10 mg, 15%) as a pow-
(64H, br s), 1.31 (3H, s), 1.42 (3H, s), 1.50–1.62 (4H, m), 2.45 (1H,
br s, OH), 2.61–2.80 (3H, m, OH), 3.42 (1H, t, J 10.5 Hz), 3.65 (1H, t,
J 2.7 Hz), 3.68–3.75 (3H, m), 3.83–3.87 (2H, m), 3.92 (1H, m), 4.08
(1H, m), 4.12 (1H, m), 4.21 (2H, s), 4.30 (1H, m), 4.49 (1H, m), 4.88
(1H, d, J 3.4 Hz, anomeric H), 6.46 (1H, d, J 10.0 Hz, NH). ESMS: m/z
1289.01 [M+H]+, 1290.02, 1291.02. HRESMS: calcd for C71H146NO12-
Si3: 1289.0153; observed: 1289.0162.
der. Physical data of 68: IR m .
max(KBr) 3496 (br), 2925, 3854 cmꢁ1
1H NMR (500 MHz, CDCl3): d 0.11 (3H, s), 0.13 (3H, s), 0.87–0.89
(12H, m), 1.25–1.34 (40H, m), 1.35 (3H, s), 1.43 (3H, s), 1.50–1.58
(2H, m), 2.27 (1H, dd, J 3.7, 9.1 Hz, OH), 3.68–3.77 (2H, m), 3.83
(1H, m), 4.07 (1H, dd, J 5.7, 8.2 Hz), 4.13 (1H, m). ESMS: m/z
607.51 [M+Na]+. HRESMS: calcd. for C35H72O4SiNa: 607.5098; ob-
served, 607.5090. Physical data of 69: IR
mmax(KBr) 3355, 2917,
2850, 1472 cmꢁ1 1H NMR (500 MHz, CDCl3) d 0.88 (3H, t, J
.
4.1.61. (2S,3S,4R)-2-[(20R,30R,40R)-20,30,40-
7.0 Hz), 1.24–1.75 (48H, m, containing two 3H singlets at 1.34
and 1.42 ppm), 1.91 (1H, m, OH), 2.18 (1H, d, J 5.6 Hz, OH),
3.72–3.76 (2H, m), 3.82 (1H, m), 3.96 (1H, m), 4.20 (1H, m). ESMS:
m/z 493.42 [M+Na]+. HRESMS: calcd for C29H58O4Na: 493.4233;
observed, 493.4227.
Trihydroxyhexacosanoylamino]-3,4-dihydroxyoctadecyl
galactopyranoside (73) (RCAI-160)
a-D-
Compound 72 (46 mg, 0.04 mmol) was treated as described for
the formation of 63 from 62 to give 73 (16 mg, 48%) as a powder.
The silica gel column chromatography was performed by elution
with CHCl3–Mel3–MeOH (9:1, then 6:1). ½a D25
ꢄ
+56.2 (c 0.75, pyri-
4.1.58. (2R,3R,4R)-2-tert-Butyldimethylsilyloxy-3,4-dihydroxy-
3,4-O-(isopropylidene)hexacosanoic acid (70)
dine). IR m .
max(KBr) 3372 (br), 2920, 2851, 1638, 1541, 1469 cmꢁ1
1H NMR (500 MHz, pyridine-d5, one drop of 1% TMS in CDCl3): d
0.88 (6H, t, J 6.9 Hz), 1.20–1.45 (60H, m), 1.58–1.69 (2H, m), 1.79
(1H, m), 1.85–1.97 (3H, m), 2.20–2.29 (2H, m), 4.26–4.35 (3H,
m), 4.36–4.41 (2H, m), 4.43–4.52 (4H, m), 4.55 (1H, s), 4.66 (1H,
dd, J 3.8, 9.9 Hz), 4.70 (1H, dd, J 5.8, 10.7 Hz), 5.05 (1H, s), 5.32
(1H, m), 5.60 (1H, d, J 3.7 Hz, anomeric H), 6.11 (1H, br s, OH),
6.16 (1H, br s, OH), 6.35 (1H, br s, OH), 6.56 (1H, br s, OH), 6.66
(1H, br s, OH), 6.67 (1H, br s, OH), 6.73 (1H, br s, OH), 7.05 (1H,
br s, OH), 7.66 (1H, br s, OH), 8.70 (1H, d, J 9.0 Hz, NH). 13C NMR
(126 MHz, pyridine-d5, one drop of 1% TMS in CDCl3): d 14.39,
23.05, 26.45, 26.58, 29.71, 29.73, 30.02, 30.04, 30.07, 30.09,
30.10, 30.11, 30.14, 30.26, 30.46, 30.51, 32.22, 32.23, 34.63,
34.81, 51.15, 62.74, 67.84, 70.34, 71.09, 71.70, 72.39, 73.06,
73.14, 74.37, 76.16, 77.77, 101.24, 174.29. ESMS: m/z 928.70
[M+Na]+. HR ESI-MS: calcd for C50H99NO12Na: 928.7065; observed:
928.7056.
A
solution of 68 (66 mg, 0.11 mmol), NaIO4 (242 mg,
1.13 mmol), and RuCl3ꢀnH2O (3 mg) in CCl4–CH3CN–H2O (2:2:3,
7 mL) was stirred for 3 h at room temperature, and diluted with
CHCl3, washed with water, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to give a residue, which was chro-
matographed on a silica gel column. Elution with hexane–EtOAc
(19:1, then 4:1) gave 70 (52 mg, 77%) as a solid. IR
mmax(KBr)
2924, 2853, 1725, 1465 cmꢁ1 1H NMR (500 MHz, CDCl3): d 0.11
.
(3H, s), 0.14 (3H, s), 0.88 (3H, t, J 6.8 Hz), 0.92 (9H, s), 1.25 (40H,
br s), 1.34 (3H, s), 1.45 (3H, s), 1.54–1.66 (2H, m), 4.19–4.20 (2H,
m), 4.30 (1H, d, J 6.1 Hz). ESMS: m/z 621.49 [M+Na]+. HRESMS:
calcd for C35H70O5SiNa: 621.4890; observed: 621.4877.
4.1.59. (2S,3S,4R)-2-[(20R,30R,40R)-20-(tert-
Butyldimethylsilyloxy)-30,40-dihydroxy-30,40-O-
(isopropylidene)hexacosanoylamino]-3,4-bis(tert-
butyldimethylsilyloxy)octadecyl 2,3,4,6-tetra-O-benzyl-
a
-D
-
4.2. Methods for measurement of biological activity
galactopyranoside (71)
To a solution of 60 (200 mg, 0.19 mmol), DMAP (320 mg,
2.62 mmol), and 70 (336 mg, 0.56 mmol) in dry THF–CH2Cl2 (1:1,
20 mL) was added EDAC (503 mg, 2.61 mmol). The mixture was
stirred for 16 h at room temperature, and concentrated in vacuo
to give a mixture, which was diluted with CH2Cl2. 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 hex-
ane–EtOAc (19:1, then 4:1) gave 71 (116 mg, 38%) as a gum. IR
4.2.1. Bioassay (mouse in vivo)16
In vivo experiment. The stock solutions (1.0 mg/mL in DMSO) of
a
GalCer and synthesized samples were diluted to 10
becco’s phosphate buffered saline (Sigma, Product No. D8537) just
before injection into mice. Each glycolipid solution (10 g/mL,
200 L) was administered intravenously. Peripheral blood was col-
lg/mL in Dul-
l
l
lected from the retro-orbital plexus of mice at indicated time
points, using heparin-coated capillary tubes (Funakoshi Pharma-
ceutical, Japan), and plasma was prepared.
m
max(KBr) 2925, 2854, 1685 cmꢁ1 1H NMR (500 MHz, CDCl3): d
.
0.05 (3H, s), 0.06 (3H, s), 0.068 (3H, s), 0.072 (3H, s), 0.10 (6H, s),
0.87–0.90 (33H, m), 1.25 (67H, br s), 1.40 (3H, s), 1.44–1.54 (4H,
m), 3.48 (1H, m), 3.58 (1H, t, J 8.5 Hz), 3.71–3.77 (3H, m), 3.90
(1H, dd, J 2.8, 10.3 Hz), 3.98–4.07 (5H, m), 4.22–4.27 (2H, m),
4.33 (1H, m), 4.37, 4.88 (2H, AB-q, J 11.7 Hz), 4.49, 4.53 (2H, AB-
q, J 11.5 Hz), 4.69, 4.74 (2H, AB-q, J 12.0 Hz), 4.69, 4.75 (2H, AB-q,
J 12.0 Hz), 4.87 (1H, d, J 3.4 Hz, anomeric H), 6.62 (1H, d, J 8.3 Hz,
NH), 7.24–7.35 (20H, m). ESMS: m/z 1649.20 [M+H]+. HRESMS:
calcd for C99H170NO12Si3: 1649.2031; observed: 1649.2056.
4.2.2. Cytokine measurement
The cytokine concentrations in plasma were quantified by cyto-
metric bead array (CBA) (BD Bioscience) for IL-4, and mouse IFN
ELISA kit (Thermo Scientific Endogen, Rockford, IL, USA) for IFN
according to the manufacturer’s protocol.
c
c
4.2.3. Experimental autoimmune encephalomyelitis (EAE)
induction by active immunization in C57BL/6 (B6) mice, and
clinical score5a,14e
EAE is induced in B6 female mice by immunization with an emul-
sion of MOG35-55 peptide in complete Freund’s adjuvant (CFA).
4.1.60. (2S,3S,4R)-2-[(20R,30R,40R)-20-(tert-
Butyldimethylsilyloxy)-30,40-dihydroxy-30,40-O-
(isopropylidene)hexacosanoylamino]-3,4-bis(tert-
MOG in CFA (Hooke Lab) solution (200
jected at 2 sites on the lower back (one injection over each hip/base
of tail) with 100 L of emulsion at each site on day 1. 5 ng/mL pertus-
sis toxin (200 L in PBS) was intraperitoneally injected on day 1 and
lL) was subcutaneously in-
butyldimethylsilyloxy)octadecyl
a
-D
-galactopyranoside (72)
l
Compound 71 (116 mg, 0.04 mmol) was hydrogenated as de-
scribed for the formation of 62 from 61 using 20% Pd(OH)2/C
(200 mg) as a catalyst to give 72 (49 mg, 54%) as a gum after silica
gel column chromatography by eluting with hexane–EtOAc (2:1,
l
day 3. Mice were observed daily until day 30 for clinical score as 0–5
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 paralysis + forelimb
weakness or paralysis, 5: moribund or deceased. In order to analyze
the effect of glycolipid on EAE, each glycolipid-pulsed GM-CSF
then 1:2). IR m
max(KBr) 3419 (br), 2925, 2854, 1684, 1465 cmꢁ1. 1H
NMR (500 MHz, CDCl3): d 0.08 (3H, s), 0.09 (3H, s), 0.12–0.13 (12H,
m), 0.88 (6H, t, J 7.1 Hz), 0.90 (9H, s), 0.92 (9H, s), 0.93 (9H, s), 1.25

66
M. Shiozaki et al. / Carbohydrate Research 370 (2013) 46–66
induced DC (GM-DC) (5 ꢃ 105 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 (5 ꢃ 106/mL) for 24 h.
Chem. Lett. 2007, 17, 2781–2784; (h) Tashiro, T.; Hongo, Y.; Nakagawa, T.;
Seino, K.; Watarai, H.; Ishii, Y.; Taniguchi, M.; Mori, K. Bioorg. Med. Chem. 2008,
16, 96–8906.
6. Shiozaki, M.; Tashiro, T.; Koshino, H.; Nakagawa, R.; Inoue, S.; Shigeura,
T.; Watarai, H.; Taniguchi, M.; Mori, K. Carbohydr. Res. 2010, 345, 1663–
1684.
7. (a) Zajonc, D. M.; Cantu, C., III; Mattner, J.; Zhou, D.; Savage, P. B.; Bendelac, A.;
Wilson, I. A.; Tayton, L. Nat. Immunol. 2005, 6, 810–818; (b) Koch, M.; Stronge,
V. S.; Shepherd, D.; Gadola, S. D.; Mathew, B.; Ritter, G.; Fersht, A. R.; Besra, G.
S.; Schmidt, R. R.; Jones, E. Y.; Cerundolo, V. Nat. Immunol. 2005, 6, 819–826; (c)
Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.; Pellicci, D. G.; Koh, R.;
Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.; McCluskey, J.; Rossjohn, J. Nature
2007, 448, 44–49; (d) Pellicci, D. G.; Patel, O.; Kjer-Nielsen, L.; Pang, S. S.;
Sullivan, L. C.; Kyparissoudis, K.; Brooks, A. G.; Reid, H. H.; Gras, S.; Lucet, I. S.;
Koh, R.; Smyth, M. J.; Mallevaey, T.; Matsuda, J. L.; Gapin, L.; McCluskey, J.;
Godfrey, D. I.; Rossjohn, J. Immunity 2009, 31, 47–59.
8. Wun, K. S.; Cameron, C.; Patel, O.; Pang, S. S.; Pellicci, D. G.; Sullivan, L. C.;
Keshipeddy, S.; Young, M. H.; Uldrich, A. P.; Thakur, M. S.; Richardson, S. K.;
Howell, A. R.; Illarionov, P. A.; Brooks, A. G.; Besra, G. S.; McCluskey, J.; Gapin, L.;
Porcelli, S. A.; Godfrey, D. I.; Rossjohn, J. Immunity 2011, 34, 327–339.
9. Eby, R.; Sondheimer, S. J.; Schuerch, C. Carbohydr. Res. 1979, 73, 273–276.
10. Chang, C.-W.; Chen, Y.-N.; Adak, A. K.; Lin, K.-H.; Tzou, D.-L. M.; Lin, C.-C.
Tetrahedron 2007, 63, 4310–4318.
11. Mansour, T.; Tse, A. H. L. U.S. Patent, No. 5756,706 (May 26, 1998).
12. Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless, K. B. J. Org. Chem. 1981, 46,
3936–3938.
13. Tashiro, T.; Hongo, N.; Nakagawa, R.; Seino, K.; Watarai, H.; Ishii, Y.; Taniguchi,
M.; Mori, K. Bioorg. Med. Chem. 2008, 16, 8896–8906.
14. (a) Rivers, T. M.; Spunt, D. H.; Berry, G. P. J. Exp. Med. 1933, 58, 39–53; (b)
Miller, S. D.; Carpus, W. J. Curr. Protoc. Immunol. 2007, Chapter 15, Unit 15.1.;
(c) Qian, G.; Xia Qin, X.; Zang, Y. Q.; Ge, B.; Guo, T. B.; Wan, B.; Fang, L.; Zhang, J.
Z. Cell Res. 2010, 20, 480–492; (d) Mars, L. T.; Gautron, A.-S.; Lucie, J. N. J.
Immunol. 2008, 181, 2321–2329; cf. (e) Marusic, S.; Leach, M. W.; Pelker, J. W.;
Azoitei, M. L.; Uozumi, N.; Cui, J.; Shen, M. W. H.; DeClercq, C. M.; Miyashiro, J.
S.; Carito, B. A.; Thakker, P.; Simmons, D. L.; Leonard, J. P.; Shimizu, T.; Clark, J.
D. J. Exp. Med. 2005, 202, 841–851.
Acknowledgments
We thank Drs. Takemichi Nakamura and Yayoi Hongo, The
Institute of Physical and Chemical Research (RIKEN), for measure-
ments of high-resolution mass spectra.
References
1. (a) Natori, T.; Koezuka, Y.; Higa, T. Tetrahedron Lett. 1993, 34, 5591–5592;
(b) Akimoto, K.; Natori, T.; Morita, M. Tetrahedron Lett. 1994, 35, 5593–
5596; (c) Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994,
50, 2771–2784.
2. Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.;
Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem. 1995, 38, 2176–2187.
3. Taniguchi, M.; Harada, M.; Kojo, S.; Nakayama, T.; Wakao, H. Annu. Rev.
Immunol. 2003, 21, 483–513.
4. (a) Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.;
Nakagawa, R.; Sato, H.; Kaneko, E.; Koseki, H.; Taniguchi, M. Science 1997, 278,
1626–1629; (b) Zeng, Z.; Castano, A. R.; Segelke, B. W.; Stura, E. A.; Peterson, P.
A.; Wilson, I. A. Science 1997, 277, 339–345.
5. (a) Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413, 531–534; (b)
Yamamura, T.; Miyamoto, K.; Illes, Z.; Paul, E.; Araki, M.; Miyake, S. Curr. Top.
Med. Chem. 2004, 4, 561–567; (c) Oki, S.; Chiba, A.; Yamamura, T.; Miyake, S. J.
Clin. Invest. 2004, 113, 1631–1640; (d) Ndonye, R. M.; Izmirian, D. P.; Dunn, M.
F.; Yu, K. O. A.; Porcelli, S. A.; Khurana, A.; Kronenberg, M.; Richardson, S. K.;
Howell, A. R. J. Org. Chem. 2005, 70, 10260–10270; (e) Murata, K.; Toba, T.;
Nakanishi, K.; Takahashi, B.; Yamamura, T.; Miyake, S.; Annoura, H. J. Org. Chem.
2005, 70, 2398–2401; (f) Toba, T.; Murata, K.; Futamura, J.; Nakanishi, K.;
Takahashi, B.; Takemoto, N.; Tomino, M.; Nakatsuka, T.; Imajo, S.; Goto, M.;
Yamamura, T.; Miyake, S.; Annoura, H. Bioorg. Med. Chem. 2012, 20, 2850–2859;
(g) Toba, T.; Murata, K.; Nakanishi, K.; Takahashi, B.; Takemoto, N.; Akabane,
M.; Nakatsuka, T.; Imajo, S.; Yamamura, T.; Miyake, S.; Annoura, H. Bioorg. Med.
15. The IL-4 production in sera after injection of glycolipid ligand is transient (only
around 3 h). This is the reason why we compare level of IL-4 with KRN7000 as a
positive control in same experimental settings.
16. Morgan, E.; Varro, R.; Sepulveda, H.; Ember, J. A.; Apgar, J.; Wilson, J.; Lowe, L.;
Chen, R.; Shivraj, L.; Agadir, A.; Campos, R.; Ernst, D.; Gaur, A. Clin. Immunol.
2004, 110, 252–266.