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

Article abstract of DOI:10.1016/j.carres.2010.05.003

These three ester analogues showed activity for IFNγ, and IL-4 production of iNKT cells. α-Galactosylceramide (αGalCer, KRN7000) has been identified as a modulator of immunological processes through its capacity to bind iNKT cells mediated by CD1d molecules. Some analogues in while the amide group in αGalCer is replaced with ester or ether groups were synthesized from d-arabinitol or l-ribose to evaluate their ability to activate iNKT cells. Ester analogues 30a, 31a, and 61 showed activity for IFNγ and IL-4 production of iNKT cells, while ether (31b) and 4-methoxy ester (76) analogues of α-galactosylceramide were not active for iNKT cells.

Full text of DOI:10.1016/j.carres.2010.05.003

Carbohydrate Research 345 (2010) 1663–1684
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and biological activity of ester and ether analogues of
a-galactosylceramide (KRN7000)
a
b
c
c
Masao Shiozaki a, , Takuya Tashiro , Hiroyuki Koshino , Ryusuke Nakagawa , Sayo Inoue ,
*
Tomokuni Shigeura c, Hiroshi Watarai c, Masaru Taniguchi c, Kenji Mori a
a Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
b Advanced Science Institute, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
c Laboratory for Immune Regulation, Research Center for Allergy and Immunology, RIKEN, Yokohama Institute, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama-shi,
Kanagawa 230-0045, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
a
-Galactosylceramide (
cesses through its capacity to bind iNKT cells mediated by CD1d molecules. Some analogues in while
the amide group in GalCer is replaced with ester or ether groups were synthesized from -arabinitol
or -ribose to evaluate their ability to activate iNKT cells. Ester analogues 30a, 31a, and 61 showed activ-
ity for IFN and IL-4 production of iNKT cells, while ether (31b) and 4-methoxy ester (76) analogues of
galactosylceramide were not active for iNKT cells.
aGalCer, KRN7000) has been identified as a modulator of immunological pro-
Received 29 March 2010
Received in revised form 28 April 2010
Accepted 6 May 2010
a
D
L
Available online 12 May 2010
c
a-
Keywords:
Glycolipid
Ó 2010 Elsevier Ltd. All rights reserved.
Glycosylation
Ester analogues of
KRN7000
a-galactosylceramide
iNKT cells
1. Introduction
antitumor drug, because
a
GalCer causes rapid secretion of both
Th1 and Th2 cytokines by iNKT cells. The production of IL-4 may
mask the beneficial effects of IFN . Therefore, we need to develop
more effective antigens that bias the production of Th1 and Th2
Agelasphins are a series of glycosphingolipids isolated from a
marine sponge (Agelas mauritianus that show potent effects on
the immune system of mice.1 This discovery spurred on the syn-
c
cytokines to either type. As a result, many candidates such as
thesis of agelasphin derivatives.
a
-Galactosylceramide (
a
GalCer),
a a a
-C-GalCer,5 -carba-GalCer,6 or 60-derivatized GalCer analogues7,8
known as KRN7000,2 as a ligand for the activation of CD1d-medi-
ated invariant natural killer T cells (iNKT cells)3a was developed
by Kirin Brewery Co. through the structural modification of agelas-
phin-9b (the main component of agelasphins). iNKT cells display
characteristics of both T cells and NK cells and play a crucial role
in diverse immune responses. A major histocompatibility complex
have been synthesized to evaluate their effectiveness (see Fig. 1).
This time, we tried to synthesize several analogues in which the
amide group in
aGalCer was replaced with an ester or ether group
as bioisosteres of
aGalCer. The reason for the synthesis of these
analogues simply comes from the fact that we previously synthe-
sized a diester analogue (RSM480)9 instead of the diamide com-
pound (E5564:10 eritoran, LPS antagonist) developed by the Eisai
Company as a lipid A related anti-sepsis drug, and RSM480 showed
ca. 50 times stronger in vitro LPS antagonistic activity toward hu-
man whole blood cells than E5564. However, a question comes to
mind about the biological activities of ester or ether analogues of
(MHC) class I-like protein3b named CD1d presents
surface of antigen-presenting cells and thereby constructs a binary
structure (CD1d/ GalCer complex). The T cell receptors (TCRs) of
the iNKT cells recognize the CD1d/
GalCer complex4a,b and con-
struct a ternary structure (CD1d/
GalCer/iNKT TCR complex).4c
Then the iNKT cells release both T helper 1 (Th1)-type cytokines
such as interferon- (IFN ) and T helper 2 (Th2)-type cytokines
aGalCer on the
a
a
a
a
GalCer toward iNKT cells. Can they still show the activities for
iNKT cells? Because, judging from the X-ray crystallographic anal-
ysis,4 the CD1d/
GalCer complex forms a hydrogen bond between
the amide N–H hydrogen on the ceramide of GalCer and the oxy-
c
c
such as interleukin-4 (IL-4). However, it is thought that the balance
of Th1 and Th2 cytokine production is important for therapeutic
a
a
use. The effectiveness of
aGalCer may be limited to its use as an
gen on the hydroxyl group of the adjacent threonine-156 residue of
mouse CD1d4a (mCD1d) (or threonine-154 residue of human
CD1d4b) as one of the several hydrogen bonds. The ester and ether
* Corresponding author.
analogues of
aGalCer have no N–H hydrogen. Therefore, they
E-mail address: shiozaki@rcai.riken.jp (M. Shiozaki).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.05.003

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M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
Figure 1. Structures of agelasphin-9b,
a-galactosylceramide, a
-C-galactosylceramide, RCAI-56, RCAI-61, and 60-(400-chloro-300-trifluoromethyl)benzoylamino-60-deoxy-
aGalCer.
cannot form this kind of hydrogen bond with threonine-156 resi-
due of mCD1d (or threonine-154 residue of human CD1d).
As a major objective of our studies, we synthesized two ana-
The galactosyl 2,3-diols of 1 were protected with 4-methoxy-
benzyl (PMB) groups in 95% yield by using 4-methoxybenzyl chlo-
ride and NaH as the base in N,N-dimethylformamide (DMF) to give
2 in good yield. The anomeric S-tolyl group of 2 was deprotected
with N-bromosuccinimide (NBS) in acetone at ꢀ20 °C to give 3 in
96% yield. Treatment of 3 with trichloroacetonitrile in dichloro-
methane using cesium carbonate as a base gave imidate 4, which
was employed for the next reaction without purification.
logues 31a and 31b by replacing the amide group in aGalCer with
an ester or ether group to determine the value of hydrogen bond-
ing and to evaluate their ability to activate iNKT cells. In addition,
we synthesized the acetylenic compounds 30a and 30b both to
evaluate their activity and to examine the synthetic procedures
for compounds having unsaturated bonds. The C60–OMe derivative
61, an ester analogue of RCAI-61, was synthesized because
Meanwhile, diol 19 as a glycosyl acceptor was prepared from
cuprate 11 derived from bromide 513 and p-methoxybenzyl ether
10 of the known compound 9.12
RCAI-617 showed 8.2 times better in vivo activity than
toward IFN production in the mouse, and also because it was
known from X-ray crystallographic analysis4 that the C60–OH
group on the -galactose unit of GalCer showed no hydrogen
aGalCer
c
The bromide 513 was converted to 11 as follows (Schemes 2 and
3). Treatment of 5 with 1-octyne and n-butyllithium in tetrahydro-
furan (THF) containing hexamethylphosphoramide (HMPA) gave 6.
The tetrahydropyanyl ether of 6 was deprotected to alcohol 7 by
use of p-toluenesulfonic acid monohydrate (p-TsOHꢁH2O) in meth-
anol, and 7 was further converted to the iodo compound 8 by
refluxing with sodium iodide in acetone via the mesylate of 7.
Treatment of 8 with 2.25 equiv of tert-butyllithium in Et2O–pen-
tane at ꢀ78 °C, and then with 0.5 equiv of copper(I) iodide gave a
solution of lithium dialkynylcuprate (11).
D
a
bonding interaction with CD1d. Moreover, the C4–OMe compound
76 was synthesized, because the C4-hydroxyl group on the
ceramide part of aGalCer also showed no hydrogen bonding inter-
action.4 These derivatives that lack the amide N–H group were
synthesized to examine the hypotheses as to whether or not the
hydrogen bonding between the amide N–H and CD1d would be
important for activity.
The hydroxy group of 9 was reacted with 4-methoxybenzyl
chloride and NaH as a base in DMF to give 10 (93% yield). A solu-
tion of 10 in THF was added to a solution of 11 (obtained above)
to give alcohol 12 in 94% yield as a floc (mp 80–81 °C). The benzyl-
idene group of 12 was deprotected by p-TsOHꢁH2O in MeOH to
yield triol 13. The primary alcohol of 13 was protected as a tert-
butyldimethylsilyl (TBDMS) ether to afford diol 14, which was fur-
ther converted to the isopropylidene derivative 15. Removal of the
p-methoxybenzyl (PMB) group of 15 with 2,3-dichloro-5,6-dicy-
ano-p-benzoquinone (DDQ) in CH2Cl2 containing H2O gave alcohol
2. Results and discussion
2.1. Synthesis
Firstly, we chose compounds 30a, 30b, 31a, and 31b as the tar-
gets of the ester and ether analogues of aGalCer. Compounds 30a
and 30b have an acetylenic unsaturated bond in their structures.
Therefore, the hydrogenolytic procedure could not be used in their
synthesis, and so we attempted to use imidate 4 as a glycosyl do-
nor, which was obtained from the already reported diol 111
(Scheme 1). Diol 19 served as the glycosyl acceptor, which was
For details of some alternate reactions that led to different products, see the
Supplementary data section.
synthesized from D
-arabinitol.12

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1665
Scheme 1. Reagents and conditions: Tol = 4-methylphenyl; PMB = 4-methoxybenzyl; (a) 4-methoxybenzyl chloride, NaH, cat. (n-Bu)4NI, DMF, 65 °C, 1 h, 95%; (b) NBS,
acetone, ꢀ20 °C, 45 min, 96%; (c) CC13CN, Cs2CO3, CH2C12, rt, 16 h.
Scheme 2. Reagents and conditions: (a) 1-octyne, n-BuLi, HMPA, THF, rt, 16 h, 52%; (b) p-TsOHꢁH2O, MeOH, rt, 6 h, 97%; (c) (1) MsCl, Et3N, CH2C12, 0 °C, 30 min; (2) NaI,
acetone, reflux, 16 h, two steps 81%.
Scheme 3. Reagents and conditions: (a) 4-methoxybenzyl chloride, NaH, DMF, rt, 3 h, 93%; (b) t-BuLi, Et2O–pentane, ꢀ78 °C?rt, 1 h, then CuI, ꢀ30 °C, 30 min; (c) 10, THF,
ꢀ20 °C, 2 h, rt, 16 h, 94%; (d) p-TsOHꢁH2O, MeOH, rt, 2 h, 85%; (e) t-BuMe2SiCl, DMAP, CH2C12, rt, 3 h, 93%; (f) Me2C(OMe)2, p-TsOHꢁH2O, rt, 1 h, 95%; (g) DDQ, 10:1 CH2C12–
H2O, rt, 30 min, 93%; (h) DEAD, Ph3P, PhCOOH, THF, ꢀ20 °C?rt, 16 h, 74%; (i) NaOMe, MeOH, rt, 16 h, 77%; (j) (n-Bu)4NF, THF, rt, 30 min, 85%.
16 in 93% yield. Mitsunobu reaction of 16 was performed to invert
the configuration from R to S at the C2–OH position. Treatment of
16 with diethyl azodicarboxylate (DEAD), triphenylphosphine, and
benzoic acid in THF afforded benzoate 17 in 73% yield. The benzoyl
group of 17 was removed by transesterification with sodium meth-
oxide in MeOH to give alcohol 18 in 77% yield. Reaction of 18 with
tetrabutylammonium fluoride afforded diol 19 in 85% yield.
We also developed an alternative route to synthesize the diol 19
-ribose (Scheme 4). -Ribose was converted to 20 according
from
L
L
to the reported method.14 Wittig reaction of 20 with the phosphor-
ane derived from (5-benzyloxypentyl)triphenylphosphonium
bromide15 and n-BuLi in THF gave 21 (42% yield) as a mixture of
E- and Z-geometrical isomers. The secondary alcohol of 21 was
protected as the TBDMS ether to give 22 quantitatively, and the

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M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
Scheme 4. Reagents and conditions: (a) Ph3P(Br)CH2(CH2)3CH2OBn, n-BuLi, THF, ꢀ78 °C?10 °C over 2 h, then ꢀ78 °C, 20, ꢀ78 °C?rt over 2 h, 42%; (b) TBDMSOTf, 2,6-
lutidine, CH2C12, rt, 1 h, 100%; (c) H2, 10% Pd/C, hexane, 88%; (d) CBr4, imidazole, PPh3, CH2C12, rt, 2 h, 92%; (e) 1-octyne, n-BuLi, HMPA, THF, ꢀ40 °C, 1 h, then ꢀ78 °C, 24,
ꢀ78 °C?ꢀ15 °C over 2 h; (f) (n-Bu)4NF, THF, rt, 50 min, two steps 36% from 24.
double bond reduction and concomitant debenzylation of 22 under
hydrogen using Pd-on-carbon gave alcohol 23. Reaction of 23 with
carbon tetrabromide, Ph3P, and imidazole gave the bromo com-
pound 24. Treatment of 24 with 1-octyne and n-BuLi in THF con-
taining HMPA at ꢀ78 °C?ꢀ15 °C gave a mixture of 25 and 18
containing a trace amount of 19. The TBDMS groups of this mixture
were removed to give diol 19 (four steps, 29% yield from 22). The
1H NMR data of 19 were identical with those of 19 derived from
Compound 33, a common starting material for 1, was protected
as 4,6-O-di-tert-butylsilylidene 34 using di-tert-butylsilyl bis(tri-
fluoromethanesulfonate) and pyridine as the base. p-Methoxyben-
zylation of 34 using 4-methoxybenzyl chloride and NaH as the
base in DMF gave di-tert-butylsilylidene ether 35 and silanol 3617
in 74% and 11% yields, respectively. Treatment of 35 with N-bro-
mosuccinimide in acetone gave 37 in 58% yield. The imidoyl deriv-
ative of 37 was treated with diol 19 as described in the formation
D-arabinitol.
of 26 from imidate 4 and diol 19 to give only the a anomer 38 (89%
The glycosidation reaction of glycosyl acceptor 19 and glycosyl
yield), which was further converted to the diol 40 (two steps, 51%
yield) via the hexacosanoyl ester 39. The silylidene group of 40 was
removed with the HFꢁpyridine complex in pyridine to yield tetraol
41, which was treated with 46% aq HF in 1:1 CH2Cl2–MeCN con-
taining H2O at room temperature for 25 min to give 30a (25% yield)
and a 3:1 mixture of 30a and 32 (6% yield), together with starting
material 41 (24% recovery). The saturated ester 31a was also ob-
tained easily by the catalytic hydrogenation of 30a.
donor 4 was performed by use of silver trifluoromethanesulfonate
(AgOTf) as a Lewis acid catalyst to give anomer 26 (61%) contain-
a
ing a small amount of inseparable unknown by-product, as well as
the b anomer 27 (31%) (Scheme 5). The secondary alcohol of 26
was treated with n-hexacosanoic acid, 4-dimethylaminopyridine
(DMAP), and N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride (EDAC) as a dehydrating agent to give ester 28a
(35%) and the starting material 26 (recovery 25%). The ether
compound 28b was obtained in 39% yield by the reaction of 26
with n-hexacosanyl methanesulfonate (n-C26H53OMs) and sodium
hydride. Compounds 28a and 28b were each treated with DDQ in
CH2Cl2 containing H2O to give 29a (78%) and 29b (48%), respec-
tively. Both benzylidene and isopropylidene groups of 29b were re-
moved with aqueous hydrofluoric acid (46 wt % HF in H2O, aq 46%
HF) in MeCN–CH2Cl2 to yield 30b (46%). However, treatment of 29a
under the same conditions gave only a trace amount of 30a, and
the compound obtained was 32 (44%), namely, the compound in
which the acyl group migrated from C2–O to C4–O (determined
by 2D COSY analysis).
The saturated ether 31b was easily obtained by the addition of
hydrogen to the acetylenic triple bond of 30b using Pd(OH)2 on
carbon as a catalyst in a solution of chloroform and methanol.
Removal of the benzylidene group of 29a without causing the
acyl migration under the prolonged acidic condition was difficult,
because this protecting group was relatively stable to the acidic
conditions. Therefore, the benzylidene group of 29a was changed
to di-tert-butylsilylene group, which should be cleaved by fluoride
L-Ribose derivative 20 was also converted to the 1,2-di-OTBDMS
ether 44 through two intermediates 42 and 43 (Scheme 7). Partial
deprotection of the TBDMS group of 44 with HFꢁpyridine in CH2Cl2
gave 45 (45%) and 46 (22%) with recovered 44 (33%). Diol 46 was
also prepared by desilylation of 43 or catalytic hydrogenation of
19, and also converted to 44 in good yield. This 46 {mp
44.5–45 °C, ½a 2D3
ꢂ
+6.2 (c 1.26, CHCl3)} prepared from
compared with 46 obtained by hydrogenation of the
L
-ribose was
-arabinitol-
value was +6.4 (c 1.51, CHCl3), and also the
D
derived 19. The ½a D25
ꢂ
other physical and spectral data (mp, 1H NMR, IR, and MS) were
identical with those of 46 obtained from L-ribose. Thus ester 31a
should also be obtained by coupling of 45 with the imidate of 47
(Scheme 8).
The known imidoyl derivative of 4711 reacted with 45 as de-
scribed in the formation of 26 from compounds 4 and 19 (see
Scheme 5) to give
(Scheme 8). Compound 48 was converted to ester 51 via alcohol
50. The benzylidene and two benzyl groups on the -galactose
a anomer 48 (46%) and b anomer 49 (39%)
D
moiety of 51 were hydrogenolyzed using Pd(OH)2-on-carbon in
THF to give 52 (quantitatively), which was converted to 31a in
42% yield by treatment with 46% aq HF in 1:1 CH2Cl2–MeCN and
H2O at room temperature for 20 min. In this reaction, the product
anion, and in addition should give preference to the
a anomer over
the b anomer in the glycosidation reaction16 (Scheme 6).

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1667
Scheme 5. Reagents and conditions: (a) AgOTf, MS 4 Å, CH2C12, 0 °C, 3 h, and rt, 3 h, 26 (61%) and 27 (31%); (b) a: n-C25H51COOH, EDAC, DMAP, 1:1 THF–CH2C12, rt, four days,
28a (35%) and 26 (25% recovery); b: n-C26H53OMs, NaH, 6:1 DMF–THF, rt, 16 h, and 55 °C, 2 h, 28b (39%); (c) DDQ, 10:1 CH2C12–H2O, rt, 2 h, 29a (78%) and 29b (48%); (d) aq
46% HF, H2O, 1:1 MeCN–CH2Cl2, rt, 4 h, 30b (46%); 30a (trace) and 32 (44%); (e) H2, 20% Pd(OH)2/C, 1:1 CHCl3–MeOH, rt, 2 h, 32%.
31a precipitated from solution. On the other hand, treatment of 52
with hydrochloric acid (1 M solution in EtOAc) in CHCl3 at room
temperature for 5 min gave 53 (72% yield), in which the acyl group
migration from C2–O to C4–O was confirmed by 2D COSY analysis.
The relative configurations of 53 at the C2, C3, and C4 positions
were confirmed by the J-based configuration analysis method.18
The stereochemistry at C2 and C3 positions should remain the
same as those of 52 in the reaction from 52 to 53. Therefore, con-
figurational analysis was focused on the relative stereochemistry
of the C3 and C4 positions. The medium J-value of 5.0 Hz between
H3 and H4 suggested a conformational mixture of anti and gauche
relationships. Heteronuclear coupling constant values were mea-
sured by HETLOC18 experiments. Assignment of the gauche rela-
tionship between H3 and C4–O was determined by J of 6 Hz
observed from H3 to C4. In addition to these data, small couplings
and C4 positions of 53 with a mixture of a C2/C5 anti and C2/C4–
O anti conformations. Therefore, the absolute configuration of 53
was determined to be 2S,3S,4R.
Secondly, we chose compound 61 (Scheme 9) as another target
of the ester analogues of
aGalCer. Compound 61 has no unsatu-
rated bond; therefore, the hydrogenolytic procedure can be applied
in the synthesis. Thus we could obtain 61 using the imidate of 56 as
the glycosyl donor, which was obtained from methyl a-D-galacto-
pyranoside (54), and the above-mentioned diol 46 as the glycosyl
acceptor.
Methyl
a-D-galactopyranoside 54 was converted to 55 in 28%
yield in a one-pot procedure by (1) methylation of the C60 primary
alcohol with 1 equiv of MeI in DMF using NaH as the base at ꢀ40 °C
and then at room temperature and (2) successive benzylation of
the C20, C30, and C40 secondary alcohols with benzyl bromide using
NaH as the base. Treatment of 55 in acetic anhydride (Ac2O) using
2
3
of JH4C3 and JH4C2 suggested erythro stereochemistry for the C3

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M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
Scheme 6. Reagents and conditions: (a) t-Bu2Si(OTf)2, pyridine, rt, 30 min, 74%; (b) 4-methoxybenzyl chloride, NaH, DMF, rt, 1 h, 35 (74%) and 36 (11%); (c) NBS, acetone,
ꢀ20 °C, 40 min, 58%; (d) (1) CC13CN, Cs2CO3, CH2C12, rt, 16 h, (2) 19, AgOTf, MS 4 Å, CH2C12, ꢀ20 °C, 30 min, then ꢀ20 °C?rt over 2.5 h, 89%; (e) n-C25H51COOH, EDAC, DMAP,
1:1 CH2C12–THF, rt, 4.5 days, 65%; (f) DDQ, 10:1 CH2C12–H2O, rt, 2 h, 78%; (g) HFꢁpyridine, pyridine, THF, rt, 1 h, 89%; (h) aq 46% HF, 1:1 CH2Cl2–MeCN, H2O, rt, 25 min, 41
(24%, recovery), 30a (25%) and a 3:1 mixture (6%) of 30a and 32; (i) H2, Pd(OH)2/C, THF, rt, 1 h, 44%.
concd sulfuric acid as the catalyst gave an anomeric mixture of
acetates, which was converted to 56 in 45% yield by a transesteri-
fication reaction in MeOH using KOH as the catalyst. Compound 56
was changed to the corresponding imidate, which was treated with
diol 46 as described in the formation of 26 from imidate 4 and diol
Thirdly, we chose compound 76, in which the substituent on the
main lipid chain was changed to a C4-methoxy group instead of a
C4-hydroxyl group. The glycosyl acceptor 70 was synthesized from
compound 62, which was prepared from L-ribose according to the
reported procedure19 (Scheme 10). Compound 62 was converted to
64 (two steps, 69% yield) via the TBDMS ether 63. Treatment of 64
in acetic anhydride using concd sulfuric acid as a catalyst gave an
anomeric mixture of 1,5-diacetates, which was converted to a
19 to yield the
a anomer 57 and b anomer 58 in 47% and 20% yield,
respectively. Compound 57 was converted to triol 61 (three steps,
38% yield from 57) via intermediates 59 and 60. It was obvious by
2D COSY analysis that the hexacosanoyl group of 61 was on the
C2–O position of the octadecane chain.
mixture of
L-ribofuranose and L-ribopyranose derivatives by
transesterification with a catalytic amount of KOH in MeOH. The

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1669
Scheme 7. Reagents and conditions: (a) 3 equiv Ph3P(Br)CH2(CH2)11Me, 3 equiv n-BuLi, THF, ꢀ10 °C, 30 min, then 20, 16 h, rt, 86%; (b) H2, Pd(OH)2/C, EtOAc, rt, 1 h, 95%; (c)
TBDMSOTf, 2,6-lutidine, CH2C12, rt, 1 h, 99%; (d) HF–pyridine, THF–pyridine, 0 °C?rt, 4 h, 44 (recovery 33%), 45 (45%), and 46 (22%); (e) (n-Bu)4NF, THF, rt, 1 h, 80%; (f) H2,
Pd(OH)2/C, EtOAc, rt, 3 h, 81%.
Scheme 8. Reagents and conditions: (a) (1) CC13CN, Cs2CO3, CH2C12, rt, 16 h; (2) 45, 4 Å MS, AgOTf, CH2C12, rt, 48 (46%) and 49 (39%); (b) (n-Bu)4NF, THF, rt, 50 min, 87%; (c)
n-C25H51COOH, EDAC, DMAP, 1:1 THF–CH2C12, rt, four days, 93%; (d) H2, Pd(OH)2/C, THF, rt, 20 h, 99% (from 51) and 82% (from 29a); (e) H2, Pd(OH)2/C, THF, rt, 2 h, quant; (f)
aq 46% HF, H2O, 1:1 CH2Cl2–MeCN, rt, 20 min, 42%; (g) HC1, 1:10 (1 M solution in EtOAc)-CHCl3, rt, 5 min, 72%.
resulting
L
-ribose derivatives were re-protected with the TBDMS
ture of 67 and 68 (84% yield) as a mixture of E- and Z-geometrical
isomers. Each alcohol of the mixture (67 and 68) was protected as
the TBDMS ether, and the double bond of the resulting product was
reduced by catalytic hydrogenation to give 69 (91% yield). The
group to give a mixture of 65 and 66 in 45% yield from 64. Wittig
reaction of this mixture with the phosphorane derived from
n-tridecyltriphenylphosphonium bromide and n-BuLi gave a mix-

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M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
Scheme 9. Reagents and conditions: (a) (1) 1 equiv MeI, 2.5 equiv NaH, DMF, ꢀ40 °C, 15 min, then rt, 16 h, and (2) 5 equiv NaH, 4 equiv BnBr, rt, 2 h, then 60 °C, 1 h, two steps
28%; (b) (1) Ac2O, cat. concd H2SO4, rt, 1 h, and (2) cat. KOH, MeOH, rt, 16 h, a l:l mixture of and b anomers, two steps 45%; (c) (1) CC13CN, Cs2CO3, CH2C12, rt, 16 h; (2) 46,
a
AgOTf, MS 4 Å, CH2C12, rt, 30 min, 57 (47%) and 58 (20%); (d) n-C25H51COOH, EDAC, DMAP, 1:1 THF–CH2C12, rt, four days, 77%; (e) H2, Pd(OH)2/C, THF, rt, 20 h, 96%; (f) aq 46%
HF, 1:1 CH2Cl2–MeCN, H2O, rt, 40 min, 52%.
Scheme 10. Reagents and conditions: (a) t-BuMe2SiCl, imidazole, CH2C12, rt, 45 min, 84%; (b) BnBr, NaH, DMF, 0 °C, 1 h, then rt, 16 h, 82%; (c) (1) Ac2O, cat. H2SO4, rt, 1 h, (2)
MeOH, cat. KOH, rt, 1 h, two steps 60%, (3) t-BuMe2SiCl, imidazole, CH2C12, rt, 30 min, 75%; (d) 3 equiv Ph3P(Br)CH2(CH2)nMe, 3 equiv n-BuLi, THF, ꢀ10 °C, 30 min, then a
mixture of 65 and 66 in THF, 3 h, rt, 84%; (e) (1) TBDMSOTf, 2,6-lutidine, CH2C12, rt, 1 h, 91%; (2) H2, Pd/C, hexane, rt, 20 min, quant; (f) HFꢁpyridine, THF–pyridine, rt, 16 h, 70
(61%) and 71 (23%).
reaction of 69 with HFꢁpyridine in THF–pyridine afforded 70 and
71 in 61% and 23%, respectively. Reaction of the imidoyl derivative
of 4711 and alcohol 70 as described in the formation of 26 from
in vivo was investigated using aGalCer as a positive standard. The
ether compounds 30b and 31b lacking the carbonyl oxygen on the
C2 side chain and the ester 76, which was a C4–OMe analogue, did
imidate 4 and diol 19 gave
a
anomer 72 (39% yield) and b anomer
not show the activity for both of production IFN
the other hand, while the three ester compounds 30a, 31a, and 61
showed the cytokine-producing activity toward both IFN (Th1 re-
sponse) and IL-4 (Th2 response), the level (ca. 10% of that of Gal-
Cer) was much weaker than that of GalCer toward IFN , and was
one- to two-thirds of that of GalCer toward IL-4, as shown in
c and IL-4 at all. On
73 (44% yield). Compound 72 was converted to pentaol 76 (three
steps, 40% yield) through alcohol 74 and hexacosanoyl ester 75
as described in the synthetic route to 31a from 48.
Thus, we could synthesize six compounds 30a, 30b, 31a, 31b,
61, and 76 for evaluation of their biological activities (Scheme 11).
c
a
a
c
a
Figures 2 and 3. These results, which were completely reversed
in the case of RSM480, could be interpreted as changing the amide
system on the ceramide to an ester actually enhanced the Th2
effect of the series since the modification had less effect on the
IL-4 cytokine profile vs. the almost lack of activity toward the
2.2. Biological activity
The cytokine-producing (IFN
compounds 30a, 30b, 31a, 31b, 61, and 76 from iNKT cells of mice
c and IL-4) activity of the synthetic

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1671
Scheme 11. Reagents and conditions: (a) (1) CC13CN, Cs2CO3, CH2C12, rt, 16 h, and (2) 70, 4 Å MS, AgOTf, CH2C12, 0 °C, 30 min and rt, 2 h, 72 (39%) and 73 (44%); (b) (n-
Bu)4NF, THF, rt, 50 min, 80%; (c) n-C25H51COOH, EDAC, DMAP, 1:1 THF–CH2C12, rt, four days, 90%; (d) H2, Pd(OH)2/C, THF, rt, 20 h, 56%.
to the CO–NCH3 type ceramide lose the activity toward IFN
response) and bias to Th2 response?
c (Th1
IFNγ in sera (2 μg/mouse, in vivo)
αGalCer
150
100
50
3. Conclusions
30a
31a
61
Three ester compounds 30a, 31a, and 61 were almost devoid of
activity toward the production of IFN . However, they still had the
activity of one- to two-thirds of that of GalCer toward IL-4. These
results have tended to bias cytokine secretion relatively to the Th2
response compared to that of GalCer. On the other hand, ether
compounds 30b and 31b did not show the activity for production
of both IFN and IL-4 at all. The ether compounds 30b and 31b,
c
a
a
c
0
lacking both carbonyl oxygen and N–H hydrogen on the C2 side
chain, could not form a hydrogen bond with the threonine-156
of mCD1d, which was held together by hydrogen bonding with
0
12
24
hr
36
48
the amide N–H hydrogen on the ceramide of
a
GalCer.4a This fact
Figure 2. The concentrations of IFNc in sera of mice (iv).
showed that the electron-withdrawing carbonyl oxygen on the
C2 side chain was absolutely essential for activity toward IL-4
(Th2 response).
However, the C4–OMe compound 76 lacked the activity for
cytokine production from iNKT cells, even though compound 76
had the acyl carbonyl oxygen on the C2 side chain. It is known from
X-ray crystallographic analysis4 that the C4-hydroxyl group on the
IL-4 in sera (2 μg/mouse, in vivo)
800
600
400
200
0
αGalCer
30a
ceramide moiety of
aGalCer forms no hydrogen-bonding interac-
31a
61
tion with CD1d. Therefore, it is estimated that the activity should
be less affected by the modification of this alcohol. In fact the
C4-dehydroxylated ceramide analogue of
aGalCer still showed
activity.20 Hence, the C4–OMe group of 76 may not fit in the cavity
of mCD1d.
4. Experimental
4.1. General
0
3
6
hr
9
12
IR spectra were measured on a Jasco FT/IR-460 plus spectrome-
ter. 1H NMR spectra (TMS as an internal standard) and 13C NMR
spectra were recorded on either a Jeol JNM-A400 or Varian
VNMRS-500 spectrometer using tetramethylsilane (TMS) as the
internal standard. Mass spectra and high resolution MS were
recorded on either a Jeol JMS-SX102A or Bruker BioAPEX II 70e
FT-ICR mass spectrometer. Optical rotation values were measured
on a Jasco P-1010 polarimeter. Column chromatography was
Figure 3. The concentrations of IL-4 of sera of mice (iv).
production of IFN
c
. In other words, it could be said that these ester
compounds have tended to bias the cytokine secretion relatively to
the Th2 response compared to that of GalCer. Therefore, the
a
amide CO–NH bond on the C2 side chain in the ceramide moiety
should be essential for Th1 response. Do the compounds modified

1672
M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
carried out using silica gel 60 N (70–230 mesh ASTM, using ca.
20–50 times the weight of substrate if there is no indication) under
a slightly elevated pressure for elution. Preparative TLC was carried
out on a PLC plate (E. Merck, Silica Gel 60-F254, 0.5 mm). Commer-
cially available anhyd THF, DMF, and CH2Cl2 were used for the
reactions.
ically on a silica gel column. Elution with 1:1 hexane–EtOAc
containing 1% Et3N gave 4 (51 mg, 71% yield) as a gum. IR vmax(KCl)
3337 (w), 3000–2840 (w), 1732, 1672, 1613, 1586, 1514 cmꢀ1 1H
.
NMR (270 MHz, CDCl3): d 3.80 (6H, s), 3.88–4.24 (6H, m), 4.68–
4.74 (4H, m), 5.50 (1H, s), 6.59 (1H, d, J 3.2 Hz, anomeric H),
6.81–6.85 (4H, m), 7.23–7.32 (7H, m), 7.50–7.53 (2H, m), 8.55
(1H, s).
4.1.1. 4-Methylphenyl 4,6-O-benzylidene-2,3-di-O-(4-
methoxybenzyl)-1-thio-b-
D-galactopyranoside (2)
4.1.4. 1-(Tetrahydro-20H-pyran-20-yloxy)-6-tridecyne (6)
To a solution of 1 (13.28 g, 35.47 mmol), 4-methoxybenzyl chlo-
ride (11.65 g, 73.39 mmol), and (n-Bu)4NI (0.60 g, 1.62 mmol) in
dry DMF (100 mL) was gradually added NaH (60% oil dispersion,
3.12 g, 78.0 mmol) at room temperature with stirring. The mixture
reacted exothermically, and it was then stirred at 65 °C for 45 min
and at 70 °C for 15 min. After cooling to room temperature, the
reaction was quenched with ice (100 g) and the mixture was ex-
tracted 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 crude solid, which was dissolved in hot EtOAc
(30 mL), and recrystallized from hexane (50 mL). The crystalline
solid was filtered off and washed with hexane to give 2 (17.60 g).
The filtrate was chromatographed on a silica gel column. Elution
with 2:1, then 1:1 hexane–EtOAc gave another amount of 2
(3.10 g, total 95% yield); mp 143–145 °C (once changed the crystal
form at 110–113 °C). IR vmax(KBr): 3000–2850, 1614, 1586 (w),
To a solution of 1-octyne (12.50 g, 113.4 mmol) in dry THF
(300 mL) containing HMPA (150 mL) was added dropwise n-BuLi
(1.6 M solution in hexane, 85.0 mL, 136 mmol) at ꢀ40 °C. After stir-
ring for 1 h at ꢀ40 °C, a solution of 1-bromo-5-(tetrahydro-2H-pyr-
an-2-yloxy)pentane (5, 28.3 g, 113 mmol) in THF (50 mL) was
slowly added. Then this solution was brought to room temperature
over 2 h, and stirred for 16 h. The reaction was quenched with satd
aq NH4Cl solution, and the mixture was extracted with Et2O
(twice). The combined extracts were washed with water and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a mixture that was chromatographed on a silica gel col-
umn. Elution with 19:1 hexane–EtOAc gave 6 (20.60 g, 65%). IR
vmax(KCl) 2934, 2859 cmꢀ1 1H NMR (270 MHz, CDCl3): d 0.89
.
(3H, t, J 6.8 Hz), 1.21–1.90 (20H, m), 2.10–2.16 (4H, m), 3.38 (1H,
m), 3.48 (1H, m), 3.75 (1H, m), 3.87 (1H, m), 4.58 (1H, dd, J 3.0,
3.8 Hz). EIMS: m/z 195, 280 [MÅ]+. HREIMS: Calcd for C18H32O2,
280.2402; observed, 280.2384.
1515 cmꢀ1 1H NMR (270 MHz, CDCl3): d 2.30 (3H, s), 3.39 (1H, br
.
s), 3.58 (1H, dd, J 3.2, 9.2 Hz), 3.78–3.85 (7H, m, containing two
3H singlets at 3.79 and 3.81 ppm), 3.97 (1H, d, J 12.2 Hz), 4.10
(1H, d, J 2.5 Hz), 4.36 (1H, d, J 12.2 Hz), 4.54 (1H, d, J 9.2 Hz),
4.62–4.65 (4H, m), 5.47 (1H, s), 6.80–6.90 (4H, m), 7.00 (2H, d,
J 7.8 Hz), 7.25–7.55 (9H, m), 7.60 (2H, d, J 7.8 Hz). EIMS; m/z 614
[MÅ]+. HREIMS: Calcd for C36H38O7S, 614.2338; observed, 614.2339.
4.1.5. 6-Tridecyn-1-ol (7)
A solution of 6 (7.40 g, 26.39 mmol) and p-TsOHꢁH2O (350 mg,
1.84 mmol) in MeOH (200 mL) was stirred for 6 h at room temper-
ature, quenched with satd aq NaHCO3 (40 mL), and concentrated in
vacuo, and then diluted with hexane. The solution was washed with
water and brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give an oil that was chromatographed
on a silica gel column. Elution with 19:1 hexane–EtOAc gave 7
4.1.2. 4,6-O-Benzylidene-2,3-di-O-(4-methoxybenzyl)-a,b-D-
galactopyranose (3)
To a solution of 2 (24.00 g, 39.04 mmol) in acetone (800 mL) was
added NBS (8.40 g, 47.20 mmol) at ꢀ20 °C. After stirring for 45 min
at the same temperature, the solution was quenched with satd aq
NaHCO3 (100 mL) and concentrated in vacuo to give a mixture,
which was extracted with EtOAc. The solution was washed with
satd aq NaHCO3 and brine, dried over MgSO4, filtered, and concen-
trated in vacuo to give a mixture that was chromatographed on a
silica gel (250 g) column. Elution with 1:1, then 1:2 hexane–EtOAc
gave 3 as a solid, which was recrystallized from 1:1 hexane–EtOAc.
The crystalline solid was filtered and washed with 2:1 hexane–
EtOAc to give 3. The filtrate was concentrated in vacuo, and the
crude product chromatographed on a silica gel column. Elution
with 1:1, then 1:2 hexane–EtOAc gave an additional amount of 3
(total 19.20 g, 96% yield); mp 130–134 °C. IR vmax(KBr): 3419 (br),
(5.04 g, 97%) as an oil. IR vmax(KCl) 3338, 2931, 2859 cmꢀ1 1H
.
NMR (270 MHz, CDCl3): d 0.89 (3H, t, J 6.8 Hz), 1.20–1.63 (14H, m,
containing OH), 2.10–2.20 (4H, m), 3.60–3.70 (2H, m).
4.1.6. 1-Iodo-6-tridecyne (8)
To a solution of 7 (5.05 g, 25.7 mmol) and Et3N (6.50 g, 64.
3 mmol) in CH2Cl2 (50 mL) was added MsCl (3.68 g, 32.2 mmol)
with stirring at 0 °C. The mixture was stirred for 30 min at 0 °C un-
der argon, diluted with CH2Cl2, washed with satd aq NaHCO3 and
brine, dried over MgSO4, filtered, and concentrated in vacuo to give
a crude mesylate, which was treated with NaI (7.70 g, 51.4 mmol)
in boiling acetone (250 mL) for 16 h. The reaction mixture was con-
centrated in vacuo and diluted with hexane. The solution was
washed with satd aq NaHCO3 and brine, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a crude iodo
compound that was chromatographed on a silica gel column. Elu-
tion with 19:1 hexane–EtOAc gave 8 (6.54 g, 83%). IR vmax(KBr):
3000–2835, 1710, 1613, 1586 (w), 1514 cmꢀ1 1H NMR (400 MHz,
.
CDCl3): d 2.94 (1H, br s, OH), 3.80 (6H, s), 3.83–4.23 (6H, m),
4.60–4.82 (4H, m), 5.31 (1H, br s, anomeric H), 5.48 (1H, s), 6.85–
6.87 (4H, m), 7.26–7.38 (7H, m), 7.51–7.54 (2H, m). EIMS: m/z
508 [MÅ]+. HREIMS: Calcd for C29H32O8, 508.2097; observed,
508.2094.
2930, 2857 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.89 (3H, t, J
.
6.8 Hz), 1.23–1.54 (12H, m), 1.81–1.87 (2H, m), 2.11–2.20 (4H,
m), 3.19 (1H, t, J 7.0 Hz). EIMS: m/z 306 [MÅ]+. HREIMS: Calcd for
C13H23I, 306.0835; observed: 306.0834.
4.1.3. Trichloroacetimidoyl 4,6-O-benzylidene-2,3-di-O-(4-
methoxybenzyl)-a-D-galactopyranoside (4)
4.1.7. 4,5-Anhydro-1,3-O-benzylidene-2-O-(4-methoxybenzyl)-
To a solution of 3 (509 mg, 1.00 mmol) in dry CH2Cl2 (10 mL)
were added Cl3CCN (1.44 mg, 10.0 mmol) and Cs2CO3 (480 mg,
1.47 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 4 (627 mg),
which was employed for the next reaction without further purifi-
cation. A part of this crude 4 (69 mg) was purified chromatograph-
D
-arabinitol (10)
To a solution of 4,5-anhydro-1,3-O-benzylidene-D-arabinitol (9;
2.23 g, 10.0 mmol) and 4-methoxybenzyl chloride (2.04 g, 13.0
mmol) in DMF (10 mL) was added NaH (60% oil dispersion,
560 mg, 14.0 mmol) at 0 °C. The mixture was stirred for 3 h at
room temperature, and the reaction was quenched with ice. The
mixture was extracted with EtOAc, the extract was washed with
water and brine, dried over MgSO4, and filtered. The filtrate was

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1673
concentrated in vacuo to give a solid that was washed with 3:1
hexane–EtOAc (10 mL) and hexane to give 10 (2.91 g). The wash li-
quid was chromatographed on a silica gel column. Elution with 9:1
and 3:1, then 3:2 hexane–EtOAc gave another amount (0.27 g) of
10 (total 3.18 g, 93%), mp 108–109 °C. IR vmax(KCl) 1613, 1584,
DMAP (716 mg, 5.80 mmol). After stirring for 3 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 a mixture that was
chromatographed on a silica gel column. Elution with 2:1 hex-
ane–EtOAc gave 14 (2.47 g, 93%) as a gum. IR vmax(KCl): 3504
1514 cmꢀ1 1H NMR (270 MHz, CDCl3): d 2.83 (1H, dd, J 2.4,
.
5.1 Hz), 2.91 (1H, dd, J 4.1, 4.9 Hz), 3.34 (1H, m), 3.50 (1H, d, J
1.6 Hz), 3.64 (1H, dd, J 1.6, 5.9 Hz), 3.81 (1H, s), 3.91 (1H, d, J
12.1 Hz), 4.40 (1H, d, J 12.1 Hz), 4.60 (1H, d, J 11.9 Hz), 4.78 (1H,
d, J 11.9 Hz), 6.82 (2H, d, J 8.6 Hz), 7.32–7.35 (5H, m), 7.49–7.53
(2H, m). FABMS: m/z 342 [MÅ]+. HRFABMS: Calcd for C20H22O5,
342.1467; observed, 342.1468. Anal. Calcd for C20H22O5: C, 70.16;
H, 6.48. Found: C, 69.58; H, 6.66.
(br), 2934, 2859, 1614, 1515 cmꢀ1 1H NMR (400 MHz, CDCl3): d
.
0.09 (6H, s), 0.89 (3H, t, J 6.8 Hz), 0.91 (9H, s), 1.25–1.60 (18H,
m), 2.14 (4H, t, J 7.0 Hz), 2.30 (1H, d, J 7.2 Hz, OH), 2.80 (1H, d, J
7.6 Hz, OH), 3.50 (1H, m), 3.61 (1H, m), 3.72 (1H, m), 3.81 (3H, s),
3.84–3.89 (2H, m), 4.52, 4.71 (2H, AB-q, J 11.2 Hz), 6.89 (2H, d, J
8.8 Hz), 7.24 (2H, d, J 8.8 Hz). FABMS: m/z 571 [M+Na]+ (on addi-
tion of NaI). HRFABMS: Calcd for C32H56O5SiNa, 571.3794; ob-
served, 579.3753.
4.1.8. (2R,3R,4R)-1,3-O-[(S)-Benzylidene]-2-(4-
methoxybenzyloxy)-11-octadecyn-4-ol (12)
4.1.11. (2R,3R,4R)-1-(tert-Butyldimethylsilyloxy)-3,4-O-
To a solution of 8 (4.55 g, 14.9 mmol) in 1:1 Et2O–pentane
(35 mL) was added t-BuLi (1.5 M in pentane, 24 mL, 36 mmol) at
ꢀ78 °C under argon. After stirring for 5 min, the cooling bath was
removed, and the solution was stirred for 1 h at room temperature.
This solution was added to a suspension of CuI (1.42 g, 7.43 mmol)
in dry THF (15 mL) with stirring at ꢀ40 °C under argon to yield
dialkynyl cuprate 11. This mixture was stirred for 30 min at
ꢀ30 °C, and a solution of 10 (2.54 g, 7.43 mmol) in THF (20 mL)
was added dropwise at ꢀ30 °C. The mixture was stirred for 2 h at
ꢀ20 °C, then gradually elevated to room temperature. After stirring
for 16 h at room temperature, the reaction mixture was diluted
with EtOAc. The solution was washed with water and brine, dried
over MgSO4, and filtered. The filtrate was concentrated in vacuo to
give a crude solid that was chromatographed on a silica gel (100 g)
column. Elution with 4:1, then 2:1 hexane–EtOAc gave 12 (3.63 g,
94%) as a floc, mp 80–81 °C (3:1 hexane–EtOAc). IR vmax(KCl) 3451,
isopropylidene-2-(4-methoxybenzyloxy)-11-octadecyne (15)
A solution of 14 (2.25 g, 4.10 mmol) in 2,2-dimethoxypropane
(30 mL) containing p-TsOHꢁH2O (60 mg, 0.32 mmol) was stirred
for 1 h at room temperature and diluted with EtOAc. The solution
was washed with satd aq NaHCO3 and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give an oil
that was chromatographed on a silica gel column. Elution with
9:1 hexane–EtOAc gave 15 (2.30 g, 95%) as an oil. IR vmax(KCl):
2934, 2856, 1615, 1515, 1249 cmꢀ1 1H NMR (400 MHz, CDCl3): d
.
0.06 (6H, s), 0.89 (3H, t, J 6.8 Hz, and 9H, s), 1.25–1.65 (24H, m, con-
taining two 3H singlets at 1.35 and 1.46 ppm), 2.12–2.14 (4H, m),
3.51 (1H, m), 3.68 (1H, m), 3.75 (1H, m), 3.80 (3H, s), 4.14 (1H,
m), 4.15 (1H, m), 4.63, 4.68 (2H, AB-q, J 11.6 Hz), 6.86 (2H, d, J
8.8 Hz), 7.30 (2H, d, J 8.8 Hz). FABMS: m/z 611 [M+Na]+ (on addi-
tion of NaI). HRFABMS: Calcd for C35H60O5SiNa, 611.4108; ob-
served, 611.4108.
3000, 2932, 2854, 1610, 1585, 1511 cmꢀ1 1H NMR (400 MHz,
.
CDCl3): d 0.88 (3H, t, J 7.0 Hz), 1.15–1.72 (18H, m), 1.76 (1H, d, J
6.0 Hz, OH), 2.10–2.15 (4H, m), 3.56 (1H, s), 3.62 (1H, d, J 8.0 Hz),
3.81 (3H, s), 3.90 (1H, m), 3.92 (1H, d, J 12.6 Hz), 4.42 (1H, d, J
12.0 Hz), 4.52 (1H, d, J 12.6 Hz), 4.82 (1H, d, J 12.0 Hz), 5.56 (1H,
s), 6.91 (2H, d, J 8.0 Hz), 7.30–7.38 (5H, m), 7.52 (2H, d, J 8.0 Hz).
FABMS: m/z 522 [MÅ]+. HRFABMS: Calcd for C33H46O5: 522.3345;
observed: 522.3345. Anal. Calcd for C33H46O5: C, 75.83; H, 8.87.
Found: C, 75.68; H, 8.92.
4.1.12. (2R,3S,4R)-1-(tert-Butyldimethylsilyloxy)-3,4-O-
isopropylidene-11-octadecyn-2-ol (16)
To a solution of 15 (2.02 g, 3.68 mmol) in CH2Cl2 (60 mL) and
H2O (6 mL) was added DDQ (2.02 g, 8.90 mmol). The mixture was
stirred for 1 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 an oil that was chromatographed on a silica gel
column. Elution with 9:1 hexane–EtOAc gave 16 (1.61 g, 93%) as
4.1.9. (2R,3R,4R)-2-(4-Methoxybenzyloxy)-11-octadecyn-1,3,4-
triol (13)
an oil. IR vmax(KCl): 3570, 2932, 2858, 1463, 1370 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 0.07 (6H, s), 0.88 (3H, t, J 7.0 Hz), 0.90 (9H,
s), 1.23–1.58 (23H, m, containing two 3H singlets at 1.34 and
1.49 ppm), 1.77 (1H, m), 2.12–2.15 (4H, m), 2.30 (1H, d, J 5.2 Hz,
OH), 3.56–3.67 (3H, m), 4.10–4.19 (2H, m). EIMS: m/z 453
[MꢀCH3]Å+, 468 [MÅ]+. HREIMS: Calcd for C27H52O4Si, 468.3635; ob-
served, 468.3617.
A solution of 12 (3.05 g, 5.83 mmol) in MeOH (180 mL) contain-
ing p-TsOHꢁH2O (300 mg, 1.58 mmol) was stirred for 2 h at room
temperature, neutralized with excess satd aq NaHCO3, concen-
trated in vacuo, and then extracted with EtOAc. The solution was
washed with water and brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to give a mixture that was
chromatographed on a silica gel column. Elution with 2:1, then
1:1, and finally 1:9 hexane–EtOAc gave 12 (recovery, 400 mg,
13%) and 13 (2.16 g, 85%) as an amorphous solid. IR vmax(KBr):
4.1.13. (2S,3R,4R)-2-Benzoyloxy-1-(tert-butyldimethylsilyloxy)-
3,4-O-isopropylidene-11-octadecyne (17)
To a solution of 16 (1.48 g, 3.15 mmol), Ph3P (4.13 g, 15.8
mmol), and PhCOOH (1.69 g, 13.9 mmol) in dry THF (40 mL) was
added DEAD (2.2 M solution in toluene, 6.30 mL, 13.9 mmol) at
ꢀ20 °C. After stirring for 30 min sequentially at ꢀ20 °C, ꢀ10 °C,
and 0 °C, the mixture was stirred for 16 h at room temperature,
and then concentrated in vacuo to give a mixture that was directly
chromatographed on a silica gel (100 g) column. Elution with
100:0, then 19:1 hexane–EtOAc gave a 1:6 mixture of olefinic by-
product (0.21 g) and 17 (1.35 g, 74%) as an oil. A part of this mix-
ture was purified chromatographically on a preparative silica gel
TLC plate by development with 9:1 hexane–EtOAc. Physical data
3323 (br), 2929, 2855, 1613, 1585 (w), 1513 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 0.89 (3H, t, J 6.8 Hz), 1.25–1.55 (18H, m),
2.12–2.16 (4H, m), 2.26 (1H, d, J 7.6 Hz, OH), 2.53 (1H, m, OH),
2.84 (1H, d, J 7.6 Hz, OH), 3.57–3.60 (2H, m), 3.71 (1H, m), 3.80
(1H, m), 3.81 (3H, s), 3.95 (1H, m), 4.51, 4.70 (2H, AB-q, J
11.2 Hz), 6.90 (2H, d, J 8.6 Hz), 7.27 (2H, d, J 8.6 Hz). FABMS (posi-
tive-ion): m/z 433, 434, 435 [M+H]+. HRFABMS (positive-ion):
Calcd for C26H43O5: 435.3110; observed: 435.3110.
4.1.10. (2R,3R,4R)-1-(tert-Butyldimethylsilyloxy)-2-(4-
methoxybenzyloxy)-11-octadecyn-3,4-diol (14)
for 17: IR vmax(KBr): 2932, 2844, 1724 cmꢀ1 1H NMR (400 MHz,
.
To a solution of 13 (2.10 g, 4.83 mmol) in CH2Cl2 (50 mL) were
added tert-butyldimethylsilyl chloride (901 mg, 5.85 mmol) and
CDCl3): d ꢀ0.03 (3H, s), ꢀ0.01 (3H, s), 0.84 (9H, s), 0.88 (3H, t, J
7.0 Hz), 1.12–1.55 (24H, m, containing two 3H singlets at 1.36

1674
M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
and 1.45 ppm), 2.01 (1H, t, J 7.0 Hz), 2.12 (2H, t, J 7.2 Hz), 3.93 (1H,
dd, J 4.8, 11.6 Hz), 3.99 (1H, dd, J 2.4, 11.6 Hz), 4.18 (1H, m), 4.42
(1H, dd, J 5.2, 8.8 Hz), 5.15 (1H, m), 7.45 (2H, t, J 7.6 Hz), 7.57
(1H, t, J 7.6 Hz), 8.04 (2H, d, J 7.6 Hz). EIMS: m/z 557, 572 [MÅ]+.
HREIMS: Calcd for C34H56O5Si, 572.3897; observed, 572.3896.
4.05–4.08 (2H, m). The 1H NMR data for 18 were identical with
those of 18 derived from -arabinitol.
D
(2) A solution of a 1:4 mixture of 25 and 18 (1.13 g, 2.32 mmol)
in THF (22 mL) containing (n-Bu)4NF (1 M in THF, 5.0 mL,
5.00 mmol) was stirred for 50 min at room temperature, concen-
trated in vacuo, and 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 that was chromatographed
on a silica gel column. Elution with 3:2 hexane–EtOAc gave 19
(650 mg, 79%, two steps 36%) as a powder. The 1H NMR data of
4.1.14. (2S,3S,4R)-1-(tert-Butyldimethylsilyloxy)-3,4-O-
isopropylidene-11-octadecyn-2-ol (18)
A solution of a mixture of olefinic by-product (0.21 g) and 17
(1.34 g, 2.34 mmol) in MeOH (72 mL) and NaOMe (1 M in MeOH,
8.0 mL, 8.0 mmol) was stirred for 24 h at room temperature, con-
centrated in vacuo to one-third of the original volume, and then di-
luted with EtOAc. The solution was washed with H2O and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a mixture that was chromatographed on a silica gel col-
umn. Elution with 39:1, then 9:1 hexane–EtOAc gave 18 (0.84 g,
19 were identical with those of 19 derived from
D-arabinitol.
4.1.16. (2S,3S,4R,5EZ)-10-Benzyloxy-1-tert-
butyldimethylsilyloxy-3,4-O-isopropylidene-5-decene-2-ol (21)
To solution of (5-benzyloxypentyl)triphenylphosphonium
a
bromide15b (7.80 g, 15.0 mmol, 3 equiv) in dry THF (40 mL) was
added a solution of n-BuLi (1.59 M in hexane, 15.0 mL, 23.9 mmol)
at ꢀ78 °C under argon with stirring. After stirring for 10 min, the
temperature was gradually elevated to ꢀ10 °C over 1 h, and then
the mixture was stirred for 30 min at ꢀ10 °C. This solution was
cooled to ꢀ78 °C, and a solution of 20 (1.54 g, 5.06 mmol) in THF
(10 mL) was added dropwise. The temperature was gradually ele-
vated to ꢀ10 °C over 1 h, and stirred for 30 min at ꢀ10 °C, then fi-
nally at room temperature for 1 h. The reaction was quenched with
MeOH (1 mL), and the mixture was diluted with excess Et2O. The
ether solution was washed with H2O and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a resi-
due that was chromatographed on a silica gel column. Elution with
9:1, then 6:1 hexane–EtOAc gave a 1:1 mixture of E- and Z-isomers
(21) (980 mg, 42%) as an oil. This mixture (20 mg) was separated
using a preparative silica gel TLC plate. Development with 6:1 hex-
ane–EtOAc gave Z-isomer (7 mg) and E-isomer (7 mg). Physical
data for the Z-isomer: Rf 0.32 (6:1 hexane–EtOAc); IR vmax(KBr):
77%) as an oil. IR vmax(KCl) 3575 (w), 2932, 2858, 1461 cmꢀ1 1H
.
NMR (400 MHz, CDCl3): d 0.09 (6H, s), 0.89 (3H, t, J 7.2 Hz), 0.91
(9H, s), 1.25–1.73 (24H, m, containing two 3H singlets at 1.32
and 1.39 ppm), 2.12 (4H, t, J 7.2 Hz), 2.58 (1H, d, J 4.4 Hz, OH),
3.64–3.67 (2H, m), 3.83 (1H, m), 3.91 (1H, m), 4.17 (1H, m). EIMS:
m/z 453, 468 [MÅ]+. HREIMS: Calcd for C27H52O4Si, 468.3635; ob-
served, 468.3635.
4.1.15. (2S,3S,4R)-3,4-O-Isopropylidene-11-octadecyn-1,2-diol
(19)
Method (A): Preparation from 18. A solution of 18 (4.34 g,
9.26 mmol) in THF (85 mL) and (n-Bu)4NF (1 M solution in THF,
15.0 mL, 15.00 mmol) was stirred for 45 min at room temperature,
concentrated in vacuo to one-third of the original volume, and then
diluted with EtOAc. The solution was washed with H2O and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a mixture that was chromatographed on a silica gel col-
umn. Elution with 3:1, then 1:1 hexane–EtOAc gave 19 (2.49 g,
3490 (br), 2934, 2861 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.083
.
76%) as an oil. IR vmax(KBr): 3426 (br), 2932, 2858 cmꢀ1
.
1H NMR
(3H, s), 0.086 (3H, s), 0.91 (9H, s), 1.35 (3H, s), 1.45 (3H, s), 1.46–
1.56 (4H, m), 1.62–1.67 (2H, m), 2.11–2.21 (2H, m), 2.46 (1H, d, J
4.4 Hz, OH), 3.47 (2H, t, J 6.5 Hz), 3.66–3.70 (2H, m), 3.81 (1H,
m), 4.01 (1H, m), 4.49 (2H, s), 4.99 (1H, dd, J 6.2, 9.5 Hz), 5.55
(1H, m), 5.69 (1H, m), 7.25–7.29 (2H, m), 7.32–7.36 (3H, m). ESIMS:
m/z 487.3 [M+Na]+. HRESIMS: Calcd for C26H44O5SiNa: 487.2850;
observed, 487.2851. Physical data for the E-isomer: Rf 0.31 (6:1
(500 MHz, CDCl3): d 0.89 (3H, t, J 7.0 Hz), 1.25–1.72 (24H, m, con-
taining two 3H singlets at 1.34 and 1.42 ppm), 1.88 (1H, t, J 5.6 Hz,
OH), 2.14 (1H, t, J 6.1 Hz), 2.17 (1H, d, J 5.7 Hz, OH), 3.73–3.77 (2H,
m), 3.83 (1H, m), 3.97 (1H, dd, J 5.8, 8.2 Hz), 4.20 (1H, m). EIMS: m/z
339, 354 [MÅ]+. HREIMS: Calcd for C21H38O4, 354.2770; observed,
354.2766.
Method (B): Preparation from 24. (1) To a solution of 1-octyne
(2.40 g, 21.8 mmol) in THF (40 mL) were added n-BuLi (1.65 M in
hexane, 14.2 mL, 23.4 mmol) and HMPA (16 mL) at ꢀ40 °C under
Argon. After stirring for 1 h at ꢀ40 °C, the solution was cooled to
ꢀ78 °C. To this solution, a solution of 24 (2.00 g, 3.61 mmol) in
THF (20 mL) was added dropwise. The temperature was elevated
gradually from ꢀ78 °C to ꢀ15 °C over 2 h, the reaction was
quenched with satd aq NH4Cl solution (25 mL), and the mixture
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 mixture that was chromatographed on a silica gel
column. Elution with 19:1 hexane–EtOAc gave 1-tert-butyldimeth-
ylsilyl-1-octyne (409 mg), (2S,3R,4R)-1,2-di-(tert-butyldimethylsil-
yloxy)-3,4-O-isopropylidene-11-octadecyne (25, 228 mg, 9%), 18
(922 mg, 45%), and an unknown mixture (640 mg) containing diol
19. Physical data for 1-tert-butyldimethylsilyloctyne: IR vmax(KBr):
hexane–EtOAc); IR vmax(KBr): 3510 (br), 2932, 2861 cmꢀ1 1H
.
NMR (500 MHz, CDCl3): d 0.082 (3H, s), 0.085 (3H, s), 0.91 (9H,
s), 1.34 (3H, s), 1.45 (3H, s), 1.48–1.56 (4H, m), 1.61–1.67 (2H,
m), 2.11 (2H, q, J 7.0 Hz), 2.49 (1H, d, J 4.6 Hz, OH), 3.47 (2H, t, J
6.5 Hz), 3.65–3.70 (2H, m), 3.80 (1H, m), 4.00 (1H, dd, J 6.1,
8.8 Hz), 4.49 (2H, s), 4.64 (1H, t, J 7.0 Hz), 5.62 (1H, dd, J 7.8,
15.4 Hz), 5.81 (1H, td, J 7.0, 15.4 Hz), 7.26–7.29 (2H, m), 7.32–
7.36 (3H, m). ESIMS: m/z 487.3 [M+Na]+. HRESIMS: Calcd for
C26H44O5SiNa: 487.2850; observed, 487.2850.
4.1.17. (2S,3R,4R,5EZ)-10-Benzyloxy-1,2-di-tert-
butyldimethylsilyloxy-3,4-O-isopropylidenedecene (22)
To a solution of 21 (2.407 g, 5.18 mmol) in CH2Cl2 (100 mL)
were added 2,6-lutidine (2.77 g, 25.9 mmol, 5 equiv) and TBDM-
SOTf (4.11 g, 15.5 mmol, 3 equiv). After stirring for 1 h at room
temperature, the reaction mixture was diluted with CH2Cl2,
washed with satd aq NaHCO3 and brine, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a residue that
was chromatographed on a silica gel column. Elution with 19:1
hexane–EtOAc gave 22 (3.00 g, quant, a 1:1 mixture of E- and Z-iso-
2955, 2930, 2858, 2174 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.08
.
(6H, s), 0.89 (3H, t, J 7.0 Hz), 0.93 (9H, s), 1.26–1.32 (4H, m),
1.38–1.41 (2H, m), 1.48–1.54 (2H, m), 2.22 (2H, t, J 7.0 Hz). EIMS:
m/z 605.4 [M+Na]+. HREIMS: Calcd for C33H66O4Si2Na: 605.4397;
observed, 605.4395. Physical data for 25. IR vmax(KCl): 2931,
mers) as an oil. IR: vmax(KBr): 2930, 2857 cmꢀ1 1H NMR (as a 1:1
.
2858 cmꢀ1
.
1H NMR (500 MHz, CDCl3): d 0.06 (6H, s), 0.08 (3H,
mixture of E- and Z-isomers) (500 MHz, CDCl3): d 0.05–0.08 (12 ꢃ
2H, m), 0.86–0.90 (18 ꢃ 2H, m), 1.30 (3H, s), 1.33 (3H, s), 1.34 (3H,
s), 1.45 (3H, s), 1.47–1.52 (2 ꢃ 2H, m), 1.62–1.67 (2 ꢃ 2H, m), 2.06–
2.22 (2 ꢃ 2H, m), 3.45–3.49 (2 ꢃ 2H, m), 3.62–3.73 (2 ꢃ 2H, m),
s), 0.11 (3H, s), 0.86 (9H, s), 0.89 (3H, t, J 7.1 Hz), 0.91 (9H, s),
1.24–1.53 (24H, m, containing two 3H singlets at 1.31 and
1.40 ppm), 2.13 (4H, t, J 7.1 Hz), 3.67 (1H, m), 3.77–3.80 (2H, m),

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1675
3.79–3.84 (1 ꢃ 2H, m), 4.17–4.19 (1 ꢃ 2H, m), 4.50 (2 ꢃ 2H, s), 4.53
(1H, m), 4.92 (1H, m), 5.60–5.74 (2 ꢃ 2H, m), 7.27–7.34 (5 ꢃ 2H,
m). ESIMS: m/z 601.3 [M+Na]+. HRESIMS: Calcd for C32H58O5Si2Na:
601.3715; observed, 601.3711.
4.1.21. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-benzylidene-2,3-di-O-(4-methoxybenzyl)-
-galactopyranoside (28a)
A suspension of 26 (594 mg, 0.703 mmol) containing a small
a
-
D
amount of by-product, n-hexacosanoic acid (1.29 g, 3.25 mmol),
DMAP (1.80 g, 14.7 mmol) and EDAC (2.87 g, 15.0 mmol) in dry
1:1 THF–CH2Cl2 (50 mL) was stirred for 5 days at room temper-
ature. The mixture was concentrated in vacuo and then diluted
with CHCl3. The solution was washed with H2O (ꢃ2) and brine,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a residue that was chromatographed on a silica gel
(20 g) column. Elution with 5:1, then 1:2 hexane–EtOAc gave 28a
(270 mg, 31%) as a wax, a 3:4 mixture of 28a and an unknown
by-product (102 mg), and 26 (148 mg, 25% recovery). A part of
the 3:4 mixture of 28a and the by-product (20 mg) was separated
on a preparative TLC plate. Development with 3:1 hexane–EtOAc
gave 28a (6 mg) Rf 0.40 (4:1 hexane–EtOAc) and the by-product
(8 mg) Rf 0.34 (4:1 hexane–EtOAc). Physical data for 26: IR
4.1.18. (2S,3R,4R)-1,2-Di-tert-butyldimethylsilyloxy-3,4-O-
isopropylidenedecan-10-ol (23)
A solution of a 1:1 mixture of 22 (2.81 g, 4.853 mmol) in hexane
(380 mL) was stirred for 16 h at room temperature under hydrogen
using 10% Pd-on-carbon (1.42 g) as a catalyst. The reaction mixture
was filtered, and the filtrate was concentrated in vacuo to give a
residue that was chromatographed on a silica gel column. Elution
with 9:1, then 3:1 hexane–EtOAc gave 23 (2.09 g, 88%) as an oil.
IR vmax(KBr): 3440 (br), 2931, 2858 cmꢀ1 1H NMR (500 MHz,
.
CDCl3–D2O): d 0.06 (6H, s), 0.08 (3H, s), 0.12 (3H, s), 0.86 (9H, s),
0.91 (9H, s), 1.30–1.60 (16H, m, containing two 3H singlets at
1.31 and 1.40 ppm), 3.61–3.67 (2H, m), 3.68 (1H, m), 3.77–3.80
(2H, m), 4.06–4.08 (2H, m). ESIMS: m/z 513.3 [M+Na]+. HRESIMS:
Calcd for C25H54O5Si2Na: 513.3402; observed, 513.3407.
vmax(KBr): 3501 (br), 2930, 2850, 1613, 1514 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 0.86–0.91 (3H, m), 1.22–1.71 (24H, m, con-
taining two 3H singlets at 1.31 and 1.38 ppm), 2.12–2.16 (4H, m),
3.30 (1H, br s, OH), 3.44 (1H, m), 3.67 (1H, s), 3.77 (1H, m), 3.80
(3H, s), 3.81 (3H, s), 3.89 (1H, m), 3.95–4.07 (4H, m), 4.13–4.22
(3H, m), 4.60, 4.80 (2H, AB-q, J 11.4 Hz), 4.66, 4.72 (2H, AB-q, J
11.8 Hz), 4.96 (1H, d, J 3.6 Hz, anomeric H), 5.47 (1H, s), 6.83–
6.88 (4H, m), 7.24–7.27 (2H, m), 7.31–7.37 (5H, m), 7.50–7.53
(2H, m). FABMS: m/z 867 [M+Na]+. HRFABMS: Calcd for
4.1.19. (2S,3R,4R)-10-Bromo-1,2-di-tert-butyldimethylsilyloxy-
3,4-O-isopropylidenedecane (24)
To a solution of 23 (2.20 g, 4.48 mmol) in dry CH2Cl2 (50 mL)
were added imidazole (460 mg, 6.76 mmol), Ph3P (1.77 g,
6.75 mmol) and CBr4 (2.33 g, 6.72 mmol). This solution was stirred
at room temperature for 2 h, concentrated in vacuo to give a mix-
ture that was chromatographed on a silica gel column. Elution with
39:1, then 19:1 hexane–EtOAc gave 24 (2.28 g, 92%) as an oil. IR
C
50H68O11Na, 867.4654; observed, 867.4657. Physical data for
28a: IR vmax(KBr): 2920, 2851, 1741, 1614, 1588 (w), 1515,
1469 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.85–0.91 (6H, m),
.
vmax(KBr): 2930, 2858 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.06
.
1.20–1.60 (70H, m, containing two 3H singlets at 1.33 and
1.41 ppm), 2.13 (4H, t, J 6.8 Hz), 2.24 (1H, dt, J 2.7, 8.0 Hz), 3.63
(1H, s), 3.71 (1H, dd, J 6.0, 12.0 Hz), 3.80 (6H, s), 3.87–4.04 (4H,
m), 4.09–4.25 (4H, m), 4.60, 4.72 (2H, AB-q, J 11.2 Hz), 4.64, 4.73
(2H, AB-q, J 11.4 Hz), 4.95 (1H, d, J 3.2 Hz, anomeric H), 5.02 (1H,
m), 5.46 (1H, s), 6.83–6.86 (4H, m), 7.26–7.35 (7H, m), 7.51 (2H,
d, J 5.6 Hz). FABMS: m/z 1245 [M+Na]+. HRFABMS: Calcd for
(6H, s), 0.08 (3H, s), 0.12 (3H, s), 0.86 (9H, s), 0.91 (9H, s), 1.30–
1.57 (16H, m, containing two 3H singlets at 1.31 and 1.40 ppm),
1.86 (2H, quintet, J 7.1 Hz), 3.40 (2H, t, J 7.1 Hz), 3.67 (1H, m),
3.77–3.80 (2H, m), 4.05–4.10 (2H, m). ESIMS: m/z 575.3 [M+Na]+.
HRESIMS: Calcd for C25H53O4Si2BrNa: 575.2558; observed, 575.
2562.
C76H118O12Na, 1245.8515; observed, 1245.8510.
4.1.20. (2S,3S,4R)-2-Hydroxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-benzylidene-2,3-di-O-(4-methoxybenzyl)-
-galactopyranoside (26) and (2S,3S,4R)-2-hydroxy-3,4-O-
isopropylidene-11-octadecynyl 4,6-O-benzylidene-2,3-di-O-(4-
methoxybenzyl)-b- -galactopyranoside (27)
To a solution of imidate 4 (purity ca. 90%, 2.35 g, 3.23 mmol)
and diol 19 (0.80 g, 2.26 mmol) in dry CH2Cl2 (80 mL) was added
4 Å MS (dry powder, 6 g). After stirring for 30 min at room temper-
ature, the mixture was cooled to at 0 °C, and AgOTf (1.40 g,
5.45 mmol) was added. After stirring for 2 h at 0 °C and for 3 h at
room temperature, the mixture was diluted with CH2Cl2. The solu-
tion was washed with satd aq NaHCO3 and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a mixture that was chromatographed on a silica gel column. Elu-
a-
4.1.22. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-benzylidene- -galactopyranoside (29a)
D
a-D
To a solution of 28a (265 mg, 0.216 mmol) in 10:1 CH2Cl2–H2O
(22 mL) was added DDQ (265 mg, 1.17 mmol), and the solution
was stirred for 3 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 that was chromatographed on a silica gel
column. Elution with 3:1, then 3:2 hexane–EtOAc gave 29a
(167 mg, 78%) as a wax. IR vmax(KBr): 3395 (br), 2921, 2851,
D
1725 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.86–0.91 (6H, m),
.
1.20–1.63 (70H, m, containing two 3H singlets at 1.34 and
1.43 ppm), 2.13 (4H, t, J 7.2 Hz), 2.30 (2H, dt, J 4.4, 7.2 Hz), 2.39
(2H, br s, OH), 3.73–3.77 (2H, m), 3.82 (2H, br s), 4.03–4.28 (6H,
m), 5.01 (1H, s, anomeric H), 5.05 (1H, dt, J 1.5, 7.0 Hz), 5.55 (1H,
s), 7.36–7.37 (3H, m), 7.48–7.49 (2H, m). FABMS: m/z 1005
[M+Na]+. HRFABMS: Calcd for C60H102O10Na, 1005.7365; observed,
1005.7387.
tion with 3:1, then 2:1 hexane–EtOAc gave
a anomer 26 (1.16 g,
61%) containing a small amount of unknown by-product (insepara-
ble chromatographically from 26 at this stage) and b anomer 27
(0.60 g, 31%) as a gum. Physical data for 27: IR vmax(KBr): 3439,
2931, 2859, 1614, 1515 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.86–
.
0.91 (3H, m), 1.28–1.63 (24H, m, containing two 3H singlets at
1.33 and 1.41 ppm), 2.11–2.15 (4H, m), 3.33 (1H, br s, OH), 3.35
(1H, s), 3.55 (1H, dd, J 3.6, 9.6 Hz), 3.73–3.89 (11H, m, containing
4.1.23. (2S,3R,4R)-2-Hexacosyloxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-benzylidene-2,3-di-O-(4-methoxybenzyl)-
-galactopyranoside (28b)
To a solution 26 (594 mg, 0.703 mmol) containing a small
a-
6H singlet at 3.80 ppm), 3.94–4.10 (3H, m), 4.28 (1H, d,
J
D
12.0 Hz), 4.41 (1H, d, J 7.6 Hz), 4.68 (2H, s), 4.75, 4.80 (2H, AB-q, J
10.8 Hz), 5.48 (1H, s), 6.84–6.88 (4H, dd, J 6.8, 8.4 Hz), 7.26–7.30
(4H, m), 7.34–7.38 (3H, m), 7.55 (2H, d, J 6.0 Hz). FABMS: m/z
867 [M+Na]+. HRFABMS: Calcd for C50H68O11Na, 867.4654; ob-
served, 867.4671.
amount of by-product, and n-hexacosyl methanesulfonate (518
mg, 1.13 mmol; soluble in DMF by warming) in 6:1 DMF–THF
(35 mL) was added NaH (60% oil dispersion, 400 mg, 10.0 mmol)
under argon. After stirring for 16 h at room temperature, the mixture

1676
M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
was warmed to 55 °C for 2 h and then diluted with EtOAc. The solu-
tion was washed with water and brine, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a mixture that
was chromatographed on a silica gel column. Elution with 6:1 hex-
ane–EtOAc gave 28b (334 mg, 39%) as a wax. IR vmax(KCl) 2920,
4.90 (1H, d, J 3.6 Hz, anomeric H). 13C NMR (100 MHz, 5:1 CDCl3–
CD3OD): d 13.80, 22.44, 25.52, 25.94, 29.14, 29.30, 29.45, 29.50,
29.60, 29.66, 31.69, 61.85, 65.04, 68.84, 69.73, 69.78, 69.96,
69.99, 71.97, 72.20, 79.14, 98.95. FABMS: m/z 845 [M+H]+, 867
[M+Na]+. HRFABMS: Calcd for C50H101O9: 845.7446; observed,
845.7448.
2851, 1615, 1585, 1516, 1469 cmꢀ1 1H NMR (400 MHz, CDCl3): d
.
0.88 (3H, t, J 6.8 Hz), 0.89 (3H, t, J 6.8 Hz), 1.22–1.60 (72H, m, con-
taining two 3H singlets at 1.31 and 1.38 ppm), 2.11–2.17 (4H, m),
3.27 (1H, m), 3.42 (1H, m), 3.56 (1H, dd, J 4.6, 11.0 Hz), 3.67 (1H,
s), 3.78 (1H, m), 3.80 (6H, s), 3.97–4.08 (5H, m), 4.14–4.21 (3H, m),
4.60, 4.74 (2H, AB-q, J 11.6 Hz,), 4.66, 4.74 (2H, AB-q, J 11.6 Hz,),
4.94 (1H, d, J 3.2 Hz, anomeric H), 5.47 (1H, s), 6.84 (4H, d,
J 8.4 Hz), 7.27–7.36 (7H, m), 7.52 (2H, m). FABMS: m/z 1231
[M+Na]+. HRFABMS: Calcd for C76H120O11Na, 1231.8723; observed,
1231.8733.
4.1.27. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-dihydroxy-11-octade-
cynyl
a-D-galactopyranoside (30a) and/or (2S,3S,4R )-4-hexaco-
sanoyloxy-2,3-dihydroxy-11-octadecynyl
a-D-galactopyranoside
(32)
Method (A): To a solution of 41 (25 mg, 0.028 mmol) in 1:1
CH2Cl2–CH3CN (20 mL) were added H2O (175 mg) and 46% aq HF
(112 mg) at 25 °C. The solution was stirred for 25 min, and the
reaction was quenched with satd aq NaHCO3. The mixture was
then diluted with CHCl3, washed with satd aq NaHCO3, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue that was chromatographed on a silica gel (3 g) column.
Elution with 19:1, then 12:1 CHCl3–MeOH gave 30a (6.0 mg,
25%) as a wax, a 3:1 mixture of 30a (Rf 0.25, 19:1 EtOAc–MeOH)
and 32 (1.4 mg, 5.8%, Rf 0.13, 19:1 EtOAc–MeOH), and 41
(6.0 mg, recovery 24%, Rf 0.50, 19:1 EtOAc–MeOH). Physical data
4.1.24. (2S,3R,4R)-2-Hexacosyloxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-benzylidene-a-D-galactopyranoside (29b)
Compound 28b (338 mg, 0.279 mmol) was treated as described
in the formation of 29a from 28a to give 29b (129 mg, 48%) as a
wax. IR vmax(KBr): 3453 (br), 2920, 2851, 1615, 1588 (w), 1516,
1469 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.86–0.91 (6H, m),
.
for 30a: ½a 2D4
ꢂ
+56.8 (c 0.85, 9:1 CHCl3–MeOH). IR vmax(KBr): 3400
1.22–1.65 (72H, m, containing two 3H singlets at 1.33 and
1.39 ppm), 2.12–2.16 (4H, m), 2.41 (1H, d, J 8.0 Hz, OH), 2.71 (1H,
br s, OH), 3.28 (1H, dd, J 7.1, 14.8 Hz), 3.40 (1H, d, J 8.8 Hz), 3.61
(1H, dd, J 6.0, 14.8 Hz), 3.69 (1H, dd, J 3.4, 10.6 Hz), 3.86 (1H, s),
3.88–3.95 (2H, m), 4.06–4.14 (3H, m), 4.17 (1H, m), 4.25–4.28
(2H, m), 5.04 (1H, d, J 3.2 Hz, anomeric H), 5.56 (1H, s), 7.36–7.38
(3H, m), 7.49–7.51 (2H, m). FABMS: m/z 897, 991 [M+Na]+.
HRFABMS: Calcd for C60H104O9Na, 991.7572; observed, 991.7590.
(br), 2921, 2851, 1730, 1468 cmꢀ1
.
1H NMR (500 MHz, 12:1
CDCl3–CD3OD): d 0.88 (3H, t, J 7.0 Hz), 0.89 (3H, t, J 7.0 Hz), 1.25–
1.61 (64H, m), 2.12–2.15 (4H, m), 2.31–2.35 (2H, m), 3.61 (1H, m,
C4–H), 3.72–3.86 (7H, m, containing C1–H2), 3.99 (1H, d,
J
3.2 Hz), 4.03 (1H, dd, J 3.9, 11.2 Hz, C3–H), 4.92 (1H, d, J 3.9 Hz,
anomeric H), 5.10 (1H, m, C2-H). 13C NMR (125 MHz, 12:1
CDCl3–CD3OD): d 14.09, 14.15, 18.77, 18.78, 22.62, 22.74, 24.96,
25.81, 28.61, 28.93, 29.18, 29.21, 29.24, 29.28, 29.39, 29.41,
29.57, 29.71, 29.72, 29.75, 29.78, 31.42, 31.98, 34.44, 62.29,
67.08, 68.92, 70.06, 70.27, 70.31, 71.79, 72.20, 73.29, 80.18,
80.35, 99.34, 173.63. ESIMS: m/z 877.7 [M+Na]+. HRESIMS: Calcd
for C50H94O10Na, 877.6739; observed, 877.6742.
Method (B): To a solution of 29a (180 mg, 0.183 mmol) in 1:1
MeCN–CH2Cl2 (70 mL) containing H2O (508 mg) was added 46%
aq HF (391 mg, 8.99 mmol). The mixture was stirred for 4 h at
room temperature and diluted with CHCl3, and the solution was
washed with satd aq NaHCO3 and brine, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a residue that
was chromatographed on a silica gel (8 g) column. Elution with 9:1
CHCl3–MeOH gave a trace amount of 30a in a mixture with 32 (by
TLC and 1H NMR) and pure 32 (69 mg, 44%) as a wax.
4.1.25. (2S,3R,4R)-3,4-Dihydroxy-2-hexacosyloxy-11-
octadecynyl a-D-galactopyranoside (30b)
To a solution of 29b (122 mg, 0.126 mmol) in 1:1 MeCN–CH2Cl2
(54 mL) containing H2O (332 mg) was added 46% aq HF (264 mg,
6.047 mmL). The mixture was stirred for 4 h at room temperature
and then diluted with CHCl3, and the solution was washed with
satd aq NaHCO3 and brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to give a residue that was chro-
matographed on a silica gel (6 g) column. Elution with 9:1
CHCl3–MeOH gave 30b (59 mg, 56%) as a wax. ½a D21
ꢂ
+62.8 (c 1.1,
CHCl3). IR vmax(KBr): 3387 (br), 2920, 2851, 1468 cmꢀ1
.
1H NMR
(400 MHz, 6:1 CDCl3–CD3OD): d 0.88 (3H, t, J 6.8 Hz), 0.89 (3H, t,
J 6.8 Hz), 1.20–1.65 (66H, m), 2.14 (4H, t, J 7.2 Hz), 3.50 (1H, m),
3.60–3.72 (4H, m), 3.72–3.90 (6H, m), 3.97–4.05 (2H, m), 4.90
(1H, d, J 3.2 Hz, anomeric H). 13C NMR (100 MHz, CDCl3): d 14.08,
14.13, 18.79, 18.82, 22.59, 22.70, 26.11, 26.22, 28.61, 29.09,
29.18, 29.37, 29.48, 29.63, 29.66, 29.73, 29.84, 31.41, 31.93,
61.63, 64.81, 68.89, 69.71, 69.89, 70.07, 72.01, 72.16, 77.21,
79.01, 80.03, 80.16, 99.13. FABMS: m/z 841 [M+H]+, 863 [M+Na]+.
HRFABMS: Calcd for C50H97O9, 841.7127; observed, 841.7126.
Method (C): To a solution of 29a (20 mg, 0.022 mmol) in CHCl3
(4 mL) was added a solution of 1.0 M HCl solution in EtOAc
(0.4 mL). The solution was stirred for 5 min at room temperature
and concentrated in vacuo to give a residue that was chromato-
graphed on a silica gel (3 g) column. Elution with 19:1, then 9:1
CHCl3–MeOH gave 32 (13 mg, 68%) as a powder. Physical data for
32: ½a 2D5
ꢂ
+54.5 (c 0.92, CHCl3). IR vmax(KBr): 3405 (br), 2920,
2851, 1717, 1468 cmꢀ1
.
1H NMR (500 MHz, 10:1 CDCl3–CD3OD):
4.1.26. (2S,3R,4R)-3,4-Dihydroxy-2-hexacosyloxy-1-octadecyl
d 0.88 (3H, t, J 7.0 Hz), 0.89 (3H, t, J 7.1 Hz), 1.26 (52H, br s),
1.31–1.70 (12H, m, containing C5–H2), 2.13 (4H, t, J 7.1 Hz),
2.32–2.35 (2H, m), 3.41 (1H, dd, J 7.2, 10.7 Hz), 3.52 (1H, dd, J
6.4, 10.5 Hz), 3.65 (1H, dd, J 1.2, 6.6 Hz), 3.73 (1H, dd, J 4.9,
6.6 Hz), 3.75–3.85 (5H, m, containing C3–H), 3.93 (1H, dd, J 2.2,
10.3 Hz), 3.97 (1H, d, J 2.4 Hz), 4.88 (1H, d, J 3.4 Hz, anomeric H),
5.07 (1H, m, C4–H). 13C NMR (100 MHz, 10:1 CDCl3–CD3OD): d
13.87, 13.93, 18.57, 22.42, 22.54, 24.91, 25.22, 28.39, 28.66,
28.97, 29.05, 29.17, 29.22, 29.40, 29.55, 29.58, 31.21, 31.77,
34.47, 61.86, 69.00, 69.53, 69.63, 70.02, 70.19, 70.62, 72.25,
74.55, 79.93, 80.16, 99.13, 174.34. FABMS: m/z 855 [M+H]+, 877
[M+Na]+. HRFABMS: Calcd for C50H95O10, 855.6925; observed,
855.6931.
a-
D
-galactopyranoside (31b)
A solution of 30b (32 mg, 0.038 mmol) in 1:1 CHCl3–MeOH
(6 mL) containing Pd(OH)2/C (20 wt % Pd-on-carbon, wet, 32 mg)
was stirred for 1 h at room temperature under hydrogen. The reac-
tion mixture was filtered, and the catalyst was washed thoroughly
with 1:1 CHCl3–MeOH. The combined filtrate was concentrated to
give a residue that was chromatographed on a silica gel (4 g) col-
umn. Elution with 9:1 CHCl3–MeOH gave 31b (13 mg, 32%) as a
wax. ½a 2D5
ꢂ
+42.3 (c 0.7, 13:1 CHCl3–MeOH). IR vmax(KBr): 3417
(br), 2919, 2850, 1469 cmꢀ1
.
1H NMR (400 MHz, 7:1 CDCl3–
CD3OD): d 0.88 (6H, t, J 6.8 Hz), 1.20–1.34 (70H, m), 1.50–1.61
(4H, m), 3.49 (1H, m), 3.60–3.89 (11H, m), 3.98–4.04 (2H, m),

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1677
4.1.28. 4-Methylphenyl 4,6-O-di-tert-butylsilylidene-1-thio-b-
galactopyranoside (34)
D
-
NaHCO3 and brine, dried over MgSO4, filtered, and concentrated in
vacuo to give a mixture that was chromatographed on a silica gel
column. Elution with 3:2, then 1:1 hexane–EtOAc gave 37
A solution of 33 (286 mg, 1.00 mmol), the starting material for
compound 1, and (t-Bu)2Si(OTf)2 (463 mg, 1.05 mmol) in dry pyri-
dine (2 mL) was stirred for 30 min at room temperature. The reac-
tion mixture was concentrated in vacuo, and the residue was
dissolved in 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 that was chromatographed on a silica gel col-
umn. Elution with hexane–EtOAc (3:2) gave 34 (317 mg, 74%) as an
(4.03 g, 58%, a mixture of
a
and b anomers) as a gum. IR vmax(KBr):
3437 (br), 2934, 2859, 1612, 1586 (w), 1514 cmꢀ1
.
1H NMR
(500 MHz, CDCl3): d 1.01 (9H, s), 1.06 (9H, s), 2.85–2.88 (1H, m,
OH), 3.33–3.42 (1H, m), 3.65–3.96 (8H, m), 4.11–4.23 (2H, m),
4.42–4.82 (5H, m), 5.14–5.20 (1H, m), 6.84–6.89 (4H, m), 7.27–
7.35 (4H, m). ESIMS: m/z 583.3 [M+Na]+. HRESIMS: Calcd for
C30H44O8SiNa, 583.2698; observed, 583.2703.
oil. IR vmax(KBr): 3419 (br), 2933, 2859, 1492, 1473 cmꢀ1 1H NMR
.
(500 MHz, CDCl3): d 1.03 (9H, s), 1.05 (9H, s), 2.34 (3H, s), 2.61 (1H,
d, J 1.7 Hz, OH), 2.75 (1H, d, J 10.5 Hz, OH), 3.44 (1H, m), 3.52 (1H,
m), 3.72 (1H, t, J 9.3 Hz), 4.25 (2H, m), 4.43 (1H, dd, J 1.0, 3.7 Hz),
4.47 (1H, d, J 9.8 Hz, anomeric H), 7.11–7.12 (2H, m), 7.45–7.47
(2H, m). ESIMS: m/z 449.2 [M+Na]+. HRESIMS: Calcd for C21H34O5
SSiNa: 449.1788; observed, 449.1792.
4.1.31. (2S,3S,4R)-2-Hydroxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-di-tert-butylsilylidene-2,3-di-O-(4-
methoxybenzyl)-a-D-galactopyranoside (38)
(1) Compound 37 (1.46 g, 2.60 mmol) was treated as described
in the formation of 4 from 3 to give the crude imidate, which was
employed for the next reaction without purification.
(2) To a solution of the imidate obtained above and diol 19
(710 mg, 2.00 mmol) in dry CH2Cl2 (52 mL) was added 4 Å MS
(4.2 g). After stirring for 30 min at room temperature, the mixture
was cooled to at ꢀ20 °C, and AgOTf (520 mg, 2.02 mmol) was
added. After stirring for 30 min at ꢀ20 °C, the temperature was
gradually elevated to room temperature over 2.5 h. Then the mix-
ture was stirred for 1 h at room temperature and filtered. The filter
cake was washed several times with CH2Cl2, and the combined fil-
trate was washed with satd aq NaHCO3 and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a residue that was chromatographed on a silica gel column. Elution
with 6:1, then 4:1 hexane–EtOAc gave 38 (1.60 g, 89%) as a gum. IR
4.1.29. 4-Methylphenyl 4,6-O-di-tert-butylsilylidene-2,3-di-O-
(4-methoxybenzyl)-1-thio-b-
Methylphenyl 6-O-di-tert-butyl(hydroxy)silyl-2,3-di-O-(4-
methoxybenzyl)-1-thio-b- -galactopyranoside (36)
D-galactopyranoside (35) and 4-
D
To a solution of 34 (7.10 g, 16.6 mmol) and 4-methoxybenzyl
chloride (10.4 g, 66.6 mmol) in dry DMF (20 mL) was gradually
added NaH (60% oil dispersion, 4.0 g, 100 mmol) at room tempera-
ture with stirring. The mixture reacted exothermically. After stir-
ring for 1 h at room temperature, the reaction mixture was
diluted with EtOAc (500 mL), and the reaction was quenched with
ice. The organic layer was washed with water and brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo to give
a mixture that was chromatographed on a silica gel column. Elu-
tion with 6:1, then 4:1 hexane–EtOAc gave 35 (8.23 g, 74%) as a
gum, and silanol 36 (1.22 g, 11%) as a gum. Physical data for 35:
vmax(KBr): 3444 (br), 2933, 2859, 1613, 1513, 1467 cmꢀ1 1H NMR
.
(500 MHz, CDCl3): d 0.89 (3H, t, J 7.0 Hz), 0.99 (9H, s), 1.06 (9H, s),
1.26–1.70 (24H, m, containing two 3H singlets at 1.30 and
1.37 ppm), 2.12–2.15 (4H, m), 3.40 (1H, dd, J 8.2, 10.9 Hz), 3.47
(1H, br s, OH), 3.66 (1H, s), 3.76–3.82 (8H, m, containing two 3H
singlets at 3.80 and 3.82 ppm), 3.88 (1H, dd, J 5.6, 9.0 Hz), 3.95–
3.99 (2H, m), 4.10 (1H, dd, J 1.6, 12.5 Hz), 4.14 (1H, m), 4.20 (1H,
dd, J 2.0, 12.5 Hz), 4.49 (1H, d, J 2.9 Hz), 4.61, 4.67 (2H, AB-q, J
11.2 Hz), 4.61, 4.80 (2H, AB-q, J 11.2 Hz), 4.76 (1H, d, J 3.7 Hz, ano-
meric H), 6.84 (2H, d, J 8.8 Hz), 6.89 (1H, (2H, d, J 8.8 Hz), 7.26 (2H,
d, J 8.8 Hz), 7.35 (2H, d, J 8.8 Hz). ESIMS: m/z 919.5 [M+Na]+. HRE-
SIMS: Calcd for C51H80O11SiNa, 919.5362; observed, 919.5357.
IR vmax(KBr): 2935, 2857, 1614, 1514 cmꢀ1 1H NMR (500 MHz,
.
CDCl3): d 1.07 (9H, s), 1.13 (9H, s), 2.32 (3H, s), 3.22 (1H, br s,
C5–H), 3.42 (1H, dd, J 2.9, 9.0 Hz, C3–H), 3.79 (1H, t, J 9.4 Hz),
3.80 (3H, s), 3.81 (3H, s), 4.15 (1H, dd, J 2.2, 12.3 Hz, C6–H), 4.19
(1H, dd, J 1.5, 12.3 Hz, C6–H), 4.44 (1H, d, J 2.9 Hz, C4–H), 4.56
(1H, d, J 9.8 Hz, anomeric H), 4.64, 4.70 (2H, AB-q, J 11.8 Hz),
4.80, 4.83 (2H, AB-q, J 9.7 Hz), 6.86–6.88 (4H, m), 7.07–7.09 (2H,
m), 7.32–7.38 (4H, m), 7.43–7.45 (2H, m). ESIMS: m/z 689.3
[M+Na]+. HRESIMS: Calcd for C37H50O7SSiNa: 689.2939; observed,
689.2939. Physical data for 36: IR vmax(KBr): 3482 (br), 2933,
4.1.32. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-di-tert-butylsilylidene-2,3-di-O-(4-methoxy-
2858, 1613, 1514, 1250 cmꢀ1 1H NMR (600 MHz, CDCl3): d 0.97
.
(9H, s), 1.02 (9H, s), 2.68 (1H, s, OH), 3.19 (1H, br s, OH), 2.32
(3H, s), 3.44 (1H, t, J 5.9 Hz, C5–H), 3.53 (1H, dd, J 3.2, 9.1 Hz,
C3–H), 3.65 (1H, dd, J 9.1, 9.6 Hz, C2–H), 3.80 (3H, s), 3.81 (3H,
s), 4.02 (1H, dd, J 5.9, 11.0 Hz, C6–H), 4.05 (1H, d, J 3.2 Hz, C4–H),
4.06 (1H, dd, J 5.9, 11.0 Hz, C6–H), 4.52 (1H, d, J 9.6 Hz, anomeric
H), 4.62 (2H, s, C3–O–CH2Ar), 4.66, 4.73 (2H, AB-q, J 10.1 Hz, C2–
OCH2Ar), 6.85 (2H, d, J 8.2 Hz), 6.88 (2H, d, J 8.2 Hz), 7.09 (2H, d,
J 8.2 Hz), 7.24 (2H, d, J 8.2 Hz), 7.33 (2H, d, J 8.2 Hz), 7.45 (2H, d,
J 8.2 Hz). 13C NMR (150 MHz, CDCl3): d 20.44, 20.54, 21.09, 27.38,
27.41, 55.25, 55.30, 62.91, 66.78, 71.88, 75.29, 76.50, 78.40,
81.83, 88.36, 113.78, 113.98, 129.57, 129.63, 129.66, 129.74,
129.85, 130.50, 132.62, 137.73, 159.34, 159.50. ESIMS: m/z 707.3
[M+Na]+. HRESIMS: Calcd for C37H52O8SSiNa: 707.3044; observed,
707.3041.
benzyl)-a-D-galactopyranoside (39)
A mixture of 38 (1.60 g, 1.78 mmol), n-hexacosanoic acid
(2.83 g, 7.13 mmol, 4 equiv), EDAC (6.84 g, 35.7 mmol, 20 equiv)
and DMAP (4.36 g, 35.7 mmol, 20 equiv) in dry 1:1 THF–CH2Cl2
(260 mL) was stirred for 4.5 days at room temperature and then di-
luted with CHCl3. The solution was washed with H2O (ꢃ2) and
brine, dried over MgSO4, and filtered. The filtrate was concentrated
in vacuo to give a mixture that was chromatographed on a silica gel
column. Elution with 9:1, then 6:1 hexane–EtOAc gave 39 (1.47 g,
65%) as a wax. IR vmax(KBr): 2917, 2849, 1738, 1513, 1463 cmꢀ1. 1H
NMR (500 MHz, CDCl3): d 0.88 (3H, t, J 7.0 Hz), 0.89 (3H, t, J 7.0 Hz),
0.99 (9H, s), 1.04 (9H, s), 1.24–1.60 (70H, m, containing two 3H
singlets at 1.33 and 1.41 ppm), 2.13 (4H, m), 2.23 (2H, m), 3.60
(1H, s), 3.71 (1H, dd, J 5.7, 11.6 Hz), 3.75 (1H, dd, J 2.9, 10.0 Hz),
3.80 (3H, s), 3.81 (3H, s), 3.85 (1H, dd, J 2.3, 11.6 Hz), 3.94 (1H,
dd, J 3.4, 10.0 Hz), 4.07 (1H, dd, J 1.7, 12.4 Hz), 4.11 (1H, m), 4.19
(1H, dd, J 1.9, 2.5 Hz), 4.24 (1H, dd, J 5.9, 7.8 Hz), 4.47 (1H, d, J
2.7 Hz), 4.60, 4.72 (2H, AB-q, J 11.5 Hz), 4.62, 4.64 (2H, AB-q, J
11.5 Hz), 4.77 (1H, d, J 3.4 Hz, anomeric H), 4.98 (1H, m), 6.84
(2H, d, J 8.6 Hz), 6.87 (2H, d, J 8.6 Hz), 7.28 (2H, d, J 6.8 Hz), 7.34
(2H, d, J 6.8 Hz). ESIMS: m/z 1297.9 [M+Na]+. HRESIMS: Calcd for
4.1.30. 4,6-O-Di-tert-butylsilylidene-2,3-di-O-(4-
methoxybenzyl)-D-galactopyranose (37)
To a solution of 35 (8.22 g, 12.3 mmol) in acetone (240 mL) was
added NBS (2.65 g, 14.9 mmol) at ꢀ20 °C. After stirring for 40 min,
the reaction was quenched with satd aq NaHCO3 (60 mL) at ꢀ20 °C
and then concentrated in vacuo to give a mixture that was diluted
with EtOAc. The solution was washed with 10% aq Na2S2O3, satd aq
C77H130O12SiNa, 1297.9224; observed, 1297.9226.

1678
M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
4.1.33. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-isopropylidene-11-
octadecynyl 4,6-O-di-tert-butylsilylidene-a-D-
galactopyranoside (40)
3.65–3.71 (2H, m), 3.81 (1H, m), 4.01 (1H, dd, J 6.2, 8.6 Hz), 4.64
(1H, t, J 7.0 Hz), 5.61 (1H, dd, J 8.0, 15.6 Hz), 5.81 (1H, dd, J 6.6,
15.6 Hz). EIMS: m/z 355 [MꢀtBuMe2Si]+, 395, 455 [MꢀMe]+. HRE-
IMS: Calcd for C26H51O4Si: 455.3557; observed, 455.3543. Physical
data for Z-isomer: Rf 0.46, 9:1 hexane–EtOAc; IR vmax(KBr): 3558,
To a solution of 39 (1.47 g, 1.15 mmol) in 10:1 CH2Cl2–H2O
(330 mL) was added DDQ (1.47 g, 6.48 mmol), and the solution
was stirred for 2 h at room temperature and 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 that was chromatographed on a silica gel
column. Elution with 4:1, then 2:1 hexane–EtOAc gave 40 (933 mg,
78%) as a wax. IR vmax(KBr): 3442, 2925, 2854, 1739, 1468,
2927, 2856, 1465 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.09 (6H, s),
.
0.88 (3H, t, J 6.8 Hz), 0.90 (9H, s), 1.25 (18H, br s), 1.36 (3H, s),
1.36–1.41 (2H, m), 1.45 (3H, s), 2.07–2.20 (2H, m), 2.46 (1H, d, J
4.8 Hz, OH), 3.66–3.71 (2H, m), 3.81 (1H, dd, J 6.0, 12.8 Hz), 4.02
(1H, dd, J 6.4, 8.4 Hz), 5.01 (1H, m), 5.54 (1H, dd, J 9.6, 11.0 Hz),
5.71 (1H, td, J 7.4, 11.0 Hz). EIMS: m/z 355 [MꢀtBuMe2Si]+, 395,
455 [MꢀMe]+. HREIMS: Calcd for C26H51O4Si: 455.3557; observed,
455.3572.
1168 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.88 (3H, t, J 7.0 Hz),
.
0.89 (3H, t, J 7.0 Hz), 1.02 (9H, s), 1.05 (9H, s), 1.25 (54H, br s),
1.30–1.60 (16H, m, containing two 3H singlets at 1.33 and
1.41 ppm), 2.13 (4H, t, J 6.8 Hz), 2.26–2.30 (2H, m), 2.36 (1H, d, J
10.1 Hz, OH), 2.53 (1H, d, J 10.5 Hz, OH), 3.67 (1H, dt, J 3.2,
10.2 Hz), 3.72 (1H, dd, J 5.8, 11.4 Hz), 3.76 (1H, s), 3.79 (1H, dt, J
3.8, 9.9 Hz), 4.00 (1H, dd, J 2.1, 11.4 Hz), 4.12–4.18 (3H, m), 4.27
(1H, dd, J 2.2, 12.4 Hz), 4.44 (1H, d, J 3.2 Hz), 4.91 (1H, d, J 3.7 Hz,
anomeric H), 5.02 (1H, m). ESIMS: m/z 1057.8 [M+Na]+. HRESIMS:
Calcd for C61H114O10SiNa, 1057.8073; observed, 1057.8068.
4.1.36. (2S,3S,4R)-1-tert-Butyldimethylsilyloxy-3,4-O-
isopropylideneoctadecan-2-ol (43)
A solution of a 1:1 mixture of 42E and 42Z (500 mg, 1.06 mmol)
in EtOAc (20 mL) was stirred for 1 h at room temperature under
hydrogen, using 20% Pd(OH)2-on-carbon (200 mg) as a catalyst.
The reaction mixture was filtered, and the filtrate was concen-
trated in vacuo to give a residue that was chromatographed on a
silica gel column. Elution with 19:1 hexane–EtOAc gave 43
(478 mg, 95%). IR vmax(KBr): 3535 (br), 2925, 2856, 1517, 1464
4.1.34. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-isopropylidene-11-
octadecynyl
a-D
-galactopyranoside (41)
cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.09 (6H, s), 0.88 (3H, t, J
.
To a solution of 40 (932 mg, 0.900 mmol) in dry THF (90 mL)
were added pyridine (750 mg, 9.48 mmol) and HFꢁpyridine (70%
HF in pyridine, 360 mg), and the solution was stirred for 1 h at
room temperature. The reaction mixture was diluted with CHCl3,
washed with satd aq NaHCO3 and brine, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a mixture that
was chromatographed on a silica gel column. Elution with 2:3 hex-
ane–EtOAc, EtOAc, and finally 19:1 EtOAc–MeOH gave 41 (720 mg,
89%) as a wax. IR vmax(KBr): 3419 (br), 2922, 2852, 1739, 1464,
6.8 Hz), 0.91 (9H, s), 1.25 (23H, br s), 1.32 (3H, s), 1.40 (3H, s),
1.52–1.59 (2H, m), 1.72 (1H, m), 3.64–3.70 (2H, m), 3.82 (1H, m),
3.91 (1H, dd, J 5.6, 8.8 Hz), 4.17 (1H, m). EIMS: m/z 457 [MꢀMe]+.
HREIMS: Calcd for C26H53O4Si: 457.3713; observed, 457.3717.
4.1.37. (2S,3R,4R)-1,2-Di-tert-butyldimethylsilyloxy-3,4-O-
isopropylidene-octadecane (44)
(1) To a solution of 43 (180 mg, 0.381 mmol) in CH2Cl2 (10 mL)
were added 2,6-lutidine (204 mg, 1.90 mmol, 5 equiv) and TBDM-
SOTf (302 mg, 1.14 mmol, 3 equiv). After stirring for 1 h at room
temperature, the reaction mixture was diluted with CH2Cl2,
washed with satd aq NaHCO3 and brine, dried over MgSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a residue that
was chromatographed on a silica gel (7 g) column. Elution with
19:1 hexane–EtOAc gave 44 (221 mg, 99%). IR vmax(KBr): 2927,
1264 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.88 (3H, t, J 7.0 Hz),
.
0.89 (3H, t, J 7.1 Hz), 1.25 (54H, br s), 1.31–1.62 (16H, m, containing
two 3H singlets at 1.34 and 1.43 ppm), 2.13 (4H, t, J 7.1 Hz), 2.27–
2.33 (2H, m), 2.34 (1H, br s, OH), 2.39 (1H, d, J 10.2 Hz, OH), 2.70
(1H, br s, OH), 2.92 (1H, br s, OH), 3.71 (1H, dd, J 5.8, 11.4 Hz),
3.75–3.80 (2H, m), 3.82–3.87 (2H, m), 3.95 (1H, m), 4.04 (1H, dd,
J 2.1, 11.4 Hz), 4.10 (1H, s), 4.13–4.19 (2H, m), 4.93 (1H, d, J
3.4 Hz, anomeric H), 5.04 (1H, m). ESIMS: m/z 917.7 [M+Na]+. HRE-
SIMS: Calcd for C53H98O10Na, 917.7052; observed, 917.7053.
2856 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.06 (6H, s), 0.09 (3H,
.
s), 0.11 (3H, s), 0.87 (9H, s), 0.88 (3H, t, J 7.1 Hz), 0.91 (9H, s),
1.25–1.40 (29H, m, containing 3H, s, at 1.31 ppm), 1.40 (3H, s),
3.68 (1H, m), 3.78–3.80 (2H, m), 4.06–4.07 (2H, m). EIMS: m/z
571 [MꢀMe]+. HREIMS: Calcd for C32H67O4Si2: 571.4578; observed,
571.4566.
(2) To a solution of 46 (1.62 g, 4.52 mmol) and 2,6-lutidine
(3.88 g, 36.23 mmol) in CH2Cl2 (80 mL) was added TBDMSOTf
(5.99 g, 22.7 mmol) at 0 °C. After stirring for 1 h at room tempera-
ture, the reaction mixture was diluted with CH2Cl2. The solution
was washed with satd aq NaHCO3, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo to give a residue that was
chromatographed on a silica gel column. Elution with 29:1 hex-
ane–EtOAc gave 44 (2.62 g, 99%).
4.1.35. (2S,3S,4R,5E)-1-tert-Butyldimethylsilyloxy-3,4-O-
isopropylidene-5-octadecen-2-ol (42E) and (2S,3S,4R,5Z)-1-tert-
Butyldimethylsilyloxy-3,4-O-Isopropylidene-5-octadecen-2-ol
(42Z)
To a solution of n-tridecyltriphenylphosphonium bromide21
(12.3 g, 23.4 mmol, 3 equiv) in dry THF (25 mL) was added a solu-
tion of n-BuLi (1.59 M in hexane, 14.7 mL, 23.4 mmol) at ꢀ10 °C
under argon with stirring. After stirring for 30 min at ꢀ10 °C, to
this red-colored solution was added a solution of 5-O-tert-butyldi-
methylsilyl-2,3-O-isopropylidene-b-L-ribofuranose (20, 2.37 g,
7.79 mmol) in THF (20 mL). This mixture was stirred for 3 h at
room temperature, and the reaction was quenched with MeOH.
The mixture was concentrated in vacuo and diluted with EtOAc.
The solution was washed with water and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a resi-
due that was chromatographed on a silica gel column. Elution with
19:1 hexane–EtOAc gave a 1:1 mixture of E- and Z-isomers (42E
and 42Z) (3.17 g, 86%). This mixture (20 mg) was separated to each
isomer using a silica gel (4 g) column. Physical data for E-isomer: Rf
0.42, 9:1 hexane–EtOAc; IR vmax(KBr): 3420, 2925, 2854, 1465
4.1.38. (2S,3R,4R)-2-tert-Butyldimethylsilyloxy-3,4-O-
isopropylideneoctadecan-1-ol (45) and (2S,3S,4R)-3,4-O-
isopropylideneoctadecan-1,2-diol (46)
(1) To a solution of 44 (6.41 g, 10.9 mmol, derived from L-ribose)
in pyridine (48 mL) and dry THF (80 mL) was added dropwise
HFꢁpyridine (16 mL; HF: ꢄ70%, pyridine: ꢄ30%) at 0 °C with stir-
ring, and the mixture was stirred for 4 h at room temperature.
The reaction mixture was diluted with EtOAc, and neutralized with
solid NaHCO3, and satd aq NaHCO3 by cautious dropwise addition.
The organic layer was separated, and washed with satd aq NaCl,
dried over MgSO4, and filtered. The filtrate was concentrated in va-
cuo to give a residue that was chromatographed on a silica gel
cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.09 (6H, s), 0.88 (3H, t, J
.
6.8 Hz), 0.91 (9H, s), 1.25 (18H, br s), 1.34 (3H, s), 1.35–1.42 (2H,
m), 1.45 (3H, s), 1.99 (2H, q, J 6.8 Hz), 2.37 (1H, d, J 4.4 Hz, OH),

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1679
column. Elution with 19:1, 4:1, and 1:1 hexane–EtOAc gave 44
(2.12 g, recovery 33%), 45 (2.34 g, 45%) and 46 (0.86 g, 22%). Phys-
ical data for 45: IR vmax(KBr): 3500 (br), 1465, 1369 cmꢀ1. 1H NMR
(500 MHz, CDCl3): d 0.11 (3H, s), 0.13 (3H, s), 0.88 (12H, br s), 1.26
(26H, br s), 1.35 (3H, s), 1.43 (3H, s), 2.26 (1H, t, J 4.5 Hz, OH), 3.72–
3.75 (3H, m), 3.83 (1H, m), 4.08 (1H, m), 4.12 (1H, m). EIMS: m/z
457 [MꢀMe]+. HREIMS: Calcd for C26H53O4Si: 457.3713; observed,
singlets at 1.30 and 1.41 ppm), 3.29 (1H, s), 3.55 (1H, m), 3.77
(1H, d, J 8.8 Hz), 3.87 (1H, m), 3.97–4.10 (6H, m), 4.27 (1H, d, J
12.0 Hz), 4.46 (1H, d, J 7.5 Hz, anomeric H), 4.71–4.77 (2H, m),
4.82, 4.94 (2H, AB-q, J 11.2 Hz), 5.46 (1H, s), 7.26–7.40 (13H, m),
7.53–7.55 (2H, m). ESIMS: m/z 925.6 [M+Na]+. HRESIMS: Calcd
for C54H82O9SiNa, 925.5620; observed, 925.5610.
457.3710. Physical data for 46: mp 44.5–45 °C; ½a D23
ꢂ
+6.2 (c 1.26,
4.1.40. (2S,3S,4R)-2-Hydroxy-3,4-O-isopropylideneoctadecyl
2,3-di-O-benzyl-4,6-O-benzylidene-a-D-galactopyranoside (50)
CHCl3): (derived from
L
-ribose); IR vmax(KBr): 3420–3280 (br),
1469, 1369, 1256, 1217 cmꢀ1
.
1H NMR (400 MHz, CDCl3): d 0.88
A solution of 48 (115 mg, 0.127 mmol) and (n-Bu)4NF (1.0 M
solution in THF, 0.6 mL) in THF (5 mL) was stirred for 50 min at
room temperature and concentrated in vacuo to give a mixture
that was diluted with EtOAc. The solution was washed with H2O
and brine, dried over MgSO4, and filtered. The filtrate was concen-
trated in vacuo to give a mixture that was chromatographed on a
silica gel column. Elution with 4:1 hexane–EtOAc gave 50
(87 mg, 87%) as a gum. IR vmax(KBr): 3511 (br), 2924, 2854, 1456,
(3H, t, J 6.8 Hz), 1.24–1.72 (32H, m, containing two 3H singlets at
1.34 and 1.41 ppm), 2.17 (1H, br s, OH), 2.39 (1H, br s, OH),
3.72–3.76 (2H, m), 3.82 (1H, m), 3.96 (1H, dd, J 5.8, 8.2 Hz), 4.20
(1H, ddd, J 3.6, 6.0, 9.6 Hz). ESIMS: m/z 381 [M+Na]+ (on addition
of NaI). HRESIMS: Calcd for C21H42O4Na: 381.2975; observed,
381.2971.
(2) To a solution of 43 (237 mg, 0.501 mmol) in THF (10 mL)
was added (n-Bu)4NF (1 M solution in THF, 1.0 mL). The solution
was stirred for 1 h at room temperature, concentrated in vacuo,
and 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 that was chromatographed on a silica gel
column. Elution with 2:1 hexane–EtOAc gave 46 (144 mg, 80%) as a
crystalline solid.
1373 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.88 (3H, t, J 6.8 Hz),
.
1.25–1.40 (28H, m, containing two 3H singlets at 1.31 and
1.38 ppm), 1.47–1.56 (3H, m), 1.69 (1H, m), 3.24 (1H, br s, OH),
3.47 (1H, dd, J 8.1, 10.8 Hz), 3.69 (1H, s), 3.80 (1H, m), 3.90 (1H,
dd, J 5.7, 9.1 Hz), 3.99–4.03 (3H, m), 4.09 (1H, dd, J 3.7, 10.0 Hz),
4.14 (1H, m), 4.21–4.24 (2H, m), 4.68, 4.88 (2H, AB-q, J 11.5 Hz),
4.74, 4.78 (2H, AB-q, J 12.0 Hz), 5.48 (1H, s), 7.27–7.42 (13H, m),
7.51–7.53 (2H, m). ESIMS: m/z 811.5 [M+Na]+. HRESIMS: Calcd
for C48H68O9Na: 811.4756; observed: 811.4750.
(3) A solution of 19 (100 mg, 0.282 mmol) derived from D-ara-
binitol in EtOAc (5 mL) containing Pd(OH)2 on carbon (20 wt %,
wet, Degussa type, 50 mg) was stirred for 3 h under hydrogen.
The mixture was filtered, and the filtrate was concentrated in va-
cuo to give residue that was chromatographed on a silica gel col-
umn. Elution with 2:1 hexane–EtOAc gave 46 (82 mg, 81%) as a
4.1.41. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-isopropylideneoc-
tadecyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranoside (51)
crystalline solid. mp 44.5–45 °C; ½a D25
ꢂ
+6.4 (c 1.51, CHCl3): (derived
Compound 50 (165 mg, 0.209 mmol) was treated as described
in the formation of 39 from 38 to give 51 (228 mg, 93%) as a
from D-arabinitol).
gum. IR vmax(KBr): 2918, 2850, 1740, 1471, 1170 cmꢀ1 1H NMR
.
4.1.39. (2S,3R,4R)-2-tert-Butyldimethylsilyloxy-3,4-O-
isopropylideneoctadecyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-
(500 MHz, CDCl3): d 0.88 (6H, t, J 6.8 Hz), 1.24–1.56 (78H, m, con-
taining two 3H singlets at 1.32 and 1.41 ppm), 2.23 (2H, m), 3.65
(1H, s), 3.72 (1H, dd, J 5.9, 11.6 Hz), 3.91 (1H, dd, J 2.4, 11.4 Hz),
3.97 (1H, dd, J 3.4, 10.0 Hz), 4.00 (1H, dd, J 1.5, 13.0 Hz), 4.06
(1H, dd, J 3.4, 10.0 Hz), 4.10 (1H, m), 4.19 (1H, d, J 8.1 Hz), 4.20
(1H, s), 4.23 (1H, dd, J 5.7, 8.1 Hz), 4.68, 4.80 (2H, AB-q, J
11.7 Hz), 4.72, 4.80 (2H, AB-q, J 12.3 Hz), 5.00 (1H, d, J 3.5 Hz, ano-
meric H), 5.02 (1H, m), 5.47 (1H, s), 7.26–7.40 (13H, m), 7.50–7.53
(2H, m). ESIMS: m/z 1189.9 [M+Na]+. HRESIMS: Calcd for
C74H118O10Na, 1189.8617; observed, 1189.8611.
D
-galactopyranoside (48) and (2S,3R,4R)-2-tert-
Butyldimethylsilyloxy-3,4-O-isopropylideneoctadecyl 2,3-di-O-
benzyl-4,6-O-benzylidene-b- -galactopyranoside (49)
D
(i) To a solution of 47 (449 mg, 1.00 mmol) and Cl3CCN (1.44 g,
10.0 mmol) in dry CH2Cl2 (10 mL) was added Cs2CO3 (480 mg,
1.47 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 a crude imidate that
was employed for the next reaction without purification.
4.1.42. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-
(ii) To a solution of imidate obtained above and 45 (355 mg,
0.750 mmol) in dry CH2Cl2 (20 mL) was added 4 Å MS (1.6 g). The
mixture was stirred for 30 min at room temperature and then
cooled to 0 °C. To this mixture was added AgOTf (200 mg,
0.778 mmol). After stirring for 30 min at 0 °C and for 2 h at room
temperature, the mixture was diluted with CH2Cl2, washed with
satd aq NaHCO3 and brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to give a mixture that was chro-
matographed on a silica gel column. Elution with 9:1, then 4:1
hexane–EtOAc gave 48 (315 mg, 46%) and 49 (246 mg, 39%). Phys-
ical data for 48: Rf 0.41, 4:1 hexane–EtOAc; IR vmax(KBr): 2925,
isopropylideneoctadecyl a-D-galactopyranoside (52)
(1) A solution of 51 (500 mg, 0.428 mmol) in THF (400 mL) con-
taining Pd(OH)2/C (20 wt % Pd-on-carbon, wet, 400 mg) was stirred
for 16 h at room temperature under hydrogen. The reaction mix-
ture was filtered, and the filtrate was concentrated to give a resi-
due that was chromatographed on a silica gel column. Elution
with 19:1 CHCl3–MeOH gave 52 (380 mg, 99%) as a powder. IR
vmax(KBr): 3367 (br), 2919, 2850, 1732, 1471 cmꢀ1 1H NMR
.
(500 MHz, CDCl3): d 0.88 (6H, t, J 6.8 Hz), 1.25 (70H, br s), 1.34
(3H, s), 1.43 (3H, s), 1.53–1.62 (2H, m), 2.26–2.35 (4H, m, contain-
ing two OH), 2.59 (1H, d, J 3.1 Hz), 2.83 (1H, s, OH), 3.71 (1H, dd, J
5.7, 11.3 Hz), 3.75–3.88 (4H, m), 3.95 (1H, m), 4.04 (1H, dd, J 1.7,
11.5 Hz), 4.10 (1H, br s), 4.16–4.18 (2H, m), 4.93 (1H, d, J 3.2 Hz,
anomeric H), 5.04 (1H, m). ESIMS: m/z 921.7 [M+Na]+. HRESIMS:
Calcd for C53H102O10Na, 921.7365; observed, 921.7357.
(2) Compound 29a (60 mg, 0.061 mmol) in THF (50 mL) con-
taining Pd(OH)2/C (20 wt % Pd-on-carbon, wet, 100 mg) was trea-
ted as described in the procedure (1) to give 52 (45 mg, 82%) as a
powder.
2854, 1457, 1367, 1249, 1218 cmꢀ1 1H NMR (500 MHz, CDCl3): d
.
0.06 (3H, s), 0.08 (3H, s), 0.85 (9H, s), 0.90 (3H, m), 1.26 (26+3H,
br s), 1.37 (3H, s), 1.52–1.58 (2H, m), 3.58 (1H, m), 3.62 (1H, br
s), 3.84 (1H, m), 3.86 (1H, m), 3.99–4.19 (7H, m), 4.68–4.84 (4H,
m), 5.01 (1H, d, J 3.5 Hz, anomeric H), 5.49 (1H, s), 7.25–7.40
(13H, m), 7.51–7.53 (2H, m). ESIMS: m/z 925.6 [M+Na]+. HRESIMS:
Calcd for C54H82O9SiNa, 925.5620; observed, 925.5612. Physical
data for 49: Rf 0.16, 4:1 hexane–EtOAc; IR vmax(KBr): 2925, 2854,
1456, 1367 cmꢀ1
.
1H NMR (500 MHz, CDCl3): d 0.13 (3H, s), 0.25
(3) Compound 41 (20 mg, 0.022 mmol) in THF (10 mL) contain-
ing Pd(OH)2/C (20 wt % Pd on carbon, wet, 15 mg) was stirred
(3H, s), 0.87 (12H, br s), 1.25–1.62 (32H, m, containing two 3H

1680
M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
under hydrogen for 2 h at room temperature, and the reaction mix-
ture was filtered. The filtrate was concentrated in vacuo to give 52
(20 mg, 99%) as a powder.
4.1.45. Methyl 2,3,4-tri-O-benzyl-6-O-methyl-a-D-galacto
pyranoside (55)
To a solution of methyl
a-D-galactopyranoside (54, 1.94 g,
10.0 mmol) in DMF (30 mL) containing MeI (1.42 g, 10 mmol)
was added NaH (60% oil dispersion, 1.0 g, 25 mmol) at ꢀ40 °C. This
mixture was stirred for 15 min at ꢀ40 °C and then for 16 h at room
temperature. The mixture was diluted with additional DMF
(20 mL), and another portion of NaH (60% oil dispersion, 2.0 g,
50 mmol) was added. BnBr (6.84 g, 40 mmol) was then cautiously
added at room temperature (exothermic). After addition of BnBr,
the mixture was warmed at 60 °C for 1 h and allowed to stand at
room temperature, and diluted with EtOAc. The solution was
washed with cold water (three times) and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a mix-
ture that was chromatographed on a silica gel column. Elution with
4:1 hexane–EtOAc gave 55 (1.34 g, 28%) and methyl 2,3,4,6-tetra-
4.1.43. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-dihydroxyoctadecyl
a-
D-galactopyranoside (31a)
(1) To a solution of 52 (50 mg, 0.056 mmol) in 1:1 CH2Cl2–
CH3CN (80 mL) were added H2O (460 mg) and 46% aq HF
(275 mg) at 25 °C. The solution was stirred for 20 min at room
temperature (25 °C). The precipitate of 31a appeared gradually.
The reaction was quenched with satd aq NaHCO3, diluted with
CHCl3, and washed with satd aq NaHCO3, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a res-
idue that was purified on a preparative TLC plate or on a silica gel
(4 g) column. Development with 7:1 CHCl3–MeOH gave 31a
(18 mg, 38%) as a powder. This compound 31a showed no UV
absorption. However, we could see the band of the product by
the difference of the density on the TLC plate through light when
the plate was wet by the developing solvent. The band was
scratched off, then charged on a column and eluted with 7:1
CHCl3–MeOH to give 31a, or elution with 19:1, then 9:1
CHCl3–MeOH for column chromatography gave 31a in 42% yield
O-benzyl-a-D
-galactopyranoside (1.36 g, 25%). 1H NMR data for
55 was identical with those reported.22 1H NMR (500 MHz, CDCl3):
d 3.28 (3H, s), 3.36 (1H, dd, J 6.3, 9.5 Hz), 3.37 (3H, s), 3.43 (1H, dd, J
6.3, 9.5 Hz), 3.85 (1H, t, J 6.3 Hz), 3.90 (1H, br s), 3.93 (1H, dd, J 2.7,
10.0 Hz), 4.04 (1H, dd, J 3.7, 10.0 Hz), 4.61, 4.95 (2H, AB-q, J
11.5 Hz), 4.69 (1H, d, J 3.7 Hz, anomeric H), 4.64, 4.83 (2H, AB-q,
J 12.0 Hz), 4.74, 4.85 (2H, AB-q, J 11.7 Hz), 7.27–7.40 (15H, m).
as a powder. ½a 2D8
ꢂ
+52.7 (c 0.69, 9:1 CHCl3–CH3OH). IR vmax(KBr):
3399 (br), 2919, 2850, 1736, 1468 cmꢀ1
.
1H NMR (600 MHz, 10:1
4.1.46. 2,3,4-Tri-O-benzyl-6-O-methyl-a-D-galactopyranose (56)
CDCl3–CD3OD) d 0.88 (6H, t, J 7.1 Hz), 1.26 (66H, br s), 1.31–1.54
(4H, m), 1.61 (2H, m), 2.32 (2H, m), 3.62 (1H, m, C4–H), 3.74 (1H,
dd, J 5.5, 11.1 Hz, C1–H), 3.75 (1H, m, C30–H), 3.77 (1H, m, C60–H),
3.79 (1H, m, C3–H), 3.82 (1H, dd, J 3.5, 7.6 Hz, C20–H), 3.84 (1H,
m, C60–H), 3.85 (1H, m, C50–H), 4.00 (1H, d, J 3.0 Hz, C40–H),
4.03 (1H, dd, J 4.0, 11.1 Hz, C1–H), 4.92 (1H, d, J 3.5 Hz, anomeric
H), 5.10 (1H, ddd, J 4.0, 5.5, 5.5 Hz, C2–H). 13C NMR (150 MHz,
10:1 CDCl3–CD3OD) d 14.14, 22.74, 24.98, 25.90, 29.24, 29.38,
29.41, 29.56, 29.72, 29.76, 31.98, 32.07, 62.44, 67.20, 69.08,
70.13, 70.33, 71.89, 72.27, 73.41, 99.38, 173.63. ESIMS: m/z
881.7 [M+Na]+. HRESIMS: Calcd for C50H98O10Na, 881.7052; ob-
served, 881.7048.
(2) A solution of 30a (20 mg, 0.038 mmol) in THF (6 mL) con-
taining Pd(OH)2/C (20 wt % Pd-on-carbon, wet, 32 mg) was stirred
for 1 h at room temperature under hydrogen. The reaction mixture
was filtered, and the catalyst was washed well with 1:1 CHCl3–
MeOH. The combined filtrate was concentrated to give a residue
that was chromatographed on a silica gel (2 g) column. Elution
with 9:1 CHCl3–MeOH gave 31a (9 mg, 44%).
(i) A solution of 55 (4.12 g, 8.61 mmol) in Ac2O (150 mL) con-
taining concd H2SO4 (0.15 mL) was stirred for 1 h at room temper-
ature and diluted with EtOAc (300 mL). The solution was washed
with satd aq NaHCO3 (three times) and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give an oil.
Toluene was added to this oil and evaporated in vacuo to remove
Ac2O. This procedure was repeated three times to give an oil.
(ii) To this oil was added MeOH (300 mL) containing a catalytic
amount of KOH (100 mg). This solution was stirred for 16 h at
room temperature and concentrated in vacuo to give a mixture
that was chromatographed on a silica gel column. Elution with
2:1, 3:2, then 1:1 hexane–EtOAc gave 56 (1.81 g, 45%) as a 3:1 mix-
ture of
a
and b anomers. IR vmax(KBr): 3408 (br), 3029, 2919, 1496,
.
1454 cmꢀ1
1H NMR (500 MHz, CDCl3): d 3.12 (0.75H, d, J 1.9 Hz,
OH), 3.24 (3H, s), 3.34–3.56 (3H, m, containing 0.25H, d, J 6.6 Hz,
at 3.42 ppm, OH), 3.75–4.13 (3.25H, m), 4.60–4.97 (6.25H, m, con-
taining 0.25H, dd, J 6.6, 7.4 Hz, at 4.67 ppm, changed to doublet, J
7.4 Hz, on addition of D2O, anomeric H), 5.30 (0.75H, m, changed
to doublet, J 3.4 Hz, on addition of D2O, anomeric H), 7.25–7.40
(15H, m). ESIMS: m/z 487.2 [M+Na]+. HRESIMS: Calcd for C28H32O6
Na: 487.2091; observed: 487.2090.
4.1.44. (2S,3S,4R)-2,3-Dihydroxy-4-(hexacosanoyloxy)octadecyl
a-
D
-galactopyranoside (53)
To a solution of 52 (193 mg, 0.215 mmol) in CHCl3 (37 mL) was
4.1.47. (2S,3S,4R)-2-Hydroxy-3,4-O-isopropylideneoctadecyl
added HCl (1 M solution in EtOAc, 3.7 mL). The solution was stirred
for 5 min at room temperature and was then concentrated to give a
residue that was chromatographed on a silica gel (4 g) column. Elu-
tion with 19:1, then 7:1 CHCl3–MeOH gave 53 (133 mg, 72%) as a
2,3,4-tri-O-benzyl-6-O-methyl-
(2S,3S,4R)-2-hydroxy-3,4-O-isopropylideneoctadecyl 2,3,4-tri-O-
benzyl-6-O-methyl-b- -galactopyranoside (58)
(i) Compound 56 (465 mg, 1.00 mmol) was treated as described
in the formation of 4 from 3 to give a crude imidate, which was
employed for the next reaction without purification.
a-D-galactopyranoside (57) and
D
powder. ½a 2D1
ꢂ
+48.8 (c 0.87, 6:1 CHCl3–MeOH). IR vmax(KBr): 3400
(br), 2920, 2850, 1713, 1469 cmꢀ1
.
1H NMR (600 MHz, 10:1
CDCl3–CD3OD): d 0.88 (6H, t, J 6.8 Hz), 1.24–1.36 (68H, m), 1.62
(2H, m), 1.68 (2H, m), 2.33 (2H, m), 3.53 (1H, dd, J 6.5, 10.6 Hz,
C1–H), 3.66 (1H, ddd, J 2.0, 6.5, 6.5 Hz, C2–H), 3.73 (1H, dd, J 5.0,
6.5 Hz, C3–H), 3.76 (1H, dd, J 3.0, 7.6 Hz, C30–H), 3.78 (1H, m,
C60–H), 3.81 (2H, m, C20–H and C50–H), 3.82 (1H, m, C60–H), 3.94
(1H, dd, J 2.0, 10.6 Hz, C1–H), 3.97 (1H, d, J 3.0 Hz, C40–H), 4.89
(1H, d, J 3.5 Hz, anomeric H), 5.07 (1H, ddd, J 5.0, 5.0, 7.6 Hz, C4–
H). 13C NMR (150 MHz, 10:1 CDCl3–CD3OD): d 14.17, 22.78,
25.19, 25.52, 28.78, 29.30, 29.42, 29.45, 29.64, 29.69, 29.80,
32.03, 34.76, 62.29, 69.41, 69.96, 70.40, 70.43, 70.89, 72.73,
74.93, 99.52, 174.58. ESIMS: m/z 881.7 [M+Na]+. HRESIMS: Calcd
for C50H98O10Na, 881.7052; observed, 881.7048.
(ii) To a solution of imidate obtained above and 46 (340 mg,
0.950 mmol) in dry CH2Cl2 (20 mL) was added 4 Å MS (1.6 g). The
mixture was stirred for 30 min at room temperature and then
cooled to 0 °C. To this mixture was added AgOTf (200 mg,
0.778 mmol). After stirring for 30 min at 0 °C and for 2 h at room
temperature, the mixture was diluted with CH2Cl2, washed with
satd aq NaHCO3 and brine, dried over MgSO4, and filtered. The fil-
trate was concentrated in vacuo to give a mixture that was chro-
matographed on a silica gel column. Elution with 9:1, then 7:1
hexane–EtOAc gave 57 (356 mg, 47%) and 58 (150 mg, 20%) as a
gum. Physical data for 57: Rf 0.60, 2:1 hexane–EtOAc; IR vmax(KBr):
3447 (br), 2925, 2854, 1496 (w), 1455, 1368, 1219 cmꢀ1 1H NMR
.

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1681
(500 MHz, CDCl3): d 0.88 (3H, t, J 6.8 Hz), 1.25 (23H, br s), 1.30 (3H,
s), 1.38 (3H, s), 1.51–1.55 (2H, m), 3.27 (3H, s), 1.68 (1H, m), 3.27
(3H, s), 3.33 (1H, dd, J 5.8, 9.4 Hz), 3.40 (1H, d, J 3.4 Hz, OH), 3.43
(1H, dd, J 6.8, 9.5 Hz), 3.57 (1H, dd, J 7.4, 10.9 Hz), 3.82–3.89 (2H,
m), 3.91–3.96 (3H, m), 4.00 (1H, t, J 6.4 Hz), 4.06 (1H, dd, J 3.7,
10.1 Hz), 4.14 (1H, m), 4.60, 4.95 (2H, AB-q, J 11.5 Hz), 4.68, 4.84
(2H, AB-q, J 11.8 Hz), 4.75, 4.82 (2H, AB-q, J 11.9 Hz), 4.87 (1H, d,
J 3.6 Hz, anomeric H), 7.27–7.40 (15H, m). ESIMS: m/z 827.5
[M+Na]+. HRESIMS: Calcd for C49H72O9Na, 827.5069; observed,
827.5056. Physical data for 58: Rf 0.46, 2:1 hexane–EtOAc. IR
gave 61 (10 mg, 52%) as a powder. [This compound 61 showed
no UV absorptions. However, we could see the band of the product
by the difference of the density on the TLC plate through light
when the plate was wet by the developing solvent. The band was
scratched off, then charged on a column, and eluted with 7:1
CHCl3–MeOH to give 61]. ½a D26
ꢂ
+54.5 (c 0.78, 12:1 CHCl3–CH3OH);
IR vmax(KBr): 3383 (br), 2919, 2850, 1734, 1468 cmꢀ1
.
1H NMR
(400 MHz, 19:1 CDCl3–CD3OD): d 0.88 (6H, t, J 6.8 Hz), 1.20–1.62
(72H, m), 2.32 (2H, t, J 7.6 Hz), 3.40 (3H, s), 3.62–3.67 (3H, m),
3.74–3.81 (2H, m), 3.81–3.88 (2H, m), 3.94–4.06 (3H, m), 4.93
(1H, d, J 4.0 Hz, anomeric H), 5.08 (1H, m). (The 2D COSY experi-
ment of compound 61 showed that the hexacosanoyl group existed
on the C2–O oxygen of the octadecane chain.) 13C NMR (125 MHz,
15:1 CDCl3–CD3OD) 14.07, 22.66, 24.90, 25.90, 29.19, 29.33, 29.35,
29.51, 29.63, 29.67, 29.70, 31.90, 34.34, 59.35, 67.29, 69.06, 70.02,
70.27, 71.79, 71.88, 72.52, 72.87, 99.24, 173.25. FABMS: m/z 679,
697, 873 [M+H]+. HRFABMS: Calcd for C51H101O10, 873.7395; ob-
served, 873.7398.
vmax(KBr): 3439 (br), 2924, 2854, 1496, 1454, 1368, 1218 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 0.88 (3H, t, J 6.8 Hz), 1.21–1.70
(32H, m, containing two 3H singlets at 1.30 and 1.40 ppm), 3.26
(3H, s), 3.37 (1H, dd, J 5.0, 8.2 Hz), 3.47–3.55 (4H, m, containing
OH), 3.68 (1H, m), 3.80–3.88 (4H, m), 4.11 (1H, m), 4.19 (1H, d, J
11.2 Hz), 4.36 (1H, d, J 8.0 Hz, anomeric H), 4.65, 4.95 (2H, AB-q,
J 11.4 Hz), 4.73, 4.76 (2H, AB-q, J 11.4 Hz), 4.82, 4.86 (2H, AB-q, J
10.8 Hz), 7.26–7.34 (15H, m). ESIMS: m/z 827.5 [M+Na]+. HRESIMS:
Calcd for C49H72O9Na, 827.5069; observed, 827.5057.
4.1.51. Methyl 5-O-tert-butyldimethylsilyl-2-O-methyl-b-L-
4.1.48.(2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-isopropylide-neoctadecyl
ribofuranoside (63)
2,3,4-tri-O-benzyl-6-O-methyl-
a
-D
-galactopyranoside (59)
A solution of methyl 2-O-methyl-b-L
-ribofuranoside 6219 (1.12
A mixture of 57 (182 mg, 0.226 mmol), n-hexacosanoic acid
(360 mg, 0.894 mmol, 4 equiv), EDAC (868 mg, 4.52 mmol,
20 equiv), and DMAP (554 mg, 4.52 mmol, 20 equiv) in dry THF–
CH2Cl2 (1:1, 24 mL) was treated as described for the preparation
of 51 from 50 to give 59 (206 mg, 77%) as a wax. IR vmax(KBr):
g, 6.29 mmol), TBDMS chloride (1.42 g, 9.43 mmol), and imidazole
(685 mg, 10.1 mmol) in CH2Cl2 (50 mL) was stirred for 45 min at
room temperature. The reaction mixture was diluted with CH2Cl2
and washed with satd aq NaHCO3 and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a resi-
due that was chromatographed on a silica gel column. Elution with
4:1, then 2:1 hexane–EtOAc gave 63 (1.55 g, 84%) as an oil. IR
2918, 2850, 1740, 1472 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.88
.
(6H, t, J 6.8 Hz), 1.20–1.60 (78H, m, containing two 3H singlets at
1.31 and 1.42 ppm), 2.19–2.28 (2H, m), 3.25 (3H, s), 3.35–3.42
(2H, m), 3.68 (1H, dd, J 5.0, 11.4 Hz), 3.85–3.97 (4H, m), 4.03 (1H,
dd, J 3.2, 11.6 Hz), 4.10 (1H, m), 4.28 (1H, dd, J 6.0, 8.0 Hz), 4.60,
4.94 (2H, AB-q, J 12.0 Hz), 4.68, 4.82 (2H, AB-q, J 11.6 Hz), 4.73,
4.75 (2H, AB-q, J 11.4 Hz), 4.90 (1H, d, J 3.2 Hz, anomeric H), 5.02
(1H, m), 7.26–7.39 (15H, m). ESIMS: m/z 1205.9 [M+Na]+. HRE-
SIMS: Calcd for C75H122O10Na, 1205.8930; observed, 1205.8928.
vmax(KBr): 3534 (br), 2930, 2860 cmꢀ1 1H NMR (400 MHz, CDCl3):
.
d 0.15 (6H, s), 0.97 (9H, s), 2.58 (1H, d, J 8.0 Hz, OH), 3.44 (3H, s),
3.58 (3H, s), 3.70 (1H, dd, J 1.2, 5.2 Hz), 3.77 (1H, dd, J 5.2,
10.8 Hz), 3.79 (1H, dd, J 4.4, 10.8 Hz), 3.99 (1H, ddd, J 4.6, 4.6,
4.6 Hz), 4.28 (1H, m, changed to a triplet on addition of D2O),
4.96 (1H, d, J 1.2 Hz). ESIMS: m/z 315.2 [M+Na]+. HRESIMS: Calcd
for C13H28O5SiNa: 315.1598; observed, 315.1598.
4.1.49. (2S,3R,4R)-2-Hexacosanoyloxy-3,4-O-
4.1.52. Methyl 3-O-benzyl-5-O-tert-butyldimethylsilyl-2-O-
isopropylideneoctadecyl 6-O-methyl-a-D-galactopyranoside (60)
methyl-b-L-ribofuranoside (64)
To a solution of 59 (50 mg, 0.042 mmol) in THF (16 mL) was
added Pd(OH)2-on-carbon (20 wt %, Degussa type, containing
ꢄ50% water, 25 mg), and the mixture was stirred for 20 h under
hydrogen at room temperature. The mixture was filtered to re-
move the catalyst. The filtrate was concentrated in vacuo to give
a residue that was chromatographed on a silica gel column. Elution
with 1:1 hexane–EtOAc and 9:1 EtOAc–MeOH gave 60 (37 mg,
To a solution of 63 (1.55 g, 5.30 mmol) and benzyl bromide
(1.36 g, 7.95 mmol) in DMF (8 mL) was added NaH (60% oil disper-
sion, 750 mg, 18.8 mmol) at 0 °C. The mixture was stirred for 3 h at
room temperature, and the reaction was quenched with ice. The
mixture was extracted with EtOAc and the solution was washed
with water and brine, dried over MgSO4, and filtered. The filtrate
was concentrated in vacuo to give a mixture that was chromato-
graphed on a silica gel column. Elution with 6:1, then 3:1 hex-
ane–EtOAc gave 64 (1.66 g, 82%) as an oil. IR vmax(KBr): 2927,
96%) as a wax. ½a 2D6
ꢂ
+40.9 (c 1.0, CHCl3): IR vmax(KBr): 3418 (br),
2919, 2851, 1732, 1470 cmꢀ1
.
1H NMR (400 MHz, CDCl3): d 0.86–
0.90 (6H, m), 1.25–1.59 (78H, m, containing two 3H singlets at
1.34 and 1.43 ppm), 2.26–2.33 (2H, m), 2.34 (1H, br s, OH), 2.56
(1H, br s, OH), 3.00 (1H, s, OH), 3.41 (3H, s), 3.67–3.75 (4H, m),
3.82 (1H, m), 3.93 (1H, t, J 4.8 Hz), 4.04 (1H, dd, J 1.8, 11.4 Hz),
4.07 (1H, br s), 4.13–4.20 (2H, m), 4.92 (1H, d, J 3.6 Hz), 5.04 (1H,
m). FABMS: m/z 837, 935 [M+Na]+ (on addition of NaI). HRFABMS:
Calcd for C54H104O10Na, 935.7527; observed, 935.7525.
2856, 1463, 1362, 1252, 1189, 1133 cmꢀ1 1H NMR (400 MHz,
.
CDCl3): d 0.07 (6H, s), 0.90 (9H, s), 3.39 (3H, s), 3.44 (3H, s), 3.59
(1H, dd, J 1.6, 4.8 Hz), 3.67 (1H, dd, J 4.4, 10.8 Hz), 3.72 (1H, dd, J
4.0, 10.8 Hz), 4.08 (1H, dd, J 4.4, 6.0 Hz), 4.13 (1H, m), 4.59, 4.62
(2H, AB-q, J 11.8 Hz), 4.90 (1H, d, J 1.6 Hz), 7.28–7.38 (5H, m).
ESIMS: m/z 405.2 [M+Na]+. HRESIMS: Calcd for C20H34O5SiNa,
405.2068; observed, 405.2067.
4.1.50. (2S,3R,4R)-3,4-Dihydroxy-2-hexacosanoyloxyoctadecyl
6-O-methyl-a-D-galactopyranoside (61)
4.1.53. 3-O-benzyl-5-O-tert-butyldimethylsilyl-2-O-methyl-
ribofuranose (65) and 3-O-Benzyl-5-O-tert-butyldimethylsilyl-
2-O-methyl- -ribopyranose (66)
(i) A solution of 64 (1.66 g, 6.53 mmol) in Ac2O (70 mL) contain-
ing concd H2SO4 (84 mg) was stirred for 1 h at room temperature
and then diluted with EtOAc (400 mL). The solution was washed
with satd aq NaHCO3 (three times) and brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give an oil.
Toluene was added to this oil and evaporated in vacuo to remove
Ac2O. This procedure was repeated three times.
L-
To a solution of 60 (20 mg, 0.022 mmol) in 1:1 CH2Cl2–MeCN
(8 mL) were added H2O (54 mg) and 46% aq HF (42 mg). After stir-
ring for 40 min at room temperature, the reaction was quenched
with excess satd aq NaHCO3, and the mixture was diluted with
CHCl3. The solution was washed with brine, dried over MgSO4,
and filtered. The filtrate was concentrated in vacuo to give a resi-
due that was chromatographed on a silica gel TLC plate (E. Merck,
60F254, thickness 0.5 mm). Development with 7:1 CHCl3–MeOH
L

1682
M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
(ii) To this oil (1.62 g) was added MeOH (300 mL) containing a
15.6 Hz), 7.28–7.34 (5H, m). ESIMS: m/z 557.4 [M+Na]+. HREIMS:
Calcd for C32H58O4SiNa: 557.3997; observed, 557.3993. Physical
data for Z-isomer of 68: Rf 0.27 (9:1 hexane–EtOAc); IR vmax(KBr):
catalytic amount of KOH (50 mg). This solution was stirred for
16 h at room temperature and concentrated in vacuo to give a mix-
ture that was chromatographed on a silica gel column. Elution with
1:2 hexane–EtOAc, then 9:1 EtOAc–MeOH gave a mixture (0.66 g,
60%) as an oil. ESIMS: m/z 277.1 [M+Na]+. HRESIMS: Calcd for
3447 (br), 2926, 2855, 1464, 1254, 1098 (br) cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 0.08 (3H, s), 0.10 (3H, s), 0.88 (3H, t, J
6.8 Hz), 0.91 (9H, s), 1.21–1.31 (20H, m), 1.96 (1H, m), 2.02 (1H,
m), 2.89 (1H, d, J 4.6 Hz, OH), 3.28 (3H, s), 3.53 (1H, dd, J 3.4,
7.5 Hz), 3.70 (1H, m, changed to dd, J 5.0, 11.2 Hz on addition of
D2O), 3.75 (1H, m, changed to dd, J 6.0, 11.2 Hz on addition of
D2O), 3.95 (1H, m), 4.13 (1H, dd, J 7.5, 10.0 Hz), 4.66, 4.75 (2H,
AB-q, J 12.0 Hz), 5.33 (1H, t, J 11.4 Hz), 5.69 (1H, m), 7.26–7.34
(5H, m). ESIMS: m/z 557.4 [M+Na]+. HREIMS: Calcd for C32H58O4S-
iNa: 557.3997; observed, 557.3992.
C
13H18O5Na: 277.1046; observed, 277.1049.
(iii) solution of the mixture obtained above (660 mg,
A
2.60 mmol), tert-butyldimethylsilyl chloride (782 mg, 5.19 mmol),
and imidazole (424 mg, 6.23 mmol) in CH2Cl2 (20 mL) was treated
as described for the preparation of 63 from 62 to give a mixture
(730 mg, 75%) of 65 (major) and 66 (minor) as an oil. This mixture
was inseparable chromatographically. The products (65 and 66)
should be estimated from the products of the next reaction. Mass
spectrum of this mixture: ESIMS: m/z 391.2 [M+Na]+. HRESIMS:
Calcd for C19H32O5Si: 391.1911; observed, 391.1971.
4.1.55. (2S,3R,4R)-3-Benzyloxy-1,2-di-tert-
butyldimethylsilyloxy-4-methoxyoctadecane (69)
(i) A mixture of 67 and 68 (850 mg, 1.59 mmol) was treated as
described for the preparation of 44 from 43 to give a 1:1 mixture of
E- and Z-isomers of (2S,3R,4R)-3-benzyloxy-1,2-di-tert-butyldi-
methylsilyloxy-4-methoxyoctadec-5-ene (591 mg, 91%). (ii) The
disilyl ether obtained above (100 mg, 0.154 mmol) in hexane
(10 mL) containing 10% Pd-on-carbon (30 mg) was hydrogenated
for 20 min at room temperature, and the mixture was filtered.
The filtrate was concentrated in vacuo to give 69 (100 mg, quant).
4.1.54. (2S,3S,4R,5EZ)-3-Benzyloxy-1-tert-
butyldimethylsilyloxy-4-methoxy-5-octadecen-2-ol (67) and
(2S,3R,4R,5EZ)-3-Benzyloxy-2-tert-butyldimethylsilyloxy-4-
methoxy-5-octadecen-1-ol (68)
To
a solution of n-tridecyltriphenylphosphonium bromide
(3.12 g, 5.94 mmol) in dry THF (8 mL) was added a solution of n-
BuLi (1.55 M in hexane, 3.9 mL, 6.05 mmol) at ꢀ10 °C under argon
with stirring. After stirring for 30 min at ꢀ10 °C, to this red-colored
solution was added a solution of a mixture of 65 and 66 (730 mg,
1.98 mmol) in THF (6 mL). This mixture was stirred for 3 h at room
temperature, and the reaction was quenched with MeOH. The mix-
ture was concentrated in vacuo, and diluted with EtOAc. The solu-
tion was washed with water and brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo to give a residue
that was chromatographed on a silica gel column. Elution with
19:1 hexane–EtOAc gave a mixture of E- and Z-isomers of 67
(722 mg, 68%) and also a mixture of E- and Z-isomers of 68
(168 mg, 16%), respectively. The E,Z-mixture of 67 (20 mg) was
separated on a silica gel TLC plate by development with 9:1 hex-
ane–EtOAc to give E-isomer of 67 (8.5 mg) and Z-isomer of 67
(8.5 mg), and also the E,Z-mixture of 68 (20 mg) was separated
on a silica gel TLC plate by development with 9:1 hexane–EtOAc
to give E-isomer of 68 (8.7 mg) and Z-isomer of 68 (7.2 mg). Phys-
ical data for E-isomer of 67: Rf 0.46 (9:1 hexane–EtOAc); IR
IR vmax(KBr): 2926, 2855, 1471, 1463, 1254, 1105 cmꢀ1 1H NMR
.
(400 MHz, CDCl3): d 0.03–0.09 (12H, m), 0.88 (3H, t, J 7.0 Hz),
0.90 (18H, s), 1.25 (24H, br s), 1.52–1.55 (2H, m), 3.36 (3H, s),
3.41 (1H, m), 3.62 (1H, t, J 4.6 Hz), 3.67 (1H, dd, J 5.2, 10.0 Hz),
3.75–3.84 (2H, m), 4.67 (2H, s), 7.26–7.33 (5H, m). ESIMS: m/z
673.5 [M+Na]+. HRESIMS: Calcd for C38H74O4Si2Na: 673.5018; ob-
served, 673.5015.
4.1.56. (2S,3R,4R)-3-Benzyloxy-2-tert-butyldimethylsilyloxy-4-
methoxyoctadecan-1-ol (70) and (2S,3S,4R)-3-Benzyloxy-4-
methoxyoctadecan-1,2-diol (71)
Compound 69 (170 mg, 0.261 mmol) was treated as described
in the formation of 45 from 44 to give 70 (85 mg, 61%) and 71
(25 mg, 23%). Physical data for 70: IR vmax(KBr): 3467 (br), 2925,
2854, 1464, 1253 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.09 (3H, s),
.
0.11 (3H, s), 0.88 (3H, t, J 6.8 Hz), 0.92 (9H, s), 1.25 (24H, br s),
1.50–1.56 (2H, m), 2.62 (1H, t, J 6.4 Hz, OH), 3.38 (1H, m), 3.40
(3H, s), 3.67–3.72 (3H, m), 3.78 (1H, m), 4.73, 4.74 (2H, AB-q, J
10.8 Hz), 7.28–7.35 (5H, m). ESIMS: m/z 559.4 [M+Na]+. HRESIMS:
Calcd for C32H60O4SiNa: 559.4153; observed, 559.4153. Physical
data for 71: IR vmax(KBr): 3421 (br), 2924, 2853, 1456, 1098 (br)
vmax(KBr): 3512 (br), 2925, 2855, 1464, 1255, 1101 (br) cmꢀ1 1H
.
NMR (400 MHz, CDCl3): d 0.056 (3H, s), 0.061 (3H, s), 0.88 (3H, t,
J 7.4 Hz), 0.90 (9H, s), 1.25 (18H, br s), 1.37–1.39 (2H, m), 2.03
(2H, q, J 6.8 Hz), 2.67 (1H, br s, OH), 3.28 (3H, s), 3.59 (2H, br s),
3.67 (1H, m), 3.76 (1H, dd, J 2.1, 10.0 Hz), 3.88 (1H, d, J 8.4 Hz),
4.58, 4.82 (2H, AB-q, J 11.4 Hz), 5.48 (1H, dd, J 8.6, 15.4 Hz), 5.74
(1H, td, J 6.8, 15.4 Hz), 7.26–7.33 (5H, m). ESIMS: m/z 557.4
[M+Na]+. HREIMS: Calcd for C32H58O4SiNa: 557.3997; observed,
557.3994. Physical data for Z-isomer of 67: Rf 0.50 (9:1 hexane–
EtOAc); IR vmax(KBr): 3488 (br), 2926, 2855, 1465, 1254, 1090
cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.88 (3H, t, J 6.8 Hz), 1.26
.
(24H, br s), 1.60–1.64 (2H, m), 2.38 (1H, br s, OH), 3.01 (1H, br s,
OH), 3.43 (3H, s), 3.47 (1H, m), 3.55 (1H, dd, J 5.1, 6.1 Hz), 3.77–
3.80 (3H, m), 4.63, 4.68 (2H, AB-q, J 11.4 Hz), 7.29–7.37 (5H, m).
ESIMS: m/z 445.3 [M+Na]+. HRESIMS: Calcd for C26H46O4Na:
445.3288; observed, 445.3287.
(br) cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.06 (6H, s), 0.88 (3H, t, J
.
6.8 Hz), 0.90 (9H, s), 1.25–1.36 (20H, m), 2.09 (1H, m), 2.15 (1H,
m), 2.67 (1H, d, J 4.8 Hz, OH), 3.30 (3H, s), 3.56 (1H, m, C2–H),
3.62 (1H, dd, J 2.8, 8.4 Hz), 3.67 (1H, dd, J 5.2, 10.4 Hz), 3.76 (1H,
dd, J 3.2, 10.0 Hz), 4.37 (1H, dd, J 2.4, 9.6 Hz), 4.59, 4.83 (2H, AB-
q, J 11.2 Hz), 5.45 (1H, dd, J 9.4, 11.2 Hz), 5.76 (1H, td, J 7.4,
11.2 Hz), 7.27–7.33 (5H, m). ESIMS: m/z 557.4 [M+Na]+. HREIMS:
Calcd for C32H58O4SiNa: 557.3997; observed, 557.3995. Physical
data for E-isomer of 68: Rf 0.31 (9:1 hexane–EtOAc); IR vmax(KBr):
4.1.57. (2S,3R,4R)-3-Benzyloxy-2-tert-butyldimethylsilyloxy-4-
methoxyoctadecyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranoside (72) and (2S,3R,4R)-3-Benzyloxy-2-tert-
butyldimethylsilyloxy-4-methoxyoctadecyl 2,3-di-O-benzyl-
4,6-O-benzylidene-b-
D-galactopyranoside (73)
(i) Trichloroacetimidoyl 2,3-di-O-benzyl-4,6-O-benzylidene-
D
-
galactopyranoside was prepared from 47 (224 mg, 0.50 mmol) as
described for the preparation of 48 and 49.
3465 (br), 2925, 2854, 1470–1457, 1256, 1105 (br) cmꢀ1
.
1H NMR
(ii) Compound 70 (200 mg, 0.37 mmol) and the imidate ob-
tained above were treated as described for the preparation of 48
and 49 from 45 and the above-mentioned imidate to give 72
(142 mg, 39%) and 73 (159 mg, 44%). Physical data for 72: Rf 0.61
(3:1 hexane–EtOAc); IR vmax(KBr): 2925, 2854, 1497, 1453, 1401,
(400 MHz, CDCl3): d 0.08 (3H, s), 0.10 (3H, s), 0.88 (3H, t, J 6.8 Hz),
0.92 (9H, s), 1.26 (18H, br s), 1.37–1.41 (2H, m), 2.05–2.11 (2H, m),
2.55 (1H, t, J 4.6 Hz, OH), 3.27 (3H, s), 3.63 (1H, t, J 4.6 Hz, OH),
3.67–3.76 (3H, m), 3.82 (1H, dd, J 5.6, 9.6 Hz), 4.71, 4.75 (2H, AB-
q, J 11.2 Hz), 5.42 (1H, dd, J 8.4, 15.6 Hz), 5.70 (1H, td, J 6.8,
1362, 1253, 1101 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.06 (3H, s),
.

M. Shiozaki et al. / Carbohydrate Research 345 (2010) 1663–1684
1683
0.08 (3H, s), 0.88 (3H, t, J 6.8 Hz), 0.89 (9H, s), 1.26 (22H, br s),
1.38–1.55 (4H, m), 3.30 (3H, s), 3.37 (1H, dd, J 5.3, 11.3 Hz), 3.45
(1H, dd, J 5.3, 9.6 Hz), 3.55–3.57 (2H, m), 3.78 (1H, dd, J 1.4,
12.4 Hz), 3.94–4.01 (3H, m), 4.07 (1H, dd, J 3.4, 10.0 Hz), 4.13
(1H, d, J 3.4 Hz), 4.59, 4.68 (2H, AB-q, J 11.2 Hz), 4.65, 4.84 (2H,
AB-q, J 11.9 Hz), 4.74, 4.79 (2H, AB-q, J 12.4 Hz), 4.91 (1H, d, J
3.4 Hz, anomeric H), 5.44 (1H, s), 7.22–7.44 (18H, m), 7.50–7.52
(2H, m). ESIMS: m/z 989.6 [M+Na]+. HRESIMS: Calcd for C59H86O9S-
iNa: 989.5933; observed: 989.5935. Physical data for 73: Rf 0.42
(3:1 hexane–EtOAc); IR vmax(KBr): 2925, 2854, 1454, 1364, 1251,
6.9 Hz), 1.25 (70H, m), 1.42–1.65 (4H, m), 2.30–2.34 (2H, m),
3.20 (1H, m), 3.38 (3H, s), 3.40–3.87 (6H, m), 3.95 (1H, dd, J 4.4,
7.3 Hz), 4.00–4.03 (2H, m), 4.90 (1H, d, J 3.6 Hz, anomeric H),
5.04 (1H, m). 13C NMR (72.5 MHz, 20:1 CDCl3–CD3OD) d 14.03,
22.62, 24.89, 25.49, 29.15–29.64, 31.86, 34.37, 57.44, 62.39,
68.28, 69.08, 69.77, 70.05, 70.39, 70.88, 72.39, 99.48, 173.43. FAB-
MS: m/z 837 [M+H]+, 895 [M+Na]+ (on addition of NaI). HRFABMS:
Calcd for C51H100O10Na, 895.7214; observed, 895.7212.
4.2. Methods for measurement of biological activity
4.2.1. Bioassay (mouse in vivo)23
1104 cmꢀ1 1H NMR (500 MHz, CDCl3): d 0.08 (3H, s), 0.16 (3H,
.
s), 0.88 (3H, t, J 6.8 Hz), 0.91 (9H, s), 1.25 (22H, br s), 1.45–1.58
(4H, m), 3.27 (1H, s), 3.32 (3H, s), 3.40 (1H, dd, J 5.0, 11.4 Hz),
3.56 (1H, dd, J 3.6, 9.8 Hz), 3.68 (1H, t, J 4.8 Hz), 3.75 (1H, dd, J
3.4, 9.8 Hz), 3.85 (1H, dd, J 7.7, 9.6 Hz), 3.98 (1H, dd, J 1.6,
12.4 Hz), 4.05 (1H, m), 4.07–4.11 (2H, m), 4.26 (1H, d, J 12.2 Hz),
4.44 (1H, d, J 7.9 Hz, anomeric H), 4.64 (1H, d, J 11.3 Hz),
4.71–4.77 (3H, m), 4.79, 4.95 (2H, AB-q, J 11.1 Hz), 5.48 (1H, s),
7.26–7.37 (18H, m), 7.52–7.54 (2H, m). ESIMS: m/z 989.6
[M+Na]+. HRESIMS: Calcd for C59H86O9SiNa: 989.5933; observed:
989.5936.
Stock solutions (1.0 mg/mL in DMSO) of
sized samples were diluted to 10 g/mL in Dulbecco’s phosphate
buffered saline (Sigma, Product No. D8537) just before injection
into mice. Each glycolipid solution (10 g/mL, 200 l) was admin-
aGalCer and synthe-
l
l
l
istered intravenously. Peripheral blood was collected from the
retro-orbital plexus of mice at indicated time points using hepa-
rin-coated capillary tubes (Funakoshi Pharmaceutical, Japan), and
plasma was prepared.
4.2.2. Cytokine measurement
4.1.58. (2S,3S,4R)-3-Benzyloxy-2-hydroxy-4-methoxyoctadecyl
2,3-di-O-benzyl-4,6-O-benzylidene-a-D-galactopyranoside (74)
Compound 72 (120 mg, 0.124 mmol) was treated as described
in the formation of 50 from 48 to give 74 (85 mg, 80%) as a gum.
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
IR vmax(KBr): 3482, 2924, 2854, 1498, 1453, 1401, 1369, 1345 cm
ꢀ1
.
1H NMR (500 MHz, CDCl3): d 0.88 (3H, t, J 6.8 Hz), 1.26 (23H,
Acknowledgments
br s), 1.40–1.62 (3H, m), 3.34 (1H, d, J 2.7 Hz, OH), 3.38 (3H, s),
3.43 (1H, m), 3.48 (1H, dd, J 8.4, 10.5 Hz), 3.56 (1H, dd, J 3.8,
6.7 Hz), 3.68 (1H, s), 3.87 (1H, m), 3.97–4.03 (3H, m), 4.07 (1H,
dd, J 3.7, 10.0 Hz), 4.19–4.20 (2H, m,), 4.62, 4.87 (2H, AB-q, J
11.5 Hz), 4.67, 4.70 (2H, AB-q, J 11.7 Hz), 4.74, 4.78 (2H, AB-q, J
12.0 Hz), 4.96 (1H, d, J 3.4 Hz, anomeric H), 5.47 (1H, s), 7.25–
7.41 (18H, m), 7.51–7.53 (2H, m). ESIMS: m/z 875.5 [M+Na]+. HRE-
SIMS: Calcd for C53H72O9Na, 875.5069; observed, 875.5074.
We thank Dr. Takemichi Nakamura and Dr. Yayoi Hongo, The
Institute of Physical and Chemical Research (RIKEN), for measure-
ments of high-resolution mass spectrum.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.05.003.
4.1.59. (2S,3R,4R)-3-Benzyloxy-2-hexacosanoyloxy-4-
methoxyoctadecyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranoside (75)
References
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.
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7.6 Hz), 3.24 (1H, q, J 5.4 Hz), 3.33 (3H, s), 3.62–3.64 (2H, m),
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ꢂ
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