Synthesis of lipid a analogues containing glucose instead of glucosamine and their LPS-antagonistic activities

Article abstract of DOI:10.1016/j.tet.2005.03.034

Lipid A analogues containing glucose in substitution for glucosamine on the reducing end were synthesized, and the inhibitory activities on LPS-induced TNFα production (LPS-antagonistic activity) in vitro using human whole blood cells were measured. The I

Full text of DOI:10.1016/j.tet.2005.03.034

Tetrahedron 61 (2005) 5101–5122
Synthesis of lipid A analogues containing glucose instead of
glucosamine and their LPS-antagonistic activities
b
a
b
c
Masao Shiozaki,a, Yukiko Watanabe, Yuji Iwano, Toshio Kaneko, Hiromi Doi,
*
Daisuke Tanaka,c Takaichi Shimozatoc and Shin-ichi Kurakatac
aChemistry Department, Chemtech Labo Inc., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
bMedicinal Chemistry Research Laboratories, Sankyo Co., Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
cBiological Research Laboratories, Sankyo Co., Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
Received 24 January 2005; accepted 4 March 2005
Available online 13 April 2005
Abstract—Lipid A analogues containing glucose in substitution for glucosamine on the reducing end were synthesized, and the inhibitory
activities on LPS-induced TNFa production (LPS-antagonistic activity) in vitro using human whole blood cells were measured. The IC50
values (nM) of these ten compounds, 8, 14, 21, 31, 40, 51, 57, 62, 67 and 72, were 11.2, 15.4, 2.7, 0.1, 0.4, 1.3, 3.2, 3.2, 1.4 and 14.4,
respectively. And also inhibitory activities (ID50) on TNFa production toward galactosamine loaded C3H/HeN mice in vivo of compounds
21, 31, 57, 62 and 67 were measured. The values of these compounds were 0.29, 0.50, 0.61, not dose-dependent and 0.33 mg/kg, respectively.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The study of endotoxin has proceeded extensively1 since
Shiba and Kusumoto’s2 total synthesis of lipid A, a toxic
component of endotoxin (lipopolysaccharide, LPS) existing
in the outer surface membrane of Gram-negative bacteria.
On the contrary, a nontoxic natural lipid A-related
compound (RsDPLA)3 was isolated from Rhodobacter
sphaeroides by an Eisai group. This compound unlike
lipid A has a unique structural feature, that is, it contains two
amides composed of an unsaturated fatty acid ((R)-3-(7-
tetradecenoyloxy)tetradecanoic acid) and a 3-oxotetradeca-
noic acid in its long fatty acid chains, and shows LPS-
agonistic activity toward neither human nor mouse
macrophages.4 Furthermore, the Eisai group found that
many RsDPLA-related compounds having an olefinic
double bond in their molecules behave as LPS antagonists
toward human and murine macrophages,4 and E5564,5 a
compound related to RsDPLA, has been developed as a
highly potent anti-septicemia drug (Fig. 1).
The active structures of all natural lipid A- and also
RsDPLA-related compounds are constructed with an
b(1-6)-linked glucosamine–glucosamine disaccharide
moiety, and the configuration of the anomeric phosphate
Keywords: LPS-antagonist; RsDPLA.
*
Corresponding author. Tel.: C81 3 3492 3131; fax: C81 3 5436 8581;
e-mail: shioza@shina.sankyo.co.jp
Figure 1. Structures of Lipid A, RsDPLA, and E5564.
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.034

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Scheme 1. Reagents and conditions: (a) tetradecyl methanesulfonate, NaH, DMF, rt, 16 h, 75%; (b) RuO2.xH2O, NaIO4, MeCN–CCl4–H2O (2:2:3), rt, 3 h,
then allyl bromide, Et3N, DMF, rt, 16 h, 72%; (c) AcOH–H2O (4:1), 65 8C, 2 h, 71%; (d) TMSOTf, MS 4A, CH2Cl2, K40 8C, 1 h, 43%; (e) (1) Zn, AcOH–
THF (1:1), rt, 4 h; (2) (Z)-11-octadecenoyl chloride, NaHCO3, THF–H2O (5:1), rt, 30 min, two steps 77%; (f) (PPh3)4Pd, PPh3, Et3N, HCOOH, THF, under N2,
55 8C, 4 h, 95%.
Scheme 2. Reagents and conditions: (a) RuO2 hydrate, NaIO4, MeCN–CCl4–H2O (2:2:3), rt, 3 h, then allyl bromide, Et3N, DMF, rt, 16 h, 69%; (b) AcOH–
H2O (4:1), 65 8C, 2 h, 60%; (c) 5, TMSOTf, MS 4A, CH2Cl2, K40 8C, 1 h, 45%; (d) (1) Zn, AcOH–THF (1:1), rt, 4 h; (2) (Z)-11-octadecenoyl chloride,
NaHCO3, THF–H2O (5:1), rt, 30 min, 67%; (e) (PPh3)4Pd, PPh3, Et3N–HCOOH, THF, under N2, 55 8C, 4 h, 55%.

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5103
Scheme 3. Reagents and conditions: (a) OsO4, NaIO4, THF–H2O (7:2), rt, 3 h, then NaBH4, EtOH, rt, 20 min, quenched with AcOH, 73%; (b) i-
Pr2NP(OCH2CH]CH2)2, 1H-tetrazole, CH2Cl2, rt, 30 min, then, aq 30% H2O2, THF–CH2Cl2, rt, 15 min, 99%; (c) p-TsOH, MeOH, rt, 2 h, 96%; (d) 5,
AgOTf, TMSOTf, MS 4A, under N2, CH2Cl2, rt, 16 h, 60%; (e) (1) Zn, AcOH–THF (1:1), rt, 3.5 h; (2) (Z)-11-octadecenoyl chloride, NaHCO3, THF–H2O
(5:1), rt, 2 h, two steps 75%; (f) (PPh3)4Pd, PPh3, Et3N–HCOOH, THF, under N2, 55 8C, 16 h, 45%.
of the reducing glucosamine part is a without exception.
We were interested in the structures of RsDPLA and E5564,
and synthesized some E5564-related compounds, which
were replaced with glucose analogues instead of the
glucosamine at the reducing end, to examine the LPS-
antagonistic activity. At this time, we fixed the nonreducing
glucosamine end to that of E5564 except for compound
31, and also we restricted the anomeric substituents of
the reducing glucose end to five structural groups as the
a-carboxymethyl, a-2-(phosphonooxy)ethyl, a-2-(phospho-
no)ethyl, a-3-(phosphonooxy)propyl and a-3-(phosphono)-
propyl groups such as compounds 8 and 14, and 21, 31, 40,
51 and 57, and 62, and 67, and 72, respectively.
a-carboxymethyl group at the anomeric position. For
synthesis of 8, known alcohol 16 was used as a starting
material. Compound 1 was treated with tetradecyl methane-
sulfonate using sodium hydride as a base to give 2.
Oxidative cleavage of the allyl group of 2 with ruthenium
oxide hydrate and sodium periodate gave carboxylic acid,
which was esterified with allyl bromide using triethyl amine
in N,N-dimethylformamide (DMF) to yield allyl ester 3. The
4,6-O-isopropylidene group of 3 was deprotected with
aqueous 80% acetic acid at 65 8C to yield diol 4, which was
coupled with already reported trichloromethylimidoyl 2-
deoxy-4-O-diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-
O-methyl-2-(trichloroethoxycarbonylamino)-D-glucopyra-
noside 55 at K408C using trimethylsilyl trifluoromethane-
sulfonate (TMSOTf) as a catalyst to afford b(1-6)-linked5
disaccharide 6. The 2,2,2-trichloroethoxycarbonyl group of
6 was deprotected with zinc powder in AcOH–THF (1:1) to
give amine, which was treated with (Z)-11-octadecenoyl
chloride in THF–H2O (5:1) using NaHCO3 to afford amide
7. The three allyl groups of 7 were deprotected with
2. Results and discussion
2.1. Synthesis
Firstly, we synthesized compounds 8 and 14 having an

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Scheme 4. Reagents and conditions: (a) (R)-3-methoxydecyl p-tolouenesulfonate, NaH, DMF, rt, 5 h, 77%; (b) p-TsOH, MeOH, rt, 2 h, 92%; (c) (1) t-BuOK,
DMSO, 85 8C, N2, 2 h, then H2O, 85 8C, 2 h, (2) ClCOOCH2CCl3, aq satd NaHCO3, THF, 0 8C, 30 min, 76%; (d) ClCOOCH2CH]CH2, pyridine, 0 8C,
30 min, 94%; (e) i-Pr2NP(OCH2CH]CH2)2, 1H-tetrazole, CH2Cl2, rt, 20 min, then aq. 30% H2O2, THF–CH2Cl2, 0 8C, 30 min, 86%; (f) I2, THF–H2O (5:1),
rt, 30 min, 94%.
tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4], tri-
phenylphosphine (PPh3) and triethylamine-formic acid
(Et3N–HCOOH) in THF to yield 8 (Scheme 1).
Compound 11 was coupled with imidate 5 at K40 8C
using TMSOTf to afford b(1-6)-linked disaccharide 12. The
2,2,2-trichloroethoxycarbonyl group of 12 was deprotected
with zinc powder in AcOH-THF (1:1) to give amine, which
was treated with (Z)-11-octadecenoyl chloride in THF–H2O
(5:1) using NaHCO3 to afford amide 13. The three allyl
groups of 13 were deprotected with Pd(PPh3)4, PPh3 and
Et3N–HCOOH in THF to yield 14 as mentioned above
(Scheme 2).
For the synthesis of 14, known alcohol 96 was used as a
starting material under almost the same conditions as for the
synthesis of 8 from 1. Oxidative cleavage of the allyl group
of 9 with ruthenium oxide hydrate and sodium periodate
gave carboxylic acid, which was esterified with allyl
bromide using triethyl amine in DMF to yield allyl ester
10, and the successive cleavage of 4,6-O-isopropylidene and
tert-butyldimethylsilyl groups from 10 was performed with
aqueous 80% acetic acid at 65 8C to yield triol 11.
Secondly, we synthesized compounds 21, 31, 40, 51 and 57
having an a-2-(phosphonooxy)ethyl group at the anomeric
position. Known compound 156 was used as a starting
Scheme 5. Reagents and conditions: (a) CCl3CN, Cs2CO3, CH2Cl2, rt, 1 h, then 18, TMSOTf, MS 4A, CH2Cl2, 0 8C, then rt, 16 h, 52%; (b) (1) Zn, AcOH–THF
(1:9), 24 8C, 3.5 h; (2) (Z)-11-octadecenoic acid, WSC.HCl, CH2Cl2, rt, 10 h, two steps 47%; (c) (PPh3)4Pd, PPh3, Et3N–HCOOH, THF, under N2, 55 8C, 16 h,
76%.

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5105
Scheme 6. Reagents and conditions: (a) Ac2O, pyridine, rt, 5 h, 99%; (b) (1) OsO4, NaIO4, THF–H2O (3:1), rt, 2 h; (2) NaBH4, EtOH, rt, 10 min, 71%;
(c) (1) KOH, EtOH, rt, 1.5 h; (2) i-Pr2NP(OCH2CH]CH2)2, 1H-tetrazole, CH2Cl2, rt, 20 min, then aq 30% H2O2, CH2Cl2–THF (1:1), rt, 10 min, 80%;
(d) 3-oxotetradecanoic acid, WSC HCl, CH2Cl2, rt, 40 min, 75%; (e) AcOH–H2O (v/v, 4:1), 85 8C, 1.5 h, 86%; (f) 5, AgOTf, TMSOTf, MS 4A, CH2Cl2, under
N2, rt, 16 h, 68%; (g) (1) Zn, AcOH–THF (1:1), rt, 4 h; (2) (Z)-11-octadecenoyl chloride, NaHCO3, THF–H2O (5:1), rt, 30 min, two steps 81%; (h) (PPh3)4Pd,
PPh3, Et3N–HCOOH, THF, under N2, 55 8C, 16 h, 84%.
material for the synthesis of 21. The allyl group of 15
was converted to aldehyde by OsO4–NaIO4 oxidation in
THF–H2O (7:2), and the aldehyde was successively reduced
to alcohol 16 using NaBH4 in EtOH. Treatment of 16
with diallyl diisopropylphosphoramidite and 1H-tetrazole in
THF yielded phosphite, which was contiguously oxidized to
phosphate 17 by use of H2O2. The 4,6-O-isopropylidene
protecting group of 17 was cleaved using p-toluenesulfonic
acid monohydrate in MeOH to give diol 18. Compound 18
was coupled with imidate 5 at room temperature using
AgOTf and TMSOTf to afford b(1-6)-linked disaccharide
19. In this coupling, we found that the use of one equivalent
of AgOTf and catalytic amount of TMSOTf at room
temperature improved the yield comparing with the known
method of single use of TMSOTf at K78wK40 8C. When
TMSOTf reagent was not used as a catalyst, excess AgOTf
(about 2 equiv) was needed in the formation of 60 from 5
and 59 as described later. The 2,2,2-trichloroethoxy-
carbonyl group of 19 was deprotected with zinc powder in
AcOH–THF (1:1) to give amine, which was treated with
(Z)-11-octadecenoyl chloride in THF–H2O (5:1) using
NaHCO3 to afford amide 20 according to almost the same
procedure as for the synthesis of 7 from 6. The three allyl
groups of 20 were deprotected with Pd(PPh3)4, PPh3 and
Et3N–HCOOH in THF to yield 21 as mentioned above
(Scheme 3).
Compound 227 was used as a starting material for the
synthesis of 31. Alcohol 22 was reacted with (R)-3-
methoxydecyl p-toluenesulfonate in DMF using NaH to
yield 23, which was treated with p-toluenesulfonic acid in
methanol to afford diol 24. Double bond isomerization5 of
24 was performed with potassium tert-butoxide in dimethyl-
sulfoxide (DMSO) at 85 8C for 2 h, and hydrolysis of C2
trifluoroacetamide to yield amino-free (Z)-vinyl ether,
which was successively protected with 2,2,2-trichloroethyl
chloroformate in aqueous THF using NaHCO3 to give 25.
Treatment of diol 25 with allyl chloroformate using pyridine
gave 26 being protected by the 6-O-allyloxycarbonyl group.
The remaining C5 secondary alcohol of 26 was treated with
diallyl diisopropylphosphoramidite and 1H-tetrazole, and
subsequent oxidation of the generated phosphite with aq
30% H2O2 yielded phosphate 27. Oxidative cleavage of the
vinyl ether group of 27 was accomplished by iodine in
THF–H2O to yield 28. After the activation of the anomeric
alcohol of 28 with trichloroacetonitrile using cesium
carbonate the generated imidoyl compound was coupled
with diol 18 using TMSOTf as a condensing catalyst to

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M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
Scheme 7. Reagents and conditions: PMBZ4-methoxybenzyl; (a) PhCH(OMe)2, cat. p-TsOH, DMF, rt, 16 h, 69%; (b) (R)-3-(4-methoxybenzyloxy)tetradecyl
methanesulfonate, NaH, DMF, rt, 6 h, 67%; (c) (1) OsO4, NaIO4, acetone–H2O (4:1), rt, 4 h, (2) NaBH4, EtOH, rt, 1 h, 64%; (d) i-Pr2NP(OCH2CH]CH2)2,
1H-tetrazole, THF, rt, 4 h, then aq 30% H2O2, 0 8C, 1 h, 96%; (e) DDQ, CH2Cl2–H2O (10:1), rt, 1.5 h, 90%; (f) PCC, CH2Cl2, rt, 5 h, 93%; (g) aq 80% AcOH,
65 8C, 2 h, 92%; (h) 5, cat. TMSOTf, MS 4A, CH2Cl2, N2, 0 8C, 1 h, 59%; (i) Zn, AcOH–THF (1:9), rt, 4 h, then (Z)-11-octadecenoic acid, WSC.HCl, CH2Cl2,
rt, 10 h, 56%; (j) (PPh3)4Pd, PPh3, Et3N–HCOOH, THF, N2, 55 8C, 4 h, 90%.
afford disaccharide 29. The 2,2,2-trichloroethoxycarbonyl
group of 29 was deprotected with zinc powder in
AcOH–THF (1:9) to give amine, which was treated with
(Z)-11-octadecenoic acid in CH2Cl2 using 1-[3-(dimethyl-
with NaBH4 in EtOH to give alcohol 34, which was
deacetylated by a catalytic amount of KOH in EtOH and the
primary alcohol was selectively reacted with diallyl
diisopropylphosphoramidite and 1H-tetrazole, and continu-
ous oxidation of generated phosphite with aq 30% H2O2 to
yield phosphate 35. Esterification of C2 alcohol 35 with
3-oxotetradecanoic using WSC$HCl as a dehydrating agent
gave 36. Treatment of 36 with aq 80% AcOH at 85 8C for
1.5 h yielded diol 37, which was coupled with imidate 5
using silver trifluoromethanesulfonate (AgOTf) and
TMSOTf as condensing catalysts to afford disaccharide
38. The 2,2,2-trichloroethoxycarbonyl group of 38 was
replaced with (Z)-11-octadecenoyl group using zinc powder
in AcOH–THF (1:1) and then (Z)-11-octadecenoyl chloride
in THF–H2O (5:1) using NaHCO3 to afford amide 39 in
accordance with the same procedure as that to convert 6 to
7. Five allyl groups of 39 were deprotected with Pd(PPh3)4,
PPh3 and Et3N–HCOOH in THF to yield 40 as mentioned
above (Scheme 6).
amino)propyl]-3-ethylcarbodiimide
hydrochloride
(WSC$HCl) as a dehydrating agent to afford amide 30 in
accordance with the same procedure as that to convert 49 to
50 as described later. The five allyl groups of 30
were deprotected with Pd(PPh3)4, PPh3 and Et3N–
HCOOH in THF to yield 31 as mentioned above (Schemes 4
and 5).
Compound 326 was used as a starting material for the
synthesis of 40. The C2 alcohol of 32 was acetylated with
acetic anhydride–pyridine to give 33. This protection was
necessary for OsO4 oxidation, because alcohol 32 did not
react with OsO4 under the same conditions as for the
synthesis of 34 from 33. The allyl group of 33 was treated
with OsO4–NaIO4 in THF–H2O, and subsequent reduction

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
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Scheme 8. Reagents and conditions: (a) OsO4, NaIO4, THF–H2O (7:2), rt, 2 h, and then NaBH4, EtOH, 0 8C, 20 min, 41%; (b) i-Pr2NP(OCH2CH]CH2)2,
1H-tetrazole, CH2Cl2, rt, 30 min, then aq 31% H2O2, rt, 15 min, 97%; (c) p-TsOH.HCl, MeOH, rt, 2 h, 84%; (d) 5, AgOTf, TMSOTf, MS 4A, N2, rt, 16 h, 67%;
(e) Zn, AcOH–THF (1:1), rt, 4 h, and then (Z)-11-octadecenoyl chloride, NaHCO3, THF–H2O (5:1), rt, 2 h, 82%; (f) Pd(PPh3)4, PPh3, Et3N–HCOOH, N2,
55 8C, 16 h, 67%.
Alcohol 416 was used as a starting material for the synthesis
of 51. Treatment of 41 with benzaldehyde dimethyl acetal
using p-TsOH monohydrate gave benzylidene 42, which
was treated with (R)-3-(4-methoxybenzyloxy)tetradecyl
methanesulfonate using sodium hydride to give 43.
Oxidative cleavage of the allyl group of 43 with OsO4–
NaIO4 in acetone–H2O, and subsequent reduction of the
generated aldehyde with NaBH4 gave alcohol 44, which
reacted with diallyl diisopropylphosphoramidite using
1H-tetrazole as an acid catalyst, and continuous oxidation
of generated phosphite with aq 30% H2O2 to yield
phosphate 45. Oxidative cleavage of the p-methoxybenzyl
group of the alcohol protective group of 45 with 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in CH2Cl2–
H2O gave alcohol 46. The alcohol on the branched chain of
46 was oxidized with pyridinium chlorochromate (PCC) to
afford ketone 47. The 4,6-O-benzylidene group of 47
was deprotected with aq 80% AcOH at 60 8C to yield
diol 48. Treatment of 48 and imidate 5 at 0 8C
using TMSOTf afforded b(1-6)-linked disaccharide 49.
The 2,2,2-trichloroethoxycarbonyl group of 49 was
deprotected with zinc powder in AcOH–THF (1:9) to
give amine, which was treated with (Z)-11-octadecenoic
acid using WSC$HCl as a dehydrating agent to afford
amide 50. The four allyl groups of phosphate 50 were
deprotected with Pd(PPh3)4, PPh3 and Et3N–HCOOH in
THF to yield 51 as mentioned in the formation of 8 from 7
(Scheme 7).
Compound 96 was used as a starting material for the
synthesis of 57. The allyl group of 9 was converted to
aldehyde by OsO4–NaIO4 oxidation in THF–H2O (7:2), and
the aldehyde was continuously reduced to alcohol 52 using
NaBH4 in EtOH. Treatment of 52 with diallyl diisopropyl-
phosphoramidite and 1H-tetrazole in THF yielded phos-
phite, which was immediately oxidized to phosphate 53
using aq 31% H2O2. The 4,6-O-isopropylidene and tert-
butyldimethylsilyl protecting groups of 53 were cleaved by
using p-TsOH monohydrate in MeOH to give triol 54.
Compound 54 was coupled with imidate 5 at room
temperature using AgOTf and catalytic amount of TMSOTf
to afford b(1-6)-linked disaccharide 55. The 2,2,2-trichloro-
ethoxycarbonyl group of 55 was deprotected with zinc
powder in AcOH–THF (1:1) to give amine, which was
treated with (Z)-11-octadecenoyl chloride in THF–H2O
(5:1) using NaHCO3 to afford amide 56. Three allyl groups
of 56 were deprotected with Pd(PPh3)4, PPh3 and Et3N–
HCOOH in THF to yield 57 according to almost the same
procedure as for the synthesis of 21 from 20 (Scheme 8).
Thirdly, we synthesized compound 62 having an a-2-
(phosphono)ethyl group at the anomeric position.

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M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
Scheme 9. Reagents and conditions: (a) (1) CBr4, PPh3, CH2Cl2, rt, 2 h, 99%; (b) (1) P(OCH2CH]CH2)3, 180 8C, 3 h, (2) aq. 80% AcOH, 60 8C, 1 h, two steps
60%; (c) 5, AgOTf, CH2Cl2, rt, 1 h, 74%; (d) (1) Zn, AcOH–THF (1:8), rt, 3 h; (2) (Z)-11-octadecenoic acid, WSC.HCl , rt, 20 h, two steps 52%; (e) (PPh3)4Pd,
PPh3, Et3N–HCOOH, THF, under N2, 55 8C, 4 h, 82%.
Compound 16 was used as a starting material for the
synthesis of 62. The alcohol of 16 was brominated with
carbon tetrabromide and triphenylphosphine to give 58.
Treatment of 58 with triallylphosphite at 180 8C for 3 h, and
then aq 80% AcOH at 60 8C for 1 h gave diol 59, which was
coupled with imidate 5 at room temperature using two
equivalents of AgOTf to afford b(1-6)-linked disaccharide
60. The 2,2,2-trichloroethoxycarbonyl group of 60 was
deprotected with zinc powder in AcOH–THF (1:8) to give
amine, which was treated with (Z)-11-octadecenoic acid in
CH2Cl2 using WSC$HCl as a dehydrating agent to afford
amide 61 as mentioned in the formation of 50 from 49. The
four allyl groups of 61 were deprotected with Pd(PPh3)4,
PPh3 and Et3N–HCOOH in THF to yield 62 (Scheme 9).
The four allyl groups of 66 were deprotected with Pd(PPh3)4
and Et3N–HCOOH in THF to yield 67 (Scheme 10).
Finally, we synthesized compound 72 having an a-3-
(phosphono)propyl group at the anomeric position. The
alcohol of 63 was brominated with CBr4 and PPh3 to give
68. Treatment of 68 with triallylphosphite at 180 8C for 3 h,
and then aq 80% AcOH at 60 8C for 1 h gave diol 69, which
was coupled with imidate 5 at 0 8C using TMSOTf to afford
b(1-6)-linked disaccharide 70 as mentioned for the
formation of 49 from 48. The 2,2,2-trichloroethoxycarbonyl
group of 70 was deprotected with zinc powder in AcOH–
THF (1:1) to give amine, which was treated with (Z)-11-
octadecenoyl chloride in THF–H2O (5:1) using NaHCO3 to
afford amide 71 as mentioned in the formation of 7 from 6.
The four allyl groups of 71 were deprotected with
Pd(PPh3)4, PPh3 and Et3N–HCOOH in THF to yield 72
(Scheme 11).
Fourthly, we synthesized compounds 67 having an a-3-
(phosphonooxy)propyl group at the anomeric position, and
156 was used as a starting material. Hydroboration of the
allyl double bond of 15 with 9-borabicyclo[3.3.1]nonane (9-
BBN), and subsequent oxidation of the borane compound
with aq 30% H2O2 and aq 3 M NaOH gave alcohol 63. After
the alcohol of 63 was converted diallyl phosphate, the 4,6-
O-isopropylidene group of 63 was deprotected with aq 80%
acetic acid at 60 8C to yield diol 64, which was coupled with
imidate 5 at 0 8C using TMSOTf to afford b(1-6)-linked
disaccharide 65 as mentioned for the formation of 49 from
48. The 2,2,2-tri-chloroethoxycarbonyl group of 65 was
deprotected with zinc powder in AcOH–THF (1:1) to give
amine, which was treated with (Z)-11-octadecenoic acid
using WSC$HCl as a dehydrating agent to afford amide 66.
Thus, we could synthesize ten disaccharides (8, 14, 21, 31,
40, 51, 57, 62, 67 and 72).
2.2. Biological activity
The inhibitory activities on LPS-induced TNFa production
in vitro (LPS-antagonistic activity) of ten synthetic
compounds were investigated using human whole blood
cells.8 The IC50 values (nM) of these ten compounds, 8, 14,
21, 31, 40, 51, 57, 62, 67 and 72, toward human whole blood
cells were 11.2, 15.4, 2.7, 0.1, 0.4, 1.3, 3.2, 3.2, 1.4 and 14.4,

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5109
Scheme 10. Reagents and conditions: (a) 9-BBN, THF, rt, 18 h, then aq 3 M NaOH and aq 30% H2O2, rt, 3 h, 89%; (b) (1) i-Pr2NP(OCH2CH]CH2)2,
1H-tetrazole, THF, rt, 4 h, then aq 30% H2O2, THF, 0 8C, 1 h; (2) aq 80% AcOH, 60 8C, 1 h, 71%; (c) 5, TMSOTf, MS 4A, CH2Cl2, under N2, 0 8C, 1 h, 53%;
(d) (1) Zn, AcOH–THF (1:1), rt, 4 h; (2) (Z)-11-octadecenoic acid, WSC.HCl, CH2Cl2, rt, 10 h, two steps 58%; (e) (PPh3)4Pd, PPh3, Et3N–HCOOH, THF, N2,
55 8C, 4 h, 84%.
Scheme 11. Reagents and conditions: (a) CBr4, PPh3, CH2Cl2, rt, 1 h, 72%; (b) (1) triallylphosphite, 180 8C, 3 h; (2) aq 80% AcOH, 60 8C, 1 h, two steps 40%;
(c) 5, AgOTf, TMSOTf, MS 4A, CH2Cl2, N2, rt, 16 h, 58%; (d) (1) Zn, AcOH–THF (1:1), rt, 4 h; (2) (Z)-11-octadecanoyl chloride, NaHCO3, THF–H2O (5:1),
rt, 30 min, two steps 52%; (e) (PPh3)4Pd, PPh3, Et3N–HCOOH, THF, N2, 55 8C, 4 h, 80%.

5110
M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
respectively. The activities of the a-carboxymethyl com-
pounds (8 and 14) in the anomeric position were relatively
weak, and those of compounds 21, 31, 40, 51 and 57 having
an a-2-(phosphonooxy)ethyl group, 62 having an a-2-
(phosphono)ethyl group and 67 having an a-3-(phosphono-
oxy)propyl group at the anomeric position were sufficiently
strong. Above all, the activity (IC50Z0.1 nM) of com-
pounds 31 was strongest. The difference between the C60
methoxyl group of 21 and the C60 hydroxyl group of 31
largely affected the activity. Compounds 21 (IC50Z2.7 nM)
possessing an a-2-(phosphonooxy)ethyl group and 62
(IC50Z3.2 nM) possessing an a-2-(phosphono)ethyl group
at the anomeric position showed almost the same level of
activities. On the contrary, the activity of 3-(phosphono)-
propyl 72 (IC50Z14.4 nM) was much weaker than that of
3-(phosphonooxy)propyl 67 (IC50Z1.4 nM). It is difficult to
understand that the difference of one methylene length
between compounds 62 and 72 or one oxygen existence
between compounds 67 and 72 has such an influence on the
LPS-antagonistic activity.
4. Experimental
4.1. General procedure
1H NMR spectra were recorded with a JEOL-GSX 400 or a
JNM-ECT 500 spectrometer using tetramethylsilane (TMS)
as an internal standard. IR absorption spectra were
measured with an IR A-2 spectrophotometer, and mass
spectra were obtained with a JMS-700 mass spectrometer.
Separation of compounds by column chromatography was
done with silica gel 60 (230–400 mesh ASTM) under a
slightly elevated pressure (111–182 kPa) for easy elution.
Commercially available anhydrous THF and dichloro-
methane were used for the reactions. DMF and pyridine
˚
were dried by storage over 4 A molecular sieves.
4.1.1. Allyl 3-O-dodecyl-4,6-O-isopropylidene-2-O-tetra-
decyl-a-D-glucopyranoside (2). To a solution of allyl
3-O-docecyl-a-D-glucopyranoside 1 (2.84 g, 6.63 mmol)
and tetradecyl methanesulfonate (2.33 g, 7.95 mmol) in
DMF (20 ml) was added NaH (55% oil dispersion 347 mg,
7.95 mmol). After stirring for 16 h at room temperature,
the reaction mixture was diluted with EtOAc, which was
washed with ice water and brine, dried over MgSO4 and
filtered. The filtrate was concentrated in vacuo to give a
mixture, which was chromatographed on a silica gel
column. Elution with hexane–EtOAc (9:1) gave 2 (3.10 g,
75%) as an oil. 400 MHz 1H NMR (CDCl3) 0.88 (6H, t, JZ
6.6 Hz), 1.26 (40H, bs), 1.41 (3H, s), 1.48 (3H, s), 1.50–1.61
(4H, m), 3.30 (1H, m), 3.50–3.74 (8H, m), 3.84 (1H, m),
4.07 (1H, dd, JZ6.6, 13.2 Hz), 4.18 (1H, dd, JZ5.1,
13.2 Hz), 4.91 (1H, d, JZ3.7 Hz), 5.22 (1H, m), 5.32 (1H,
m), 5.92 (1H, m)). FABMS (positive-ion) m/z, 625 [MC
H]C, 647 [MCNa]C.
In addition, inhibitory activity (ID50) on TNFa production
toward galactosamine loaded C3H/HeN mice in vivo of
compounds 21, 31, 57, 62 and 67 was measured.9 The
values were 0.29, 0.50, 0.61, not dose-dependent and
0.33 mg/kg, respectively. These compounds were suf-
ficiently strong except for anomeric 2-(phosphono)ethyl
compound 62 toward C3H/HeN mice. However, judging
from the result of C60 methoxy compound 21 and C60
hydroxy compound 31, methoxy compound 21 was a little
stronger than hydroxyl compound 31 toward C3H/HeN
mice in spite of compound 31 having been much stronger
activity than compound 21 toward human whole blood cells.
The difference of one methylene length between compounds
2-(phosphonooxy)ethyl 21 and 3-(phosphonooxy)propyl
67 did not influence largely the inhibitory activities on
LPS-induced TNFa production.
4.1.2. (Allyloxycarbonyl)methyl 3-O-dodecyl-4,6-O-iso-
propylidene-2-O-tetradecyl-a-D-glucopyranoside (3). To
a solution of 2 (625 mg, 1.00 mmol) in MeCN–CCl4–H2O
(2:2:3, 35 ml) were added RuO.2xH2O (16 mg) and NaIO4
(4.0 g). The mixture was stirred for 3 h at room temperature,
and diluted with EtOAc, which was washed with water and
brine, dried over MgSO4 and filtered. The filtrate was
concentrated in vacuo to give a carboxylic acid, which was
dissolved in DMF (10 ml). Allyl bromide (1.2 ml) and Et3N
(0.8 ml) were added to this solution, and this mixture was
stirred for 16 h at room temperature, and diluted with
EtOAc, which was washed with water and brine, dried over
MgSO4 and filtered. The filtrate was concentrate in vacuo to
give a residue, which was chromatographed on a silica gel
column. Elution with cyclohexane–EtOAc (9:1) gave 3
(490 mg, 72%) as an oil. 400 MHz 1H NMR (CDCl3) d 0.88
(6H, t, JZ6.6 Hz), 1.25 (40H, bs), 1.40 (3H, s), 1.48 (3H, s),
1.48–1.65 (4H, m), 3.18 (1H, m), 3.33 (1H, m), 3.50–3.89
(8H, m), 4.18, 4.34 (2H, AB-q, JZ16.2 Hz), 4.62–4.67 (2H,
m), 5.08 (1H, m), 5.22 (1H, m), 5.24–5.36 (2H, m), 5.91
(1H, m). FABMS (positive-ion) m/z, 683 [MCH]C, 705
[MCNa]C.
Usually, lipid A analogs having six fatty acids chains10
show LPS-agonistic (endotoxic) activity toward both human
and mouse macrophages, and lipid IVa (biosynthetic
precur of lipid A)11 having four fatty acid chains shows
LPS-antagonistic activity toward human blood cells and
adversely endotoxic activity toward mouse macrophages.12
However, the synthetic compounds, this time, showed
LPS-antagonistic activity toward both human whole blood
cells and galactosamine loaded C3H/HeN mice. This
tendency was the same as that for nontoxic natural
RsDPLA3 and synthetic E55645 containing a (Z)-double
bond in one of the fatty acids.
3. Conclusion
Thus, we could synthesize ten E5564-related disaccharides
containing a glucose instead of the glucosamine at the
reducing end. As a result, it was proved that these novel
synthetic compounds had almost the same or stronger
activities towards both human blood cells and murine
macrophages than against classic lipid A-type disaccharides
having the glucosamine-glucosamine moieties.13
4.1.3. (Allyloxycarbonyl)methyl 3-O-dodecyl-2-O-tetra-
decyl-a-D-glucopyranoside (4). A solution of 3 (480 mg,
0.703 mmol) in AcOH–H2O (4:1, 5 ml) was stirred for 2 h at
65 8C, and concentrated in vacuo to give a residue, which
was chromatographed on a silica gel column. Elution with

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5111
cyclohexane–EtOAc (3:2, then 2:3) gave triol 4 (319 mg,
71%) as a wax. IR nmax(film) 3410, 2923, 2852, 1758,
1466 cmK1. 400 MHz 1H NMR (CDCl3CD2O) d 0.88
(6H, t, JZ6.6 Hz), 1.26 (40H, bs), 1.54–1.68 (4H, m), 3.19
(1H, m), 3.33 (1H, m), 3.47 (1H, m), 3.51–3.67 (2H, m),
3.72–3.88 (4H, m), 3.91–3.98 (2H, m), 4.21, 4.33 (2H,
AB-q, JZ16.8 Hz), 4.62–4.67 (2H, m), 5.12 (1H, m), 5.25–
5.37 (2H, m), 5.91 (1H, m). FABMS (positive-ion) m/z, 665
[MCNa]C. Anal. Calcd for C37H70O8,: C, 69.12; H, 10.97.
Found: C, 69.09; H, 10.81.
m), 3.10–4.40 (29H, m, containing two 3H, s, at 3.28 and
3.38 ppm), 4.54–4.66 (6H, m), 5.09–5.39 (10H, m), 5.89–
5.98 (3H, m), 5.96–6.06 (1H, m). FABMS (positive-ion)
m/z, 1434 [MCNa]C. HRFABMS, calcd for C79H146NO17-
PNa: 1435.0226. Found: 1435.0234.
4.1.6. Carboxymethyl 6-O-{2-deoxy-3-O-[(R)-3-methoxy-
decyl]-6-O-methyl-2-[(Z)-11-octadecenoylamino]-4-O-
phosphono-b-D-glucopyranosyl}-3-O-dodecyl-2-O-tetra-
decyl-a-D-glucopyranoside (8). To a solution of 7 (104 mg,
0.074 mmol) in dry THF (6 ml) were added PPh3 (10 mg,
0.034 mmol), Et3N (37 mg, 0.365 mmol), HCOOH (34 mg,
0.728 mmol) and Pd(PPh3)4 (5 mg, 0.004 mmol) in this
sequence. The solution was stirred for 4 h at 55 8C under
nitrogen, and concentrated in vacuo to give a mixture, which
was chromatographed on a DEAE-cellulose (Whatman ion-
exchange cellulose, wet 3 g) column. The column was
prepared by preliminary consecutive washing with 30 ml
each of 0.5 M HCl, H2O, 0.5 M NaOH, and H2O, and 12 ml
each of 1 M AcOH and H2O, and 30 ml of 0.05 M AcONH4,
30 ml each of CHCl3–MeOH–H2O (2:3:1) and finally
CHCl3–MeOH (2:1). The column was eluted with 3 ml
each of CHCl3–MeOH (2:1), then 0.05 M AcONH4 in
CHCl3–MeOH–H2O (2:3:1). The fractions containing 8
were collected. To this solution were added another volume
of CHCl3 and aq 0.15 M HCl to adjust the ratio of CHCl3–
H2O–MeOH to 1:1:1, and the mixture was shaken well. The
lower CHCl3 layer was separated, and concentrated in vacuo
to give 8 (83 mg, 95%) as a powder. [The fractions were
analyzed by silica-gel TLC and a solvent system of CHCl3–
MeOH–AcOH–H2O (8:4:1:1).] IR nmax(KBr) 3500–3000
4.1.4. (Allyloxycarbonyl)methyl 6-O-{2-deoxy-4-O-dia-
llylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyrano-
syl}-3-O-dodecyl-2-O-tetradecyl-a-D-glucopyranoside
(6). To a solution of imidate 5 (249.2 mg, 0.296 mmol) and
4 (190 mg, 0.296 mmol) in CH2Cl2 (7 ml) was added MS
˚
4 A (400 mg). After stirring for 30 min at room temperature,
the mixture was cooled at K40 8C, and TMSOTf (10 mg,
0.045 mmol) was added to this mixture. The mixture was
stirred for 2 h at K40 8C under nitrogen, and diluted with
EtOAc, which was washed with satd NaHCO3 and brine,
dried over MgSO4 and filtered. The filtrate was concentrated
in vacuo to give a mixture, which was chromatographed on
a silica gel column. Elution with cyclohexane–EtOAc (1:1,
then 1:2) gave 6 (169 mg, 43%) as a gum. IR nmax(film)
3448, 3291, 3084, 2925, 2855, 1751, 1650 (w), 1546,
1465 cmK1. 400 MHz 1H NMR (CDCl3) d 0.89 (9H, t, JZ
6.6 Hz), 1.26 (50H, bs), 1.40–1.60 (8H, m), 3.25–3.91 (23H,
m, containing 3H, s, at d 3.28 ppm, and 3H, s, at d
3.39 ppm), 4.05–4.19 (2H, m), 4.28–4.35 (3H, m), 4.55–
4.81 (8H, m), 5.12 (1H, m), 5.09 (1H, d, JZ3.7 Hz), 5.24–
5.39 (6H, m), 5.90–5.99 (3H, m). FABMS (positive-ion)
m/z, 1346 [MCNa]C, 1344 [MCNa, 35Cl]C. HRFABMS,
calcd for C64H115NCl3O18PNa: 1344.6818. Found:
1344.6826.
(br), 2924, 2853, 1735, 1655, 1630, 1549, 1466 cmK1
.
400 MHz 1H NMR (CDCl3CD2O) d 0.88 (12H, t, JZ
6.6 Hz), 1.26 (72H, bs), 1.40–1.80 (8H, m), 1.99–2.02 (4H,
m), 2.20–2.40 (2H, m), 3.10–5.10 (29H, m, containing two
3H, s at d 3.25 and 3.38 ppm), 5.31–5.38 (2H, m). FABMS
(negative-ion) m/z, 1291 [MKH]K. HRFABMS, calcd for
C70H133NO17P: 1290.9336. Found: 1290.9317. Anal. Calcd
for C70H134NO17P: C, 62.35; H, 10.09; N, 1.70; P, 2.08.
Found: C, 62.78; H, 10.41; N, 1.10; P, 2.40.
4.1.5. (Allyloxycarbonyl)methyl 6-O-{2-deoxy-4-O-dia-
llylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-2-
[(Z)-11-octadecenoylamino]-b-D-glucopyranosyl}-3-O-
dodecyl-2-O-tetradecyl-a-D-glucopyranoside (7). To a
solution of 6 (154 mg, 0.116 mmol) in THF–AcOH (1:1,
6 ml) was added Zn powder (150 mg). The mixture was
stirred vigorously with a magnetic stirrer at 25 8C for 4 h,
and filtered. The filtrate was concentrated in vacuo below
30 8C, and diluted with EtOAc, which was washed with aq
satd NaHCO3 and brine, dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo to give an amine, which
was dissolved in THF (1.0 ml)–H2O (0.3 ml) containing
NaHCO3 (30 mg, 0.357 mmol). To this solution was added
a solution of (Z)-11-octadecenoyl chloride [obtained from
(Z)-11-octadecenoic acid (40 mg, 0.142 mmol) by treatment
of the excess oxalyl chloride in benzene at 25 8C for 2 h] in
THF (0.5 ml) with vigorous stirring at 25 8C. After stirring
for 0.5 h, the reaction mixture was diluted with EtOAc,
which was washed with aq satd NaHCO3 and brine, dried
over MgSO4 and filtered. The filtrate was concentrated in
vacuo to give a mixture, which was chromatographed on a
silica gel column. Elution with cyclohexane–EtOAc (1:2)
gave 7 (125 mg, 77%) as a gum. IR nmax(KBr) 3306, 3082
(w), 2925, 2854, 1760, 1659, 1634, 1545 cmK1. 400 MHz
1H NMR (CDCl3) d 0.88 (12H, t, JZ6.4 Hz), 1.25 (62H,
bs), 1.35–1.80 (14H, m), 2.00–2.02 (6H, m), 2.10–2.25 (4H,
4.1.7. (Allyloxycarbonyl)methyl 2-O-[(R)-3-tert-butyldi-
methylsilyloxytetradecyl]-3-O-dodecyl-4,6-O-isopropyl-
idene-a-D-glucopyranoside (10). Compound 9 was treated
as described for the formation of 3 from 2 to give 10 (72%
yield) as an oil. IR nmax(film) 2926, 2856, 1759, 1745
1
(shoulder) cmK1. 400 MHz H NMR (CDCl3) d 0.04 (3H,
s), 0.05 (3H, s), 0.88 (9H, s, and 6H, t, JZ6.6 Hz), 1.26
(38H, bs), 1.40 (3H, s), 1.48 (3H, s), 1.48–1.55 (2H, m),
1.74–1.79 (2H, m), 3.33 (1H, m), 3.50–3.86 (10H, m), 4.17,
4.30 (2H, AB-q, JZ12.8 Hz), 4.64–4.65 (2H, m), 5.07
(1H, d, JZ3.7 Hz), 5.24–5.36 (2H, m), 5.92 (1H, m).
FABMS (positive-ion) m/z, 813 [MCH]C, 835 [MCNa]C.
HRFABMS, calcd for C46H88O9SiNa: 835.6095. Found:
835.6084.
4.1.8. (Allyloxycarbonyl)methyl 2-O-[(R)-3-hydroxy-
tetradecyl]-3-O-dodecyl-a-D-glucopyranoside (11). Com-
pound 10 was treated as described for the formation of 4
from 3 to give triol 11 (60%) as a solid, mp 55–56 8C
(recrystallized from EtOAc/hexaneZ1/4). IR nmax(KBr)
1
3426, 2919, 2850, 1753, 1468 cmK1. 400 MHz H NMR
(CDCl3) d 0.88 (6H, t, JZ6.6 Hz), 1.26 (36H, bs), 1.40–1.78

5112
M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
(8H, m), 2.10 (1H, bs, OH)), 2.62 (1H, bs, OH), 3.34 (1H,
dd, JZ3.7, 9.5 Hz), 3.49–4.00 (10H, m), 4.20, 4.34 (2H,
AB-q, JZ16.8 Hz), 4.62–4.66 (2H, m), 5.21 (1H, d, JZ
2.9 Hz), 5.25–5.36 (2H, m), 5.91 (1H, m). Anal. Calcd for
C37H70O9: C, 67.44; H, 10.71. Found: C, 67.41; H, 10.70.
filtered. The filtrate was concentrate in vacuo to give an
aldehyde (4 g), which was dissolved in EtOH (50 ml). To
this solution was added NaBH4 (270 mg), and the solution
was stirred for 20 min, quenched with AcOH (200 mg),
diluted with EtOAc, which was washed with aq satd
NaHCO3 and brine, dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo to give a mixture, which
was chromatographed on a silica gel column. Elution with
hexane–EtOAc (3:1) gave alcohol 16 (3.00 g, 73%) as an
4.1.9. (Allyloxycarbonyl)methyl 6-O-{2-deoxy-4-O-dia-
llylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyrano-
syl}-3-O-dodecyl-2-O-[(R)-3-hydroxytetradecyl]-a-D-
glucopyranoside (12). Compound 11 and imidate 5 were
treated as described for the formation of 6 from 5 to give 12
(45%) as a gum, and imidate 5 (85 mg, 34%) and starting
triol 11 (80 mg, 42%) were recovered. IR nmax(film) 3457
(br), 3325 (br), 3085 (w), 2926, 2855, 1748, 1650 (w),
1545 cmK1. 400 MHz 1H NMR (CDCl3) d 0.88 (9H, t, JZ
6.6 Hz), 1.26 (46H, bs), 1.36–1.80 (10H, m), 3.22–4.90
(37H, m, containing 3H, s, at d 3.27 ppm, and 3H, s, at d
3.39 ppm), 5.07–5.40 (7H, m), 5.87–5.99 (3H, m). FABMS
(positive-ion) m/z, 1360 [MCNa, 35Cl]C. HRFABMS,
calcd for C64H115NCl3O19PNa,: 1360.6764. Found:
1360.6764.
1
oil. 400 MHz H NMR (CDCl3) d 0.88 (9H, t, JZ6.6 Hz),
1.26 (36H, bs), 1.40 (3H, s), 1.41–1.43 (4H, m), 1.48 (3H, s),
2.82 (1H, bs, OH), 3.30 (1H, m), 3.51–3.85 (14H, m),
4.89 (1H, d, JZ3.7 Hz). FABMS (positive-ion) m/z, 601
[MCH]C, 623 [MCNa]C.
4.1.13. 2-(Diallylphosphonooxy)ethyl 2,3-di-O-dodecyl-
4,6-O-isopropylidene-a-D-glucopyranoside (17). To a
solution of 16 (5.65 g, 9.2 mmol) in CH2Cl2 (70 ml) were
added 1H-tetrazole (1.54 g, 22 mmol), diallyl diisopropyl-
phosphoramidite (3.50 g, 1.128 mmol) and Na2SO4 (6 g).
After stirring for 30 min at room temperature, to this
reaction mixture were added THF (70 ml) and aq 30% H2O2
solution (2 ml). The mixture was stirred for 15 min at room
temperature, and diluted with EtOAc, which was washed
with aq 10% Na2S2O3, aq satd NaHCO3 and brine, dried
over MgSO4 and filtered. The filtrate was concentrated in
vacuo to give a mixture, which was chromatographed on a
silica gel column. Elution with hexane–EtOAc (2:1) gave
alcohol 17 (6.60 g, 99%) as an oil. 400 MHz 1H NMR
(CDCl3) d 0.88 (6H, JZ6.6 Hz), 1.21–1.38 (36H, m), 1.39
(3H, s), 1.47 (3H, s), 1.50–1.58 (4H, m), 3.25–3.3 (1H, m),
3.51–3.88 (11H, m) 4.23–4.27 (2H, m) 4.56–4.60 (4H, m),
4.89 (1H, d, JZ3.7 Hz), 5.25–5.41 (4H, m), 5.91–6.00 (2H,
m). FABMS (positive-ion) m/z, 761 [MCH]C, 783 [MC
Na]C.
4.1.10. (Allyloxycarbonyl)methyl 6-O-{2-deoxy-4-O-dia-
llylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-2-
[(Z)-11-octadecenoylamino]-b-D-glucopyranosyl}-3-O-
dodecyl-2-O-[(R)-3-hydroxytetradecyl]-a-D-glucopyrano-
side (13). Compound 12 was treated as described for the
formation of 7 from 6 to give 13 (67%) as a gum. IR
n
max(film) 3500–3000 (br), 2926, 2855, 1752, 1652,
1549 cmK1. 400 MHz H NMR (CDCl3) d 0.88 (12H, t,
JZ6.6 Hz), 1.26 (72H, bs), 1.37–1.85 (6H, m), 1.97–2.02
(4H, m), 2.12–2.24 (2H, m), 2.97–4.32 (30H, m, containing
two 3H, s, at 3.27 and 3.38 ppm), 4.53–4.66 (6H, m), 5.02–
5.40 (10H, m), 5.85–5.98 (3H, m), 6.09 (1H, d, JZ6.6 Hz).
FABMS (positive-ion) m/z, 1450 [MCNa]C. HRFABMS,
calcd for C79H146NO18PNa: 1451.0174. Found: 1451.0171.
1
4.1.14. 2-(Diallylphosphonooxy)ethyl 2,3-di-O-dodecyl-
a-D-glucopyranoside (18). To a solution of 17 (6.60 g,
8.67 mmol) in MeOH (60 ml) was added p-TsOH$H2O
(412 mg, 2.17 mmol). After stirring for 2 h at room
temperature, the reaction mixture was concentrated in
vacuo to give a mixture, which was chromatographed on a
silica gel column. Elution with EtOAc and then 10% MeOH
in EtOAc gave 18 (6.01 g, 96%) as a wax. 400 MHz
1H NMR (CDCl3) d 0.88 (6H, t, JZ6.6 Hz), 1.26 (36H,bs),
1.5–1.71 (4H,m), 3.27 (1H, dd, JZ3.7, 8.8 Hz), 3.41–3.91
(11H, m), 4.24–4.28 (2H, m), 4.48–4.59 (4H, m), 4.95
(1H, d, JZ3.7 Hz), 5.19–5.4 (4H,m), 5.9–6.00 (2H, m).
FABMS (positive-ion) m/z, 721. [MCH]C, 743 [MCNa]C.
HRFABMS, calcd for C38H74O10P: 721.4941. Found:
721.4950.
4.1.11. Carboxymethyl 6-O-{2-deoxy-3-O-[(R)-3-meth-
oxydecyl]-6-O-methyl-2-[(Z)-11-octadecenoylamino]-4-
O-phosphono-b-D-glucopyranosyl}-3-O-dodecyl-2-O-
[(R)-3-hydroxytetradecyl]-a-D-glucopyranoside (14).
Compound 13 (110 mg, 0.077 mmol) was treated as
described for the formation of 8 from 7 to give 14 (55 mg,
55%) as a powder. IR nmax(KBr) 3292 (br), 2925, 2854,
1
1737, 1654, 1631, 1552, 1466 cmK1. 400 MHz H NMR
(CDCl3CCD3OD, 5:1) d 0.88 (12H, t, JZ6.6 Hz), 1.27
(70H, bs), 1.40–1.80 (8H, m), 2.00–2.03 (4H, m), 2.15–2.23
(2H, m), 3.26–4.26 (28H, m, containing two 3H, s at d 3.31
and 3.41 ppm), 4.65 (1H, d, JZ6.5 Hz), 5.08 (1H, d, JZ
2.9 Hz), 5.35 (2H, m). FABMS (negative-ion) m/z, 1306
[MKH]K, 1328 [MK2HCNa]K. HRFABMS, calcd for
C70H133NO18P: 1306.9260. Found: 1306.9288. Anal. Calcd
for C70H134NO18P: C, 64.24; H, 10.32; N, 1.07; P, 2.37.
Found: C, 64.46; H, 10.46; N, 1.27; P, 2.29.
4.1.15. 2-(Diallylphosphonooxy)ethyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyra-
nosyl}-2,3-di-O-dodecyl-a-D-glucopyranoside (19). To a
solution of 5 (1.00 g, 1.18 mmol) and 18 (1.02 g,
4.1.12. 2-Hydroxyethyl 2,3-di-O-dodecyl-4,6-O-isopro-
pylidene a-D-glucopyranoside (16). To a solution of 15
(4.00 g, 6.70 mmol) in THF–H2O (7:2, 66 ml) were added
NaIO4 (6.7 g) and a 2.5% solution of OsO4 in tert-BuOH
(1.4 ml). This mixture was stirred for 3 h at room
temperature, and diluted with EtOAc, which was washed
with aq satd NaHCO3 and brine, dried over MgSO4 and
˚
1.49 mmol) in CH2Cl2 (30 ml) were added MS 4 A
(1.5 g), AgOTf (350 mg, 1.36 mmol) and TMSOTf
(20 mg, 0.090 mmol). The mixture was stirred for 16 h at
room temperature under nitrogen, and diluted with EtOAc,
which was washed with aq satd NaHCO3 and brine, dried
over MgSO4 and filtered. The filtrate was concentrated in

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5113
vacuo to give a mixture, which was chromatographed on a
silica gel column. Elution with EtOAc–Hexane (2:1) gave
19 (1.01 g, 60%) as a gum. 400 MHz H NMR (CDCl3) d
from 17 to give 24 (92%) as a powder. IR nmax(KBr) 3548,
3375, 3265, 3116, 2927, 2874, 2858, 1702, 1672 cmK1
.
400 MHz 1H NMR (CDCl3) d 0.88 (3H, t, JZ6.6 Hz), 1.27–
1.77 (14H, m), 3.01 (2H, bs, OH), 3.29 (3H, s), 3.34–3.53
(3H, m), 3.59 (1H, t, JZ9.5, 8.8 Hz), 3.67 (1H, m), 3.75–
3.85 (3H, m), 3.92 (1H, dd, JZ3.7, 11.7 Hz), 4.07 (1H, m),
4.31 (1H, m), 4.84 (1H, d, JZ8.8 Hz), 5.19–5.29 (2H, m).
FABMS (positive-ion) m/z, 508 [MCNa]C, 486 [MCH]C.
HRFABMS, calcd for C22H38F3NO7Na: 508.2494. Found:
508.2503.
1
0.88 (9H, t, JZ6.6 Hz), 1.26 (48H, bs), 1.38–1.46 (2H, m),
1.53–1.58 (4H, m), 1.69–1.77 (2H, m), 2.60 (1H, bs, OH),
3.21–3.89 (23H, m, containing two 3H at d 3.28 and
3.39 ppm), 4.09–4.32 (4H, m), 4.52–4.60 (8H, m), 4.95 (1H,
d, JZ3.7 Hz), 5.24–5.40 (8H, m), 5.89–5.99 (4H, m), 6.41
(1H, broad, NH). FABMS (positive-ion) m/z, 1422 [MCNa,
35Cl]C, 1424. HRFABMS, calcd for C65H118Cl3NO20P2Na:
1422.6468. Found: 1422.6473.
4.1.20. (Z)-1-Propenyl 2-deoxy-3-O-[(R)-3-methoxyde-
cyl]-2-(2,2,2-trichloroethoxycarbonylamino)-b-D-gluco-
pyranoside (25). A solution of 24 (5.34 g, 11.0 mmol) in
DMSO (30 ml) containing tert-BuOK (3.10 g, 27.6 mmol)
was stirred for 85 8C for 2 h under nitrogen. To this solution
was added H2O (10 ml), and this solution was stirred at
85 8C for 6 h. After cooling, the aqueous solution was
extracted with CH2Cl2 (three times), washed with H2O
and brine, dried over MgSO4 and filtered. The filtrate was
concentrated in vacuo to give an amine, which was
dissolved in THF (40 ml), and aq satd NaHCO3 (20 ml)
was added. To this mixture was added 2,2,2-trichloroethyl
chloroformate (2.58 g, 12.2 mmol), and this mixture was
stirred at 0 8C for 30 min. After another amount of aq satd
NaHCO3 was added, the reaction mixture was extracted
with CH2Cl2, which was washed with brine, dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo
to give a residue, which was chromatographed on a silica gel
column. Elution with hexane–EtOAc (2:3) gave 25 (4.70 g,
76%) as a solid. IR nmax(KBr) 3323, 3054, 2927, 2873,
2857, 1716, 1672, 1642 cmK1. 400 MHz 1H NMR (CDCl3)
d 0.89 (3H, t, JZ6.8 Hz), 1.28–1.62 (15H, m), 1.74–1.79
(2H, m), 2.36 (1H, s, OH), 3.30 (3H, s), 3.36–3.46 (3H, m),
3.58–3.71 (3H, m), 3.81 (1H, m), 3.90–3.94 (2H, m), 4.06
(1H, s, OH), 4.57 1H, m), 4.74 (2H, s), 4.86 (1H, d, JZ
6.8 Hz), 5.37 (1H, br, NH), 6.15 (1H, m). FABMS (positive-
ion) m/z, 586 [MCNa]C, 564 [MCH]C. HRFABMS, calcd
for C23H40Cl3NO8Na: 586.1726. Found: 586.1703.
4.1.16. 2-(Diallylphosphonooxy)ethyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-[(Z)-11-octadecenoylamino]-b-D-glucopyranosyl}-2,3-
di-O-dodecyl-a-D-glucopyranoside (20). Compound 19
was treated as described for the formation of 7 from 6 to
give 20 (75%) as a gum. 400 MHz 1H NMR (CDCl3) d 0.88
(12H, t, JZ6.6 Hz), 1.26 (66H, bs), 1.40–1.79 (10H, m),
1.99–2.04 (4H, m), 2.17–2.25 (2H, m), 3.01(1H, bs, OH),
3.17–3.82 (24H, m, containing two 3H, s, at 3.28 and
3.38 ppm), 3.97–4.31 (5H, m), 4.52–4.58 (8H, m), 4.88 (1H,
d, JZ3.7 Hz), 5.13 (1H, d, JZ8.1 Hz), 5.23–5.38 (10H, m),
5.90–6.00 (4H, m), 6.62 (1H, d, JZ6.6 Hz, NH). FABMS
(positive-ion) m/z, 1512 [MCNa]C. HRFABMS, calcd for
C80H149NO19P2Na: 1513.0097. Found: 1513.0121.
4.1.17. 2-(Phosphonooxy)ethyl 6-O-{2-deoxy-3-O-[(R)-3-
methoxydecyl]-6-O-methyl-2-[(Z)-11-octadecenoyl-
amino]-4-O-phosphono-b-D-glucopyranosyl}-2,3-di-O-
dodecyl-a-D-glucopyranoside (21). Compound 20 was
treated for 16 h as described for the formation of 8 from 7
to give 21 (45%) as a wax. IR nmax(KBr) 3292 (br), 2954,
2853, 1630 cmK1. 400 MHz 1H NMR (CDCl3CCD3OD) d
0.88 (12H, t, JZ6.6 Hz), 1.26 (66H, bs), 1.40–1.79 (10H,
m), 1.99–2.07 (4H, m), 2.17–2.25(2H, m), 3.20–3.90 (24H,
m, containing two 3H, s at 3.30 and 3.40 ppm), 4.00–4.21
(5H, m), 4.65(1H, d, JZ8.1 Hz), 4.88 (1H, d, JZ3.7 Hz),
5.37–5.40 (2H, m). FABMS (negative-ion) m/z, 1328
[MKH]K, 1350 [MCNa-2H]K. Anal. Calcd for
C68H133NO19P2,: C, 61.37; H, 10.07; N, 1.05; P, 4.66.
Found: C, 60.89; H, 10.11; N, 1.13; P, 4.42.
4.1.21. (Z)-1-Propenyl 6-O-allyloxycarbonyl-2-deoxy-3-
O-[(R)-3-methoxydecyl]-2-(2,2,2-trichloroethoxycarbo-
nylamino)-b-D-glucopyranoside (26). To a solution of 25
(4.60 g, 8.14 mmol) in CH2Cl2 (30 ml) were added pyridine
(1.4 ml, 17.3 mmol) and allyl chloroformate (1.1 ml,
10.4 mmol) at 0 8C. The mixture was stirred at 0 8C for
1 h, diluted with CH2Cl2, washed with aq satd NaHCO3,
H2O and brine, dried over MgSO4 and filtered. The filtrate
was concentrated in vacuo to give a residue, which
was chromatographed on a silica gel column. Elution with
hexane–EtOAc (3:2) gave 26 (4.95 g, 94%) as a wax. IR
4.1.18. Allyl 2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-
methoxydecyl]-2-(trifluoroacetylamino)-b-D-glucopyra-
noside (23). Compound 22 and (R)-3-methoxydecyl
p-toluenesulfonate were treated as described for the
formation of 2 from 1 to give 23 (77%) as an amorphous
solid. IR nmax(KBr) 3304, 3114,2995, 2930,2877, 2858,
2825, 1705, 1674 cmK1 500 MHz 1H NMR (CDCl3) d 0.88
(3H, t, JZ6.8 Hz), 1.27–1.47 (12H, m), 1.41 (3H, s), 1.50
(3H, s), 1.63–1.67 (2H, m), 3.25 (1H, m), 3.28 (3H, s), 3.32
(1H, td, JZ9.8, 4.9 Hz), 3.43 (1H, m), 3.58–3.64 (2H, m),
3.77–3.87 (3H, m), 3.93 (1H, dd, JZ4.9, 10.7 Hz), 4.06
(1H, dd, JZ5.9, 12.7 Hz), 4.31 (1H, dd, JZ4.9, 12.7 Hz),
4.88 (1H, d, JZ7.8 Hz), 5.19–5.28 (2H, m), 5.83 (1H, m),
6.56 (1H, d, JZ7.8 Hz, NH). FABMS (positive-ion) m/z,
548 (MCNa)C, 526 (MCH)C. HRFABMS, calcd for
C25H42F3NO7Na: 548.2808. Found: 548.2815.
n
max(KBr) 3518, 3310, 3086, 3059, 2932, 2885, 2857, 1728,
1709, 1674, 1652 cmK1. 400 MHz 1H NMR (CDCl3) d 0.89
(3H, t, JZ6.8 Hz), 1.25–1.62 (15H, m), 1.70–1.79 (2H, m),
3.30 (3H, s), 3.36–3.44 (2H, m), 3.50–3.71 (4H, m), 3.91–
3.94 (2H, m, containing OH), 4.38 (1H, dd, JZ4.9,
11.7 Hz), 4.50 (1H, dd, JZ2.0, 11.7 Hz), 4.56 (1H, m),
4.63 (2H, d, JZ5.9 Hz), 4.74 (2H, s), 4.85 (1H, m), 5.26–
5.38 (3H, m, containing NH), 5.93 (1H, m), 6.16 (1H,
dd, JZ2.0, 5.9 Hz). FABMS (positive-ion) m/z, 670
[MCNa]C, 648 [MCH]C. HRFABMS, calcd for
C27H44Cl3NO10Na: 670.1914. Found: 670.1959.
4.1.19. Allyl 2-deoxy-3-O-[(R)-3-methoxydecyl]-2-(tri-
fluoroacetylamino)-b-D-glucopyranoside (24). Com-
pound 23 was treated as described for the formation of 18

5114
M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
4.1.22. (Z)-1-Propenyl 6-O-allyloxycarbonyl-2-deoxy-4-
O-diallylphosphono-3-O-[(R)-3-methoxydecyl]-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranoside
(27). To a solution of 26 (4.80 g, 7.40 mmol) in THF (30 ml)
were added 1H-tetrazole (830 mg, 11.8 mmol) and diallyl
diisopropylphosphoramidite (2.50 g, 10.2 mmol) under
nitrogen at room temperature. After stirring for 20 min,
the mixture was cooled to 0 8C, and aq 30% H2O2 (10 ml)
was added to this phosphite solution. The mixture was
stirred for 30 min, and diluted with EtOAc, which was
washed with aq satd Na2S2O3 and brine, dried over MgSO4
and filtered. The filtrate was concentrated in vacuo to give a
residue, which was chromatographed on a silica gel column.
Elution with hexane–EtOAc (3:2) gave 27 (5.17 g, 86%) as
a gum. IR nmax(CHCl3) 3450, 3089, 2955, 2873, 2859,
2829, 1746, 1674, 1650 cmK1. 400 MHz 1H NMR (CDCl3)
d 0.88 (3H, t, JZ6.8 Hz), 1.27–1.56 (15H, m), 1.68–1.81
(2H, m), 3.29 (3H, s), 3.31 (1H, m), 3.47 (1H, m), 3.72–3.82
(3H, m), 3.90 (1H, m), 4.32–4.38 (2H, m), 4.52–4.63 (8H,
m), 4.73 (2H, s), 4.99 (1H, m), 5.19–5.38 (6H, m), 5.56
(1H, m, NH), 5.89–5.99 (3H, m), 6.13 (1H, m). FABMS
(positive-ion) m/z, 830 [MCNa]C, 808 [MCH]C.
HRFABMS, calcd for C33H53Cl3NO13PNa: 830.2215.
Found: 830.2231.
with aq satd NaHCO3, extracted with CH2Cl2, and
concentrated in vacuo to give a residue, which was
chromatographed on a silica gel column. Elution with
hexane–EtOAc (1:4) gave 29 (125 mg, 52%) as a gum.
400 MHz 1H NMR (CDCl3) d 0.88 (9H, t, JZ6.6 Hz), 1.26–
1.44 (39H, m), 1.53–1.60 (6H, m), 1.64–1.79 (3H, m), 2.50
(1H, bs), 3.23–3.36 (8H, m, containing 3H, s, at d
3.28 ppm), 3.45–3.92 (15H, m), 4.10 (1H, br d, JZ
8.1 Hz), 4.19–4.36 (5H, m), 4.53–4.59 (10H, m), 4.63
(2H, d, JZ5.9 Hz), 4.68 (1H, br.d, JZ11.7 Hz), 4.78 (1H, d,
JZ11.7 Hz), 4.88–4.91 (2H, m), 5.23–5.40 (10H, m), 5.88–
5.98 (5H, m), 6.54 (1H, m). FABMS (positive-ion) m/z,
1492 [MCNa]C.
4.1.25. 2-(Diallylphosphonooxy)ethyl 6-O-{6-O-allyloxy-
carbonyl-2-deoxy-4-O-diallylphosphono-3-O-[(R)-3-
methoxydecyl]-2-[(Z)-11-octadecenoylamino]-b-D-gluco-
pyranosyl}-2,3-di-O-dodecyl-a-D-glucopyranoside (30).
Compound 29 (125 mg, 0.085 mmol) was treated as
described later in the formation of 50 from 49 to give 30
(62 mg, 47%) as an oil. IR nmax(film) 3300, 3085, 2925,
2855, 1752, 1652 cmK1. 400 MHz 1H NMR (CDCl3) d 0.88
(12H, t, JZ6.8 Hz), 1.25–1.79 (77H, m), 1.99–2.01 (3H,
m), 2.16–2.24 (2H, m), 2.87 (1H, d, JZ3.9 Hz), 3.12–3.17
(1H, m), 3.24 (1H, dd, JZ9.8, 2.9 Hz), 3.26–3.38 (5H, m,
containing 3H, s, at d 3.28 ppm), 3.45–3.55 (3H, m), 3.57–
3.85 (8H, m), 4.02–4.06 (1H, m), 4.10 (1H, d, JZ10.7 Hz),
4.16–4.33 (3H, m), 4.51–4.64 (12H, m), 4.88 (1H, d, JZ
3.9 Hz), 5.19 (1H, d, JZ7.8 Hz), 5.23–5.40 (12H, m), 5.89–
5.98 (5H, m), 6.74 (1H, d, JZ6.8 Hz). FABMS (positive-
ion) m/z, 1582 [MCNa]C. HRFABMS, calcd for
C83H151O21NP2Na: 1583.0161. Found: 1583.0179.
4.1.23. 6-O-Allyloxycarbonyl-2-deoxy-4-O-diallylphos-
phono-3-O-[(R)-3-(methoxy)decyl]-2-(2,2,2-trichloro-
ethoxycarbonylamino)-D-glucopyranose (28). To a
solution of 27 (4.80 g, 5.93 mmol) in THF (30 ml) were
added I2 (3.08 g, 12.1 mmol) and H2O (6 ml). After stirring
for 30 min at room temperature, the reaction mixture was
diluted with EtOAc, washed with aq satd Na2S2O3, aq satd
NaHCO3 and brine, dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo to give a residue, which
was chromatographed on a silica gel column. Elution with
hexane–EtOAc (1:1) gave 28 (4.30 g, 94%) as a gum. IR
4.1.26. 2-(Phosphonooxy)ethyl 6-O-{2-deoxy-3-O-[(R)-3-
methoxydecyl]-2-[(Z)-11-(octadecenoyl)amino-4-O-
phosphono]-b-D-glucopyranosyl}-2,3-di-O-dodecyl-a-D-
glucopyranoside (31). Compound 30 (136 mg,
0.087 mmol) was treated as described for the formation of
8 from 7 to give 31 (67 mg, 76%) as a powder. IR nmax(KBr)
3285, 3064, 3005, 2955, 2923, 2853, 2327, 1716, 1657,
n
max(CHCl3) 3598, 3435, 3317, 3089, 2955, 2931, 2873,
2858, 1746, 1651 cmK1. 500 MHz 1H NMR (CDCl3) d 0.88
(3H, t, JZ6.8 Hz), 1.26–1.50 (12H, m), 1.67–1.78 (2H, m),
3.26 (3H, s), 3.32 (1H, m), 3.63–3.73 (2H, m), 3.86–3.94
(2H, m), 4.18 (1H, m), 4.30–4.38 (3H, m), 4.51–4.63 (7H,
m), 4.67, 4.74 (2H, AB-q, JZ11.7 Hz), 5.24–5.40 (7H, m),
5.81 (1H, d, JZ8.8 Hz, NH), 5.89–5.98 (3H, m). FABMS
(positive-ion) m/z, 790 [MCNa]C, 768 [MCH]C.
HRFABMS, calcd for C30H50Cl3NO13P: 768.2085. Found:
768.2089.
1
1632 cmK1. 500 MHz H NMR (CDCl3CCD3OD) d 0.90
(12H, t, JZ6.8 Hz), 1.29–1.46 (70H, m), 1.56–1.74 (8H,
m), 2.01–2.04 (4H, m), 2.24–2.28 (2H, m), 3.20 (1H, dd, JZ
3.9, 9.8 Hz), 3.30 (3H, s), 3.31–3.40 (2H, m), 3.45–3.49
(2H, m), 3.53–3.56 (1H, m), 3.62–3.88 (12H, m), 4.07–4.17
(4H, m), 4.49 (1H, d, JZ8.8 Hz), 4.92 (1H, d, JZ3.9 Hz),
5.33–5.35 (2H, m). ESIMS (negative-ion) m/z, 1314
[MKH]K. HRESIMS, calcd for C67H130NO19P2:
1314.8708. Found: 1314.8694. Anal. Calcd for
C67H131NO19P2: C, 61.12; H, 10.03; N, 1.06; P, 4.70.
Found: C, 60.98; H, 10.01; N, 1.10; P, 4.62.
4.1.24. 2-(Diallylphosphonooxy)ethyl 6-O-{6-O-allyloxy-
carbonyl-2-deoxy-4-O-diallylphosphono-3-O-[(R)-3-
methoxydecyl]-2-(2,2,2-trichloroethoxycarbonylamino)-
b-D-glucopyranosy}-2,3-di-O-dodecyl-a-D-glucopyrano-
side (29). To a solution of 28 (164 mg, 0.213 mmol) in
CH2Cl2 (5 ml) were added trichloroacetonitrile (0.15 ml,
1.4 mmol) and Cs2CO3 (15 mg). After stirring for 1 h at
room temperature, this solution was diluted with CH2Cl2,
and washed with aq satd NaHCO3 and brine, dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo
to give an imidate, which was dissolved in CH2Cl2 (5 ml).
To this solution were added a solution of 18 (154 mg,
4.1.27. Allyl 2-O-acetyl-3-O-decyl-4,6-O-isopropylidene-
a-D-glucopyranoside (33). A solution of 32 (1.50 g,
4.244 mmol) in pyridine (5 ml) was allowed to stand for
5 h at room temperature, and concentrated in vacuo to give a
residue, which was chromatographed on a silica gel column.
Elution with cyclohexane–EtOAc (3:1) gave 33 (1.50 g,
99%) as an oil. IR nma1x(film) 2995, 2926, 2857, 1748, 1647
(w) cmK1. 400 MHz H NMR (CDCl3) d 0.88 (3H, t, JZ
6.9 Hz), 1.26 (16H, bs), 1.41 (3H, s), 1.50 (3H, s), 2.11 (3H,
s), 3.54 (1H, m), 3.60–3.77 (5H, m), 3.84 (1H, m), 3.98 (1H,
dd, JZ6.6, 13.2 Hz), 4.16 (1H, dd, JZ5.5, 13.5 Hz), 4.77
˚
0.213 mol) and molecular sieves 4 A (150 mg) under
nitrogen. After stirring for 30 min at room temperature, to
this mixture was added TMSOTf (10 mg) at 0 8C. After
stirring for 16 h at rt, the reaction mixture was quenched

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5115
(1H, dd, JZ4.5, 9.5 Hz), 5.00 (1H, d, JZ3.7 Hz), 5.20–5.32
(2H, m), 5.88 (1H, m). FABMS (positive-ion) m/z, 443
[MCH]C, 465 [MCNa]C. HRFABMS, calcd for
C24H42O7Na: 465.2828. Found: 465.2810.
and brine, dried over MgSO4 and filtered. The filtrate was
concentrated in vacuo to give a mixture, which was
chromatographed on a silica gel column. Elution with
cyclohexane-EtOAc (1:1) gave 36 (464 mg, 75%) as a gum.
IR nmax(film) 2926, 2856, 1750, 1719, 1650 (w), 1627 (w)
cmK1. 400 MHz 1H NMR (CDCl3) d 0.88 (6H, JZ6.6 Hz),
1.26 (30H, bs), 1.39 (3H, s), 1.40–1.63 (7H, m, containing
3H, s, at d 1.49 ppm), 2.53 (2H, t, JZ7.3 Hz), 3.40–3.54
(3H, m), 3.61–3.75 (6H, m), 3.82–3.88 (2H, m), 4.19–4.23
(2H, m) 4.56–4.59 (4H, m), 4.77 (1H, dd, JZ3.7, 9.5 Hz),
5.03 (1H, d, JZ4.4 Hz), 5.26–5.41 (4H, m), 5.93–6.00 (2H,
m). FABMS (positive-ion) m/z, 789 [MCH]C, 811 [MC
Na]C. HRFABMS, calcd for C41H73O12PNa: 811.4737.
Found: 811.4724.
4.1.28. 2-Hydroxyethyl 2-O-acetyl-3-O-decyl-4,6-O-iso-
propylidene-a-D-glucopyranoside (34). To a solution of
33 (2.00 g, 4.519 mmol) in THF–H2O (3:1, 40 ml) were
added NaIO4 (5.70 g, 26.65 mmol) and a 2.5% solution of
OsO4 in tert-BuOH (900 mg). This mixture was stirred for
2 h at room temperature, and diluted with EtOAc, washed
with aq satd NaHCO3 and brine, dried over MgSO4 and
filtered. The filtrate was concentrated in vacuo to give an
aldehyde, which was dissolved in EtOH (40 ml). To this
solution was added NaBH4 (200 mg, 5.286 mmol), and the
solution was stirred for 10 min, quenched with AcOH
(300 mg), diluted with EtOAc, washed with aq satd
NaHCO3 and brine, dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo to give a mixture, which
was chromatographed on a silica gel column. Elution with
hexane–EtOAc (3:2) gave alcohol 34 (1.43 g, 71%) as an
4.1.31. 2-(Diallylphosphonooxy)ethyl 3-O-decyl-2-O-(3-
oxotetradecanoyl)-a-D-glucopyranoside (37). A solution
of 36 (460 mg, 0.583 mmol) in aq 80% AcOH (46 ml) was
heated at 85 8C for 1.5 h, and concentrated in vacuo to give a
residue, which was chromatographed on a silica gel column.
Elution with EtOAc–MeOH (19:1) gave 37 (374 mg, 86%)
as a gum. IR nmax(film) 3407 (br), 2925, 2855, 1747, 1718,
oil. IR nmax(film) 3484, 2995, 2926, 2857, 1748 cmK1
.
400 MHz 1H NMR (CDCl3CD2O) d 0.88 (3H, t, JZ
6.8 Hz), 1.26 (16H, bs), 1.42 (3H, s), 1.48–1.52 (5H, m,
containing 3H, s, at d 1.50 ppm), 2.11 (3H, s), 3.53–3.87
(11H, m), 4.80 (1H, dd, JZ3.9, 9.8 Hz), 5.00 (1H, d, JZ
3.9 Hz). FABMS (positive-ion) m/z, 447 [MCH]C, 469
[MCNa]C. HRFABMS, calcd for C23H42O8Na: 469.2777.
Found: 469.2778.
1650 (w), 1465 cmK1. 400 MHz H NMR (CDCl3) d 0.88
1
(6H, JZ6.6 Hz), 1.26 (30H, bs), 1.53–1.60 (4H, m), 2.51–
2.55 (3H, m, containing OH), 2.66 (1H, d, JZ2.9 Hz, OH),
3.46–3.90 (11H, m, containing 2H, s at d 3.50 ppm), 4.20–
4.25 (2H, m) 4.55–4.59 (4H, m), 4.72 (1H, dd, JZ3.7,
9.5 Hz), 5.08 (1H, d, JZ3.7 Hz), 5.26–5.41 (4H, m), 5.91–
5.99 (2H, m). FABMS (positive-ion) m/z, 749 [MCH]C,
771 [MCNa]C. HRFABMS, calcd for C38H69O12PNa:
771.4425. Found: 771.4418.
4.1.29. 2-(Diallylphosphonooxy)ethyl 3-O-decyl-4,6-O-
isopropylidene-a-D-glucopyranoside (35). A solution of
34 (430 mg, 0.972 mmol) in EtOH (99.5%, 15 ml) contain-
ing KOH (10 mg) was stirred for 5 h at room temperature,
concentrated in vacuo and diluted with EtOAc, which was
washed with brine, dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo to give a residue, which
was dissolved in CH2Cl2 (10 ml). To this solution Na2SO4
(300 mg), 1H-tetrazole (102 mg, 1.457 mmol) and diallyl
diisopropylphosphoramidite (262 mg, 1.069 mmol) were
added at room temperature. After stirring for 20 min, THF
(10 ml) and aq 30% H2O2 (ca. 200 mg) were added. After
stirring for 15 min at room temperature, the reaction
mixture was diluted with EtOAc, washed with aq 10%
Na2S2O3 and brine, dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo to give a mixture, which
was chromatographed on a silica gel column. Elution with
hexane–EtOAc gave alcohol 35 (430 mg, 79%) as an oil. IR
4.1.32. 2-(Diallylphosphonooxy)ethyl 3-O-decyl-6-O-{2-
deoxy-4-O-diallylphosphono-3-O-[(R)-3-methoxydecyl]-
6-O-methyl-2-(2,2,2-trichloroethoxycarbonylamino)-b-
D-glucopyranosyl}-2-O-(3-oxotetradecanoyl)-a-D-gluco-
pyranoside (38). Compound 37 (374 mg, 0.499 mmol) was
treated as described for the formation of 19 from 5 and 18 to
give 38 (486 mg, 68%) as a gum after silica gel column
chromathography with EtOAc. IR nmax(film) 3292 (br),
3085 (w), 2927, 2856, 1748, 1721, 1650 (w), 1545,
1460 cmK1. 400 MHz 1H NMR (CDCl3) d 0.88 (9H, t,
JZ6.6 Hz), 1.26 (42H, bs), 1.40–1.80 (8H, m), 2.52 (1H, t,
JZ7.7 Hz), 2.80 (1H, bs, OH), 3.24–3.84 (24H, m,
containing two 3H at d 3.29 and 3.39 ppm), 4.09–4.32
(4H, m), 4.53–4.59 (8H, m), 4.69–4.71 (3H, m), 4.87 (1H,
m), 5.03 (1H, d, JZ3.7 Hz), 5.24–5.41 (8H, m), 5.90–5.99
(4H, m), 6.36 (1H, bs, NH). FABMS (positive-ion) m/z,
1428 [MCH]C, 1450 [MCNa, 35Cl]C. HRFABMS, calcd
for C65H114Cl3NO22P2Na: 1450.6298. Found: 1450.6301.
n
max(film) 3409, 2994, 2926, 2857, 1744 cmK1. 400 MHz
1H NMR (CDCl3) d 0.88 (3H, JZ6.6 Hz), 1.26 (14H, bs),
1.40 (3H, s), 1.49 (3H, s), 1.55–1.59 (2H, m), 3.09 (1H, d,
JZ8.1 Hz, OH), 3.44–3.94 (10H, m), 4.23–4.28 (2H, m)
4.55–4.60 (4H, m), 4.88 (1H, d, JZ3.7 Hz), 5.25–5.30 (2H,
m), 5.36–5.43 (2H, m), 5.91–6.02 (2H, m).
4.1.33. 2-(Diallylphosphonooxy)ethyl 3-O-decyl-6-O-{2-
deoxy-4-O-diallylphosphono-3-O-[(R)-3-methoxydecyl]-
6-O-methyl-2-[(Z)-11-octadecenoylamino]-b-D-gluco-
pyranosyl}-2-O-(3-oxotetradecanoyl)-a-D-glucopyrano-
side (39). Compound 38 was treated as described for the
formation of 7 from 6 to give 39 (81%) as a gum. IR
nmax(film) 3302, 3086 (w), 2926, 2855, 1746, 1719, 1654,
1553, 1465 cmK1. 400 MHz 1H NMR (CDCl3) d 0.88 (12H,
t, JZ6.6 Hz), 1.26 (60H, bs), 1.40–1.80 (10H, m), 1.99–
2.02 (4H, m), 2.17–2.27 (2H, m), 2.52 (2H, m), 3.20–3.91
(26H, m, containing two 3H, s, at 3.29 and 3.38 ppm), 4.07–
4.27 (6H, m), 4.54–4.57 (8H, m), 4.70 (1H, dd, JZ3.7,
4.1.30. 2-(Diallylphosphonooxy)ethyl 3-O-decyl-4,6-O-
isopropylidene-2-O-(3-oxotetradecanoyl)-a-D-gluco-
pyranoside (36). To a solution of 35 (440 mg, 0.779 mmol)
in CH2Cl2 (30 ml) were added 3-oxotetradecanoic acid
(208 mg, 0.858 mmol) and WSC$HCl (179 mg,
0.935 mmol). The mixture was stirred for 40 min, and
concentrated in vacuo to give a residue, which was diluted
with EtOAc. The solution was washed with aq satd NaHCO3

5116
M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
9.5 Hz), 5.02 (1H, d, JZ3.7 Hz), 5.19 (1H, d, JZ8.1 Hz),
5.23–5.40 (10H, m), 5.88–5.99 (4H, m), 6.51 (1H, d, JZ
6.6 Hz, NH). FABMS (positive-ion) m/z, 1518 [MCH]C,
1540 [MCNa]C. HRFABMS, calcd for C80H145NO21P2Na:
1540.9682. Found: 1540.9653.
3.80 ppm), 3.86 (1H, dt, JZ4.9, 9.8 Hz), 4.07 (1H, dd, JZ
6.8, 12.7 Hz), 4.20 (1H, dd, JZ5.9, 12.7 Hz), 4.26 (1H, dd,
JZ4.9, 9.8 Hz), 4.43, 4.47 (2H, ABq, JZ11.7 Hz), 4.96
(1H, d, JZ3.9 Hz), 5.21–5.35 (2H, m), 5.55 (1H, s), 5.92
(1H, m), 6.87 (2H, d, JZ7.8 Hz), 7.25–7.27 (2H, m), 7.33–
7.38 (3H, m), 7.59–7.50 (2H, m). FABMS (positive-ion):
m/z 831 (MCNa)C. HRFABMS, calcd for C50H80O8Na:
831.5755. Found: 831.5731.
4.1.34. 2-(Phosphonooxy)ethyl 3-O-decyl-6-O-{2-deoxy-
3-O-[(R)-3-methoxydecyl]-6-O-methyl-2-[(Z)-11-
octadecenoylamino]-4-O-phosphono-b-D-glucopyrano-
syl}-2-O-(3-oxotetradecanoyl)-a-D-glucopyranoside (40).
Compound 39 (200 mg, 0.132 mmol) was treated for 16 h at
55 8C as described for the formation of 8 from 7 to give 40
(150 mg, 84%) as a wax. IR nmax(KBr) 3286 (br), 2825,
4.1.37. 2-Hydroxyethyl 4,6-O-benzylidene-3-O-dodecyl-
2-O-[(R)-3-(4- methoxybenzyloxy)tetradecyl]-a-D-gluco-
pyranoside (44). To a solution of 43 (1.63 g, 2.01 mmol) in
acetone (16 ml) and H2O (4 ml) were added NaIO4 (1.28 g,
5.98 mmol) and OsO4 (2.5 wt% solution in t-BuOH, 1.3 ml,
0.104 mmol). After stirring for 4 h at room temperature, the
reaction mixture was quenched with aq satd Na2S2O3,
extracted with EtOAc, washed with water and brine, dried
over MgSO4 and filtered. The filtrate was concentrated in
vacuo to give a residue, which was dissolved in EtOH
(15 ml). To this solution was added NaBH4 (102 mg,
2.71 mmol) at 0 8C. After stirring for 1 h at room
temperature, the mixture was concentrated in vacuo, diluted
with EtOAc, washed with water and brine, dried over
MgSO4, filtered, concentrated in vacuo and chromato-
graphed on a silica gel column. Elution with hexane–EtOAc
(3:2) gave 44 (1.05 g, 64%) as a gum. IR nmax(CHCl3) 3516,
2928, 2856 cmK1. 500 MHz 1H NMR (CDCl3) d 0.88 (6H,
t, JZ6.8 Hz), 1.22–1.62 (40H, m), 1.77–1.83 (2H, m), 2.82
(1H, dd, JZ5.9, 6.8 Hz), 3.35 (1H, d, JZ3.9, 9.8 Hz), 3.50
(1H, dd, JZ8.8, 9.8 Hz), 3.53 (1H, m), 3.63 (1H, m), 3.68–
3.91 (13H, m, containing 3H, s, at 3.80 ppm), 4.27 (1H, dd,
JZ4.9, 9.8 Hz), 4.42, 4.47 (2H, ABq, JZ11.7 Hz), 4.95
(1H, d, JZ3.9 Hz), 5.54 (1H, s), 6.87 (2H, d, JZ7.8 Hz),
7.25–7.27 (2H, m), 7.33–7.38 (3H, m), 7.48–7.50 (2H, m).
FABMS (positive-ion): m/z 835 (MCNa)C.
1
2854, 1742, 1716, 1629, 1552, 1466 cmK1. 400 MHz H
NMR (CDCl3–CD3OD, 5:1) d 0.88 (12H, t, JZ6.6 Hz),
1.26 (60H, bs), 1.40–1.63 (8H, m), 1.72–1.77 (2H, m), 1.99–
2.04 (4H, m), 2.18–2.22 (2H, m), 2.53–2.57 (2H, m), 3.30–
3.83 (24H, m, containing two 3H, s at 3.30 and 3.41 ppm),
4.01–4.14 (4H, m), 4.65–4.72 (2H, m), 4.98 (1H, d, JZ
3.7 Hz), 5.30–5.36 (2H, m). FABMS (negative-ion) m/z,
1356 [MKH]K. Anal. Calcd for C68H129NO21P2. H2O: C,
59.33; H, 9.59; N, 1.02; P, 4.50. Found: C, 59.24; H, 9.66;
N, 1.20; P, 4.32.
4.1.35. Allyl 4,6-O-benzylidene-3-O-dodecyl-a-D-gluco-
pyranoside (42). To a solution of 41 (2.85 g, 7.33 mmol) in
DMF (30 ml) were added benzaldehyde dimethyl acetal
(3.3 ml) and p-toluenesulfonic acid monohydrate (140 mg).
The mixture was stirred for 16 h at room temperature,
diluted with ether, washed with aq. sat. NaHCO3 and brine,
dried over MgSO4 and filtered. The filtrate was concentrated
in vacuo to give a residue, which was chromatographed on
a silica gel column. Elution with hexane–EtOAc (5:1) gave
42 (3.05 g, 87%) as a solid. IR nmax(KBr) 3441, 1371,
1072 cmK1. 500 MHz 1H NMR (CDCl3) d 0.88 (3H, t, JZ
6.8 Hz), 1.23–1.33 (18H, m), 1.57–1.61 (2H, m), 2.29 (1H,
d, JZ6.8 Hz, OH), 3.52–3.56 (2H, m), 3.66–3.75 (3H, m),
3.84–3.90 (2H, m), 4.07 (1H, dd, JZ6.8, 12.7 Hz), 4.24
(1H, dd, JZ4.9, 12.7 Hz), 4.27 (1H, dd, JZ4.9, 9.8 Hz),
4.96 (1H, d, JZ2.9 Hz), 5.24 (1H, d, JZ10.7 Hz), 5.34 (1H,
dd, JZ2.0, 15.6 Hz), 5.55 (1H, s), 5.94 (1H, m), 7.33–7.39
(3H, m), 7.47–7.49 (2H, m). FABMS (positive-ion): m/z
477 (MCH)C, 499 (MCNa)C. HRFABMS, calcd for
C28H44O6Na: 499.3036. Found: 499.3040. Anal. Calcd for
C28H44O6: C, 70.56; H, 9.30. Found: C, 70.21; H, 9.00.
4.1.38. 2-(Diallylphosphonoxy)ethyl 4,6-O-benzylidene-
3-O-dodecyl-2-O-[(R)-3-(4-methoxybenzyloxy)tetra-
decyl]-a-D-glucopyranoside (45). To a solution of 44
(930 mg, 1.14 mmol) and 1H-tetrazole (157 mg,
2.25 mmol) in THF (10 ml) was added diallyl diisopropyl-
phosphoramidite (379 mg, 1.54 mmol). After stirring for 4 h
at room temperature, aq 30% H2O2 (1 ml) was added to the
reaction mixture at 0 8C. After stirring for 1 h at 0 8C, the
mixture was quenched with aq satd Na2S2O3, extracted with
EtOAc, washed with water, aq satd NaHCO3 and brine,
dried over MgSO4, filtered, and concentrated in vacuo and
chromatographed on a silica gel column. Elution with
hexane–EtOAc (1:1) gave 45 (1.07 g, 96%) as a gum.
500 MHz 1H NMR (CDCl3) d 0.88 (6H, t, JZ6.8 Hz), 1.21–
1.59 (40H, m), 1.73–1.85 (2H, m), 3.33 (1H, dd, JZ3.9,
8.8 Hz), 3.50 (1H, t, JZ9.8 Hz), 3.55 (1H, m), 3.66–3.91
(12H, m, containing 3H, s, at 3.80 ppm), 4.22–4.29 (3H, m),
4.42, 4.47 (2H, AB-q, JZ11.7 Hz), 4.54–4.57 (4H, m), 4.94
(1H, d, JZ3.9 Hz), 5.21–5.39 (4H, m), 5.54 (1H, s), 5.87–
5.98 (2H, m), 6.86–6.91 (2H, m), 7.25–7.27 (2H, m), 7.32–
7.38 (3H, m), 7.47–7.49 (2H, m). FABMS (positive-ion):
m/z 995 (MCNa)C, 973 (MCH)C.
4.1.36. Allyl 4,6-O-benzylidene-3-O-dodecyl-2-O-[(R)-3-
(4-methoxybenzyloxy)tetradecyl]-a-D-glucopyranoside
(43). To a solution of 42 (3.54 g, 7.43 mmol) in DMF
(100 ml) was gradually added NaH (60% oil dispersion,
1.62 g, 40.5 mmol) at 0 8C with stirring. After 15 min at
0 8C, (R)-3-(4-methoxybenzyloxy)tetradecyl methanesulfo-
nate (4.02 g, 9.38 mmol) was added to the mixture. After
stirring for 6 h at room temperature, the mixture was
quenched with water, extracted with EtOAc, washed with
water and brine, dried over MgSO4 and filtered. The filtrate
was concentrated in vacuo and chromatographed on a silica
gel column. Elution with hexane–EtOAc (4:1) gave 43
(4.01 g, 67%) as a white solid. IR nmax(KBr) 3067, 3038,
2920, 2851, 1615 cmK1. 500 MHz 1H NMR (CDCl3) d 0.88
(6H, t, JZ6.8 Hz), 1.21–1.59 (40H, m), 1.76–1.87 (2H, m),
3.34 (1H, dd, JZ3.9, 8.8 Hz), 3.51 (1H, dd, JZ8.8, 9.8 Hz),
3.56 (1H, m), 3.68–3.82 (9H, m, containing 3H, s, at
4.1.39. 2-(Diallylphosphonoxy)ethyl 4,6-O-benzylidene-
3-O-dodecyl-2-O-[(R)-3-hydroxytetradecyl]-a-D-gluco-
pyranoside (46). To a solution of 45 (1.04 g, 1.07 mmol) in
CH2Cl2 (10 ml) and H2O (1 ml) was added DDQ (291 mg,

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5117
1.28 mmol). After stirring for 1.5 h at room temperature, the
solution was diluted with CH2Cl2, washed with aq satd
NaHCO3 and brine, dried over MgSO4 and filtered. The
filtrate was concentrated in vacuo, and chromatographed on
a silica gel column. Elution with hexane-EtOAc (3:7) gave
46 (821 mg, 90%) as a gum. IR nmax(CHCl3) 3502, 2927,
2855, 1732 cm K1. 500 MHz 1H NMR (CDCl3) d 0.88 (6H,
t, JZ6.8 Hz), 1.23–1.59 (40H, m), 1.65–1.75 (2H, m), 3.19
(1H, d, JZ2.9 Hz, OH), 3.37 (1H, dd, JZ3.9, 9.8 Hz), 3.52
(1H, t, JZ9.8 Hz), 3.66–3.92 (10H, m), 4.23–4.29 (3H, m),
4.55–4.59 (4H, m), 5.01 (1H, d, JZ3.9 Hz), 5.22–5.24 (2H,
m), 5.33–5.37 (2H, m), 5.53 (1H, s), 5.89–5.97 (2H, m),
7.33–7.38 (3H, m), 7.46–7.48 (2H, m). FABMS (positive-
ion): m/z 875 (MCNa)C, 853 (MCH)C. HRFABMS, calcd
for C47H81O11PNa: 875.5413. Found: 875.5426.
to give a mixture, which was chromatographed on a silica
gel column. Elution with hexane–EtOAc (1:3) gave 49
(627 mg, 53%) as an amorphous solid. IR nmax(CHCl3)
3588, 3450, 3272, 3088, 2928, 2873, 2856, 1732, 1651 cmK1
.
500 MHz 1H NMR (CDCl3) d 0.88 (9H, t, JZ6.8 Hz), 1.25–
1.78 (52H, m), 2.42 (2H, dd, JZ6.8, 7.8 Hz), 2.60–2.73
(3H, m, containing 1H, OH), 3.26–3.36 (8H, m, containing
3H, s, at 3.28 ppm), 3.39 (3H, s), 3.44 (1H, t, JZ8.8 Hz),
3.54 (1H, m), 3.60–3.64 (2H, m), 3.68–3.89 (11H, m), 4.10
(1H, d, JZ11.7 Hz), 4.20 (1H, m), 4.25–4.31 (2H, m), 4.53–
4.59 (8H, m), 4.70, 4.77 (2H, AB-q, JZ12.8 Hz), 4.85 (1H,
d, JZ7.8 Hz), 4.92 (1H, d, JZ2.9 Hz), 5.24–5.39 (8H, m),
5.90–5.99 (4H, m), 6.45 (1H, bs, NH). FABMS (positive-
ion): m/z 1464 (MCNa)C. HRFABMS, calcd for C67H120
-
Cl3NO21P2Na: 1464.6798. Found: 1464.6812.
4.1.40. 2-(Diallylphosphonoxy)ethyl 4,6-O-benzylidene-
3-O-dodecyl-2-O-(3-oxotetradecyl)-a-D-glucopyranoside
(47). To a solution of 46 (810 mg, 0.950 mmol) in CH2Cl2
(10 ml) was added PCC (413 mg, 1.92 mmol). After stirring
for 5 h at room temperature, the solution was filtered
through Celite. The filtrate was concentrated in vacuo and
chromatographed on a silica gel column. Elution with
4.1.43. 2-(Diallylphosphonoxy)ethyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-[(Z)-11-octadecenoylamino]-b-D-glucopyranosyl}-3-O-
dodecyl-2-O-(3-oxotetradecyl)-a-D-glucopyranoside (50).
To a solution of 49 (432 mg, 0.300 mmol) in THF (9 ml) and
acetic acid (1 ml) was added zinc dust (776 mg,
11.872 mmol). After vigorously stirring for 4 h at room
temperature, the solution was filtered to remove the Zn
powder and concentrated in vacuo to give a crude product.
The product was diluted with EtOAc, washed with aq satd
NaHCO3 and brine, dried over Na2SO4, filtered and
concentrated in vacuo to give a crude product, which was
dissolved in CH2Cl2 (10 ml). (Z)-11-Octadecenoic acid
(198 mg, 0.697 mmol) and WSC$HCl (167 mg,
0.873 mmol) were added to this solution. After stirring for
10 h at room temperature, the mixture was diluted with
CH2Cl2, washed with water and brine, dried over MgSO4,
filtered and concentrated in vacuo to give a mixture, which
was chromatographed on a silica gel column. Elution with
hexane–EtOAc (1:9) gave 50 (256 mg, 56%) as an amor-
phous solid. IR nmax(CHCl3) 3605, 3453, 3316, 2928, 2856,
1712, 1662 cm K1. 500 MHz 1H NMR (CDCl3) d 0.88 (12H,
t, JZ6.8 Hz), 1.18–1.80 (74H, m), 1.99–2.01 (4H, m), 2.14–
2.26 (2H, m), 2.41 (2H, dd, JZ6.8, 7.8 Hz), 2.62, 2.70 (2H,
AB-q, t, JZ16.6, 6.8 Hz), 3.07 (1H, d, JZ2.9 Hz, OH), 3.20
(1H, m), 3.25–3.32 (5H, m, containing 3H, s, at 3.28 ppm),
3.35–3.40 (4H, m, containing 3H, s, at 3.38 ppm), 3.43 (1H,
dd, JZ8.8, 9.8 Hz), 3.56–3.88 (13H, m), 3.96 (1H, dd, JZ
8.8, 9.8 Hz), 4.09 (1H, d, JZ9.8 Hz), 4.17 (1H, m), 4.22–4.29
(2H, m), 4.53–4.58 (8H, m), 4.90 (1H, d, JZ2.9 Hz),
5.15 (1H, d, JZ7.8 Hz), 5.23–5.40 (10H, m). FABMS
(positive-ion): m/z 1554 (MCNa)C. HRFABMS, calcd for
C82H151NO20P2Na: 1555.0201. Found: 1555.0200.
hexane–EtOAc (3:7) gave 47 (753 mg, 93%) as a gum. IR
n
1
max(CHCl3) 2927, 2855, 1713 cmK1. 500 MHz H NMR
(CDCl3) d 0.88 (6H, t, JZ6.8 Hz), 1.23–1.30 (34H, m),
1.52–1.56 (4H, m), 2.43 (2H, dd, JZ6.8, 7.8 Hz), 2.64, 2.74
(2H, ABqt, JZ16.6, 6.8 Hz), 3.38 (1H, dd, JZ3.9, 9.8 Hz),
3.49 (1H, dd, JZ8.8, 9.8 Hz), 3.63–3.71 (3H, m), 3.75–3.78
(2H, m), 3.83–3.92 (4H, m), 4.22–4.28 (3H, m), 4.55–4.57
(4H, m), 4.95 (1H, d, JZ3.9 Hz), 5.22–5.24 (2H, m), 5.33–
5.37 (2H, m), 5.52 (1H, s), 5.89–5.97 (2H, m), 7.34–7.37
(3H, m), 7.46–7.48 (2H, m). FABMS (positive-ion): m/z
873 (MCNa)C, 851 (MCH)C. HRFABMS, calcd for
C47H79O11PNa: 873.5257. Found: 873.5265.
4.1.41. 2-(Diallylphosphonoxy)ethyl 3-O-dodecyl-2-O-(3-
oxotetradecyl)-a-D-glucopyranoside (48). Compound 47
was treated as described for the formation of 4 from 3 to
give diol 48 (92%) as an amorphous solid. IR nmax(CHCl3)
1
3599, 3409, 2927, 2872, 2855, 1713 cmK1. 500 MHz H
NMR (CDCl3) d 0.88 (6H, t, JZ6.8 Hz), 1.26–1.31 (34H,
m), 1.51–1.56 (4H, m), 1.72 (1H, bs, OH), 2.43 (2H, t, JZ
6.8, 7.8 Hz), 2.62 (1H, bs, OH), 2.64, 2.71 (2H, ABqt, JZ
16.6, 6.8 Hz), 3.30 (1H, dd, JZ2.9, 9.8 Hz), 3.44 (1H, t, JZ
8.8 Hz), 3.50 (1H, t, JZ8.8 Hz), 3.58 (1H, m), 3.72–3.91
(8H, m), 4.20–4.30 (2H, m), 4.55–4.59 (4H, m), 4.97 (1H, d,
JZ2.9 Hz), 5.25–5.40 (4H, m), 5.91–5.99 (2H, m). FABMS
(positive-ion): m/z 785(MCNa)C. HRFABMS, calcd for
C40H75O11PNa: 785.4943. Found: 785.4951.
4.1.44. 2-(Phosphonoxy)ethyl 6-O-{2-deoxy-3-O-[(R)-3-
methoxydecyl]-6-O-methyl-2-[(Z)-11-octadecenoyl-
amino]-4-O-phosphono-b-D-glucopyranosyl}-3-O-do-
decyl-2-O-(3-oxotetradecyl)-a-D-glucopyranoside (51).
Compound 50 (204 mg, 0.133 mmol) was treated as
described for the formation of 8 from 7 to give 51
(164 mg, 90%) as a white powder. IR nmax(KBr) 3287,
3070, 2954, 2925, 2854, 2324, 1714, 1629 cm K1. 500 MHz
1H NMR (CDCl3) d 0.90 (12H, t, JZ6.8 Hz), 1.30–1.67
(72H, m), 1.72–1.76 (2H, m), 1.99–2.04 (4H, m), 2.20–2.30
(2H, m), 2.50 (2H, t, JZ7.3 Hz), 2.67–2.69 (2H, m), 3.23
(1H, dd, JZ3.9, 9.8 Hz), 3.29–3.31 (4H, m, containing 3H,
s, at 3.29 ppm), 3.35 (1H, t, JZ8.8 Hz), 3.39 (3H, s), 3.42
4.1.42. 2-(Diallylphosphonoxy)ethyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyra-
nosyl}-3-O-dodecyl-2-O-(3-oxotetradecyl)-a-D-gluco-
pyranoside (49). A solution of imidate 5 (844 mg,
1.00 mmol), diol 48 (626 mg, 0.821 mmol) and molecular
˚
sieves 4 A (590 mg) in CH2Cl2 (10 ml) was stirred at room
temperature. After stirring for 1 h, cat. TMSOTf (6 ml,
0.033 mmol) was added to the mixture at 0 8C. After stirring
for 1 h at 0 8C, the mixture was quenched with aq satd
NaHCO3, diluted with CH2Cl2, washed with water and
brine, dried over MgSO4, filtered and concentrated in vacuo

5118
M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
(1H, dd, JZ8.8, 9.8 Hz), 3.52–3.77 (11H, m), 3.79–3.91
(5H, m), 4.04–4.15 (4H, m), 4.48 (1H, d, JZ8.8 Hz), 4.93
(1H, d, JZ3.9 Hz), 5.31–5.37 (2H, m). ESIMS (negative-
ion): m/z 1370 (MKH)K. HRESIMS, calcd for
C70H134NO20P2: 1370.8966. Found: 1370.8944.
glucopyranoside (55). Compound 54 was treated as
described for the formation of 19 from imidate 5 and 18
to give compound 55 as a gum in 67% yield. IR nmax(film)
3500–3250, 2926, 2856, 1746, 1547, 1464 cmK1. 400 MHz
1H NMR (CDCl3) d 0.88 (9H, t, JZ6.6 Hz), 1.26 (51H, bs),
1.38–1.77 (8H, m), 2.72 (1H, bs, OH), 3.13 (1H, bs, OH),
3.21–3.90 (24H, m, containing two 3H, s, at d 3.28 and
3.39 ppm), 4.07–4.30 (4H, m), 4.53–4.60 (8H, m), 4.65–
4.90 (3H, m), 4.95 (1H, d, JZ3.7 Hz), 5.24–5.40 (8H, m),
5.89–5.99 (4H, m), 6.40 (1H, bs, NH). FABMS (positive-
ion): m/z 1466 (MCNa)C, 1468. HRFABMS, calcd for
C67H122Cl3NO21P2Na: 1466.6984. Found: 1466.6962.
4.1.45. 2-Hydroxyethyl 2-O-[(R)-3-(tert-butyldimethyl-
silyloxy)tetradecyl]-3-O-dodecyl-4,6-O-isopropylidene-
a-D-glucopyranoside (52). To a solution of 9 (1.45 g,
1.925 mmol) in THF (35 ml) and H2O (10 ml) were added
NaIO4 (3.00 g, 14.03 mmol) and OsO4 (2.5 wt% solution in
t-BuOH, 1.3 ml, 1.04 mmol). After stirring for 2 h at room
temperature, the reaction mixture was diluted with ether,
washed with aq satd NaHCO3 and brine, dried over MgSO4
and filtered. The filtrate was concentrated in vacuo to give a
residue, which was dissolved in EtOH (40 ml). To this
solution was added NaBH4 (150 mg, 3.965 mmol) at 0 8C.
After stirring for 20 min at 0 8C, diluted with EtOAc,
washed with aq satd NaHCO3 and brine, dried over MgSO4
and filtered. The filtrate was concentrated in vacuo to give a
residue, which was chromatographed on a silica gel column.
Elution with hexane–EtOAc (4:1) gave 52 (598 mg, 41%) as
a gum. IR nmax(film) 3468 (br), 2926, 2856, 1465, 1380,
1370 cmK1. 400 MHz 1H NMR (CDCl3) d 0.04 (6H, s), 0.88
(9H, s), 0.89 (6H, t, JZ6.3 Hz), 1.26 (36H, bs), 1.40 (3H, s),
1.41–1.43 (2H, m), 1.48 (3H, s), 1.52–1.56 (2H, m), 1.71–
1.75 (2H, m), 2.82 (1H, br, OH), 3.30 (1H, m), 3.51–3.85
(14H, m), 4.89 (1H, d, JZ3.7 Hz). FABMS (positive-ion):
m/z 759 (MCH)C, 781 (MCNa)C. HRFABMS, calcd for
C43H86O8SiNa: 781.5990. Found: 781.5977.
4.1.49. 2-(Diallylphosphonooxy)ethyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-[(Z)-11-octadecenoylamino]-b-D-glucopyranosyl}-3-O-
dodecyl-2-O-[(R)-3-hydroxytetradecyl]-a-D-glucopyra-
noside (56). Compound 55 was treated as described for
the formation of 7 from 6 to give 56 (82%) as a gum. IR
nmax(film) 3398 (br), 2925, 2855, 1640, 1555, 1465 cmK1
.
400 MHz 1H NMR (CDCl3) d 0.88 (12H, t, JZ6.6 Hz), 1.26
(68H, bs), 1.40–1.79 (10H, m), 1.99–2.04 (4H, m), 2.17–
2.21 (2H, m), 3.18 (1H, bs, OH), 3.20 (1H, m), 3.27–3.95
(26H, m, containing two 3H, s, at d 3.28 and 3.38 ppm),
4.07–4.27 (4H, m), 4.55–4.65 (8H, m), 4.94 (1H, d, JZ
3.7 Hz), 5.13 (1H, d, JZ8.1 Hz), 5.25–5.39 (10H, m), 5.90–
5.99 (4H, m), 6.58 (1H, d, JZ7.3 Hz, NH). FABMS
(positive-ion): m/z 1556 (MCNa)C. HRFABMS, calcd for
C82H153NO20P2Na: 1557.0359. Found: 1557.0341.
4.1.50. 2-(Phosphonoxy)ethyl 6-O-{2-deoxy-3-O-[(R)-3-
methoxydecyl]-6-O-methyl-2-[(Z)-11-octadecenoyl-
amino]-4-O-phosphono-b-D-glucopyranosyl}-3-O-do-
decyl-2-O-[(R)-3-hydroxytetradecyl]-a-D-glucopyrano-
side (57). Compound 56 was treated as described for the
formation of 21 from 20 to give 57 (67%) as a powder. IR
4.1.46. 2-(Diallylphosphonooxy)ethyl 2-O-[(R)-3-(tert-
butyldimethylsilyloxy)tetradecyl]-3-O-dodecyl-4,6-O-
isopropylidene-a-D-glucopyranoside (53). Compound 52
(428 mg, 0.654 mmol) was treated as described for the
formation of 17 from 16 to give 53 (504 mg, 97%) as a gum.
1
IR nmax(film) 2927, 2856, 1464 cmK1.400 MHz H NMR
nmax(KBr) 3289 (br), 2924, 2853, 1629, 1554 cmK1
.
1
(CDCl3) d 0.04 (6H, s), 0.88 (15H, bs), 1.26 (36H, bs), 1.39
(3H, s), 1.41–1.43 (2H, m), 1.48 (3H, s), 1.51–1.55 (2H, m),
1.64–1.76 (2H, m), 3.28 (1H, m), 3.41–3.89 (12H, m), 4.22–
4.27 (2H, m), 4.56–4.59 (4H, m), 4.88 (1H, d, JZ3.7 Hz),
5.21–5.41 (4H, m), 5.91–6.01 (2H, m). FABMS (positive-
ion): m/z 919 (MCH)C, 941 (MCNa)C. HRFABMS, calcd
for C49H96O11PSi: 919.6459. Found: 919.6484.
400 MHz H NMR (CDCl3) d 0.90 (12H, t, JZ6.6 Hz),
1.26 (68H, bs), 1.40–1.77 (10H, m), 1.98–2.04 (4H, m),
2.17–2.22 (2H, m), 3.23–3.85 (26H, m, containing two 3H,
s, at d 3.30 and 3.40 ppm), 4.00–4.18 (4H, m), 4.69 (1H, d,
JZ8.1 Hz), 4.94 (1H, d, JZ3.7 Hz), 5.33–5.38 (2H, m).
FABMS (negative-ion): m/z 1372 (MKH)K. HRFABMS,
calcd for C70H136NO20P2: 1372.9131. Found: 1372.9121.
Anal. Calcd for C70H137NO20P2: C, 60.36; H, 10.06; N,
1.01; P, 4.45. Found: C, 60.11; H, 10.15; N, 1.28; P, 4.34.
4.1.47. 2–(Diallylphosphonooxy)ethyl 3-O-dodecyl-2-O-
[(R)-3-hydroxytetradecyl]-a-D-glucopyranoside (54).
Compound 53 (489 mg, 0.532 mmol) was treated as
described for the formation of 18 from 17 to give triol 54
(340 mg, 84%) as a gum. IR nmax(film) 3388 (br), 2918,
2850, 1467 cmK1. 400 MHz 1H NMR (CDCl3) d 0.88 (6H,
t, JZ6.6 Hz), 1.26 (36H, bs), 1.41–1.73 (6H, m), 2.56 (1H,
m, OH), 2.68 (1H, d, JZ2.9 Hz, OH), 3.12 (1H, bs, OH),
3.29 (1H, m), 3.44–3.92 (12H, m), 4.22–4.28 (2H, m), 4.55–
4.59 (4H, m), 5.00 (1H, d, JZ3.7 Hz), 5.25–5.28 (2H, m),
5.35–5.41 (2H, m), 5.90–6.00 (2H, m). FABMS (positive-
ion): m/z 765 (MCH)C, 787 (MCNa)C. HRFABMS, calcd
for C40H77O11PNa: 787.5101. Found: 787.5118.
4.1.51. 2-Bromoethyl 2,3-di-O-dodecyl-4,6-O-isopropyl-
idene-a-D-glucopyranoside (58). To a solution of 16
(1.10 g, 1.83 mmol) in CH2Cl2 (10 mL) was added CBr4
(731 mg, 2.20 mmol) and PPh3 (671 mg, 2.56 mmol). After
stirring for 2 h at room temperature, the solution was diluted
with CH2Cl2, washed with water and brine, dried over
MgSO4 and filtered. The filtrate was concentrated in vacuo,
and chromatographed on a silica gel column. Elution with
hexane–EtOAc (9:1) gave 58 (1.20 g, 99%) as a gum. IR
n
max(CHCl3) 2927, 2855 cmK1. 500 MHz 1H NMR
(CDCl3) d 0.88 (6H, t, JZ6.8 Hz), 1.25–1.60 (46H, m,
containing two 3H, s, d 1.41 and 1.48 ppm), 3.30 (1H, dd,
JZ2.9, 8.8 Hz), 3.50–3.57 (4H, m), 3.59–3.75 (6H, m),
3.83–3.96 (3H, m), 4.91 (1H, d, JZ3.9 Hz). FABMS
(positive-ion) m/z 685 (MCNa)C. HRFABMS, calcd for
C35H67BrO6Na: 685.4002. Found: 685.4056.
4.1.48. 2-(Diallylphosphonooxy)ethyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyra-
nosyl}-3-O-dodecyl-2-O-[(R)-3-hydroxytetradecyl]-a-D-

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5119
4.1.52. 2-(Diallylphosphono)ethyl 2,3-di-O-dodecyl-a-D-
glucopyranoside (59). A mixture of 58 (1.20 g, 1.81 mmol)
and triallylphosphite (5 mL) was stirred at 180 8C for 3 h.
The mixture was cooled to room temperature, and
chromatographed on a silica gel short column. Elution
with hexane-EtOAc (2:3) gave a compound, which was
dissolved in aq 80% AcOH (15 mL) and stirred at 60 8C for
1 h. The solution was diluted with EtOAc, washed with aq
satd NaHCO3 and brine, dried over MgSO4, filtered. The
filtrate was concentrated in vacuo and chromatographed on
a silica gel column. Elution with CHCl3–EtOH (20:1) gave
59 (760 mg, 60%) as a white powder. IR nmax(CHCl3) 3598,
3420, 3089, 2927, 2855 cmK1. 500 MHz 1H NMR (CDCl3)
d 0.88 (6H, t, JZ6.8 Hz), 1.26–1.61 (40 H, m), 2.28(1H, bs,
OH), 2.58 (1H, bs, OH), 3.27 (1H, dd, JZ2.9, 9.8 Hz),
3.44–3.53 (3 H, m), 3.57–3.62 (2H, m), 3.70–3.80 (3H, m),
3.84–3.92 (2H, m), 3.98 (1H, m), 4.51–4.60 (4H, m), 4.90
(1H, d, JZ2.9 Hz), 5.24–5.38 (4H, m), 5.90–5.98 (2H, m).
FABMS (positive-ion) m/z 727 (MCNa)C; HRFABMS,
calcd for C38H73O9PNa: 727.4889. Found: 727.4892.
CH2Cl2, washed with water and brine, dried over MgSO4,
filtered and concentrated in vacuo to give a mixture, which
was chromatographed on a silica gel column. Elution with
EtOAc gave 61 (122 mg, 52%) as an amorphous compound.
IR nmax(CHCl3) 3004, 2928, 2856, 1662 cmK1. 500 MHz
1H NMR (CDCl3) d 0.88 (12H, t, JZ6.8 Hz), 1.25–1.80
(76H, m), 1.99–2.01 (4H, m), 2.16–2.33 (4H, m), 2.85 (1H,
d, JZ3.9 Hz, OH), 3.07 (1H, m), 3.23–3,25 (2H,m), 3.29
(3H, s), 3.36 (1H, m), 3.38 (3H, s), 3.44 (1H, t, JZ8.8 Hz),
3.48–3.84 (12H, m), 3.91 (1H, m), 4.09 (1H, dd, JZ9.8,
8.8 Hz), 4.17 (1H, d, JZ10.7 Hz), 4.25 (1H, dd, JZ
9.8,8.8 Hz), 4.50–4.58 (8H, m), 4.85 (1H, d, JZ3.9 Hz),
5.19 (1H, d, JZ7.8 Hz), 5.23–5.38 (10H, m), 5.89–5.98
(4H,m), 6.91 (1H, d, JZ6.8 Hz, NH). FABMS (positive-
ion) m/z 1496 (MCNa)C; HRFABMS, calcd for
C80H149NO18P2Na: 1497.0139. Found: 1497.0123.
4.1.55. 2-(Phosphono)ethyl 6-O-{2-deoxy-3-O-[(R)-3-
methoxydecyl]-6-O-methyl-2-[(Z)-11-octadecenoyl-
amino]-4-O-phosphono-b-D-glucopyranosyl}-2,3-di-O-
dodecyl-a-D-glucopyranoside (62). Compound 61
(110 mg, 0.075 mmol) was treated as described in the
formation of 8 from 7 to give 62 (80 mg, 82%) as a white
powder. IR nmax(KBr) 3284, 3218, 3178, 3116, 2955,
2924, 2853, 1655, 1630 cm K1. 500 MHz 1H NMR
(CD3OD:CDCl3Z5:1) d 0.90 (12H, t, JZ6.8 Hz), 1.28–
1.67 (74H, m), 1.73–1.77 (2H, m), 2.01–2.03 (4H, m), 2.16
(2H, dt, JZ18.6, 7.8 Hz), 2.25 (2H, t, JZ6.8 Hz), 3.22 (1H,
dd, JZ2.9, 9.8 Hz), 3.31 (3H, s), 3.34 (1H, m), 3.41 (3H, s),
3.46 (1H, dd, JZ9.8, 8.8 Hz), 3.54–3.85 (15H, m), 3.96
(1H, m), 4.07–4.12 (2H, m), 4.54 (1H, d, JZ8.8 Hz). 4.87
(1H, d, JZ3.9 Hz), 5.31–5.37 (2H, m).
4.1.53. 2-(Diallylphosphono)ethyl 6-O-{2-deoxy-4-O-dia-
llylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyrano-
syl}-2,3-di-O-dodecyl-a-D-glucopyranoside (60). To a
solution of imidate 5 (338 mg, 0.401 mmol) and diol 59
(189 mg, 0.268 mmol) were dissolved in CH2Cl2 (5 mL). To
this solution was added AgOTf (155 mg, 0.604 mmol) at
room temperature. After stirring for 1 h at room tempera-
ture, the mixture was quenched with aq. sat. NaHCO3,
diluted with CH2Cl2, washed with water and brine, dried
over MgSO4, filtered, and concentrated in vacuo to give a
mixture, which was chromatographed on a silica gel
column. Elution with hexane–EtOAc (1:9) gave 60
(273 mg, 74%) as an amorphous compound. IR
4.1.56. 3-Hydroxypropyl 2,3-di-O-dodecyl-4,6-O-isopro-
pylidene-a-D-glucopyranoside (63). To a solution of 15
(3.30 g, 5.53 mmol) in THF (25 ml) was added 9-BBN
(0.5 M solution in THF, 28 ml, 14.0 mmol). After stirring
for 18 h at room temperature, aq 3 M NaOH (50 ml) and aq
30% H2O2 (18 ml) were added to the reaction mixture at
0 8C. After stirring for 3 h at room temperature, the mixture
was extracted with EtOAc, washed with satd NH4Cl and
brine, dried over MgSO4 and filtered. The filtrate was
concentrated in vacuo, and chromatographed on a silica gel
column. Elution with hexane–EtOAc (7:3) gave 63 (3.03 g,
n
max(CHCl3) 3449, 3255, 3088, 2928, 2873, 2856,
1734 cmK1. 500 MHz 1H NMR (CDCl3) d 0.88 (9H, t,
JZ6.8 Hz), 1.26–1.80 (54H, m), 2.24–2.32 (2H, m), 2.55
(1H, s, OH), 3.21–3.36 (7H, m, containing 3H, s, d 3.29),
3.39 (3H, s), 3.42–3.66 (7H, m), 3.71–3.88 (6H, m), 3.92–
3.99 (2H, m), 4.16 (1H, d, JZ10.7 Hz), 4.30 (1H, q, JZ8.8,
9.8 Hz), 4.53–4.59 (8H, m), 4.70, 4.78 (2H, AB-q, JZ
11.7 Hz), 4.87 (1H, d, JZ2.9 Hz), 4.93 (1H, d, JZ7.8 Hz),
5.23–5.39 (8H, m), 5.88–5.98 (4H, m), 6.75 (1H, d, JZ
6.8 Hz, NH). FABMS (positive-ion) m/z 1406 (MCNa)C.
HRFABMS, calcd for C65H118Cl3NO19P2Na: 1406.6732.
Found: 1406.6720.
89%) as a gum. IR nmax(CHCl3) 3506, 2927, 2855 cmK1
.
500 MHz 1H NMR (CDCl3) d 0.88 (6H, t, JZ6.8 Hz), 1.25–
1.94 (48H, m, containing two 3H, s, at 1.40 and 1.48 ppm),
2.88 (1H, dd, JZ4.9, 6.8 Hz, OH), 3.28 (1H, dd, JZ3.9,
7.8 Hz), 3.49–3.85 (12H, m), 3.96 (1H, m), 4.87 (1H, d, JZ
3.9 Hz). FABMS (positive-ion): m/z 637 (MCNa)C, 615
(MCH)C. HRFABMS, calcd for C36H70O7Na: 637.5011.
Found: 637.5034.
4.1.54. 2-(Diallylphosphono)ethyl 6-O-{2-deoxy-4-O-dia-
llylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-2-
[(Z)-11-octadecenoylamino]-b-D-glucopyranosyl}-2,3-di-
O-dodecyl-a-D-glucopyranoside (61). To a solution of 60
(220 mg, 0.159 mmol) in THF (4 mL) and acetic acid
(0.5 mL) was added zinc dust (210 mg). After vigorously
stirring for 3 h at room temperature, the solution was filtered
to remove the Zn dust, and concentrated in vacuo to give a
crude product. The product was diluted with EtOAc, washed
with aq satd NaHCO3 and brine, dried over Na2SO4, filtered
and concentrated in vacuo to give a crude product, which
was dissolved in CH2Cl2 (5 mL), and (Z)-11-octadecenoic
acid (68 mg, 0.239 mmol) and WSC$HCl (55 mg,
0.287 mmol) were added to this solution. After stirring for
20 h at room temperature, the mixture was diluted with
4.1.57. 3-(Diallylphosphonooxy)propyl 2,3-di-O-dodecyl-
a-D-glucopyranoside (64). To a solution of 63 (981 mg,
1.59 mmol) and 1H-tetrazole (168 mg, 2.40 mmol) in THF
(8 ml) was added diallyl diisopropylphosphoramidite
(512 mg, 2.09 mmol). After stirring for 4 h at room
temperature, aq 30% H2O2 (1 ml) was added to the reaction
mixture at 0 8C. After stirring for 1 h at 0 8C, the mixture
was quenched with aq 10% Na2S2O3, diluted with EtOAc,
washed with water, aq satd NaHCO3 and brine, dried over
MgSO4, filtered, and concentrated in vacuo to give a

5120
M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
residue, which was dissolved in aq 80% AcOH (10 ml)
and stirred at 60 8C for 1 h. The solution was diluted with
EtOAc, washed with aq satd NaHCO3 and brine, dried over
MgSO4, filtered, concentrated in vacuo, and chromato-
graphed on a silica gel column. Elution with hexane–EtOAc
(1:9) gave 64 (828 mg, 71%) as a white powder. IR
dd, JZ3.9, 9.8 Hz), 3.29 (3H, s), 3.33 (1H, m), 3.39 (3H, s),
3.44 (1H, dd, JZ9.8, 8.8 Hz), 3.48–3.67 (10H, m), 3.73–
3.85 (6H, m), 4.04–4.12 (4H, m), 4.48 (1H, d, JZ8.8 Hz),
4.85 (1H, d, JZ2.9 Hz), 5.31–5.38 (2H, m). ESIMS
(negative-ion): m/z 1342 (MKH)K. HRESIMS (negative-
ion): calcd for C69H134NO19P2: 1342.9031. Found:
1342.9047.
1
nmax(CHCl3) 3600, 3393, 2927, 2855 cmK1. 500 MHz H
NMR (CDCl3) d 0.88 (6H, t, JZ6.8 Hz), 1.25–1.33 (36H,
m), 1.53–1.59 (4H, m), 1.71 (1H, bs, OH), 1.97–2.09 (3H,
m, containing OH), 3.26 (1H, dd, JZ3.9, 9.8 Hz), 3.43 (1H,
t, JZ8.8 Hz), 3.51–3.75 (7H, m), 3.83–3.91 (3H, m), 4.13
(1H, m), 4.27 (1H, m), 4.53–4.57 (4H, m), 4.89 (1H, d, JZ
3.9 Hz), 5.25–5.39 (4H, m), 5.90–5.98 (2H, m). FABMS
(positive-ion): m/z 757 (MCNa)C, 735 (MCH)C.
HRFABMS, calcd for C39H76O10P: 735.5175. Found:
735.5180.
4.1.61. 3-Bromopropyl 2,3-di-O-dodecyl-4,6-O-isopropyl-
idine-a-D-glucopyranoside (68). Compound 63 was treated
as described for the formation of 58 from 16 to give 68
(72%) as a gum. IR nmax(CHCl3) 2927, 2855 cmK1
.
500 MHz 1H NMR (CDCl3) d 0.88 (6H, t, JZ6.6 Hz),
1.21–1.32 (36H, m), 1.41 (3H, s), 1.48 (3H, s), 1.50–1.60
(4H, m), 2.13–2.19 (2H, m), 3.29 (1H, m), 3.49–3.75 (11H,
m), 3.81–3.87 (2H, m), 4.86 (1H, d, JZ4.4 Hz). FABMS
(positive-ion): m/z 699 (MCNa)C. HRFABMS, calcd for
C36H69BrO6Na: 699.4173. Found: 699.4180.
4.1.58. 3-(Diallylphosphonoxy)propyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-(2,2,2-trichloroethoxycarbonylamino)-b-D-gluco-
pyranosyl}-2,3-di-O-dodecyl-a-D-glucopyranoside (65).
Compound 64 was treated as described for the formation
of 49 from 48 to give 65 (53%) as an amorphous solid. IR
4.1.62. 3-(Diallylphosphono)propyl 2,3-di-O-dodecyl-a-
D-glucopyranoside (69). Compound 68 was treated as
described for the formation of 59 from 58 to give 69 (40%)
as a white powder. IR nmax(CHCl3) 3600, 2927, 2856 cmK1
.
nmax (CHCl3) 3450, 3088, 2928, 2873, 2856, 1737 cmK1
.
500 MHz 1H NMR (CDCl3) d 0.88 (6H, t, JZ6.8 Hz), 1.26–
1.33 (36H, m), 1.53–1.60 (4H, m), 1.83–2.00 (4H, m), 2.64–
2.75 (2H, bs, 2OH), 3.26 (1H, dd, JZ3.9, 9.8 Hz), 3.44 (1H,
dd, JZ8.8, 9.8 Hz), 3.48–3.68 (6H, m), 3.72–3.80 (2H, m),
3.84–3.93 (2H, m), 4.49–4.58 (4H, m), 4.90 (1H, d, JZ
3.9 Hz), 5.23–5.38 (4H, m), 5.90–5.98 (2H, m). FABMS
(positive-ion): m/z 741 (MCNa)C, 719 (MCH)C.
HRFABMS, calcd for C39H76O9P: 719.5222. Found:
719.5242.
500 MHz 1H NMR (CDCl3) d 0.88 (9H, t, JZ6.8 Hz), 1.25–
1.79 (54H, m), 1.95–2.06 (2H, m), 2.78 (1H, d, JZ2.9 Hz,
OH), 3.24 (1H, dd, JZ3.9, 9.8 Hz), 3.28 (3H, s), 3.30 (1H,
m), 3.39 (3H, s), 3.41–3.86 (17H, m), 4.08 (1H, d, JZ
8.8 Hz), 4.12–4.24 (2H, m), 4.29 (1H, m), 4.52–4.60 (8H,
m), 4.72, 4.76 (2H, AB-q, JZ11.7 Hz), 4.84 (1H, d, JZ
3.9 Hz), 4.87 (1H, d, JZ7.8 Hz), 5.24–5.39 (8H, m), 5.90–
5.98 (4H, m), 6.03 (1H, bs, NH). FABMS (positive-ion): m/z
1436 (MCNa)C. HRFABMS, calcd for C66H120Cl3NO20-
P2Na: 1436.6844. Found: 1436.6847.
4.1.63. 3-(Diallylphosphono)propyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyra-
nosyl}-2,3-di-O-dodecyl-a-D-glucopyranoside (70). Com-
pound 69 (470 mg, 0.654 mmol) was treated as described in
the formation of 19 from 18 to give 70 (533 mg, 58%) as an
amorphous solid. IR nmax(CHCl3) 3588, 3450, 3253, 3088,
2928, 2873, 2856, 1733 cmK1. 500 MHz 1H NMR (CDCl3)
d 0.88 (9H, t, JZ6,8 Hz), 1.26–1.79 (54H, m), 1.83–2.03
(4H, m), 2.64 (1H, s, OH), 3.22–3.27 (3H, m, containing 1H,
dd, JZ3.9, 9.8 Hz, at 3.23 ppm), 3.28 (3H, s), 3.36 (1H, m),
3.39 (3H, s), 3.44–3.88 (15H, m), 4.11 (1H, d, JZ9.8 Hz),
4.29 (1H, m), 4.48–4.59 (8H, m), 4.73, 4.75 (2H, ABq, JZ
12.7 Hz), 4.84 (1H, d, JZ2.9 Hz), 4.90 (1H, d, JZ7.8 Hz),
5.24–5.39 (8H, m), 5.90–5.98 (4H, m), 6.36 (1H, d, JZ
5.9 Hz). FABMS (positive-ion): m/z 1420 (MCNa)C.
HRFABMS, calcd for C66H120NO19Cl3P2Na: 1420.6896.
Found: 1420.6904.
4.1.59. 3-(Diallylphosphonoxy)propyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-methoxydecyl]-6-O-methyl-
2-[(Z)-11-octadecenoylamino]-b-D-glucopyranosyl}-2,3-
di-O-dodecyl-a-D-glucopyranoside (66). Compound 65
was treated as described for the formation of 50 from 49
to give 66 as an amorphous solid. IR nmax(CHCl3) 3454,
1
3319, 3089, 2928, 2856, 1665 cmK1. 500 MHz H NMR
(CDCl3) d 0.88 (12H, t, JZ6.8 Hz), 1.25–1.80 (76H, m),
1.95–2.05 (6H, m), 2.14–2.27 (2H, m), 3.17 (1H, d, JZ
3.9 Hz, OH), 3.21–3.24 (2H, m), 3.28 (3H, s), 3.30 (1H, m),
3.38 (3H, s), 3.41–3.82 (15H, m), 3.94 (1H, t, JZ9.8,
8.8 Hz), 4.07 (1H, d, JZ8.8 Hz), 4.11–4.20 (2H, m), 4.25
(1H, q, JZ8.8, 9.8 Hz), 4.52–4.58 (8H, m), 4.84 (1H, d,
JZ3.9 Hz), 5.16 (1H, d, JZ7.8 Hz), 5.23–5.39 (10H, m),
5.90–5.98 (4H, m), 6.36 (1H, d, JZ6.8 Hz, NH). FABMS
(positive-ion): m/z 1526 (MCNa)C. HRFABMS, calcd for
C81H151NO19P2Na: 1527.0261. Found: 1527.0277.
4.1.64. 3-(Diallylphosphono)propyl 6-O-{2-deoxy-4-O-
diallylphosphono-3-O-[(R)-3-(methoxy)decyl]-6-O-
methyl-2-[(Z)-11-octadecenoylamino]-b-D-glucopyrano-
syl}-2,3-di-O-dodecyl-a-D-glucopyranoside (71). Com-
pound 70 (480 mg, 0.343 mmol) was treated as described
in the formation of 7 from 6 to give 71 (266 mg, 52%) as an
amorphous solid. IR nmax(CHCl3) 3454, 3300, 3088, 2928,
2856, 1662 cmK1. 500 MHz 1H NMR (CDCl3) d 0.88 (12H,
t, JZ6.6 Hz), 1.20-1.96 (80H, m), 1.99-2.03 (4H, m), 2.14–
2.26 (2H, m), 3.08 (1H, d, JZ3.9 Hz, OH), 3.18 (1H, m),
3.22 (1H, dd, JZ3.9, 9.8 Hz), 3.26–3.32 (4H, m, containing
4.1.60. 3-(Phosphonoxy)propyl 6-O-{2-deoxy-3-O-[(R)-3-
methoxydecyl]-6-O-methyl-2-[(Z)-11-octadecenoyl-
amino]-4-O-phosphono-b-D-glucopyranosyl}-2,3-di-O-
dodecyl-a-D-glucopyranoside (67). Compound 66 was
treated as described for the formation of 8 from 7 to give
67 (84%) as a white powder. IR nmax(KBr) 3285, 3228,
3069, 3004, 2955, 2924, 2853, 2318, 1656, 1630 cmK1
.
1
500 MHz H NMR (CD3ODCCDCl3) d 0.89 (12H, t, JZ
6.8 Hz), 1.30–1.67 (74H, m), 1.72–1.75 (2H, m), 1.93–1.99
(2H, m), 2.01–2.04 (4H, m), 2.20–2.30 (2H, m), 3.17 (1H,

M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
5121
3H, s, at 3.28 ppm), 3.35–3.42 (4H, m, containing 3H, s, at
3.38 ppm), 3.46 (1H, dd, JZ8.8, 9.8 Hz), 3.48–3.83 (13H,
m), 3.98 (1H, dd, JZ7.8, 8.8 Hz), 4.09 (1H, d, JZ9.8 Hz),
4.25 (1H, t, JZ8.8 Hz), 4.48–4.58 (8H, m), 4.83 (1H, d, JZ
3.9 Hz), 5.17 (1H, d, JZ7.8 Hz), 5.23–5.38 (10H, m), 5.90–
5.98 (4H, m), 6.52 (1H, d, JZ6.8 Hz, NH). FABMS
(positive-ion): m/z 1510 (MCNa)C. HRFABMS, calcd for
C81H151NO18P2Na: 1511.0304. Found: 1511.0305.
Statistical analysis: The percentage of inhibition of TNFa
production was calculated by the following formula:
1K(concentration of TNFa in the test sampleKconcen-
tration of TNFa in the negative control sample)/(concentra-
tion of TNFa in the positive control sampleKconcentration
of TNFa in the negative control sample)!100. The
suppressive activities of test compounds are expressed as
the fifty percent inhibitory concentration (IC50) of the test
compound, the concentration at which the test compound
suppresses TNFa production by 50%. The IC50 was
calculated from the percentage of inhibition using the
SAS System for Windows. The results are expressed as
the mean IC50 of triplicate experiments.
4.1.65. 3-(Phosphono)propyl 6-O-{2-deoxy-3-O-[(R)-3-
methoxydecyl]-6-O-methyl-2-[(Z)-11-octadecenoyl-
amino]-4-O-phosphono-b-D-glucopyranosyl}-2,3-di-O-
dodecyl-a-D-glucopyranoside. (72). Compound 71
(251 mg, 0.168 mmol) was treated as described in the
formation of 8 from 7 to give 72 (177 mg, 80%) as a white
powder. IR nmax(KBr) 3286, 3069, 3004, 2954, 2925, 2854,
4.2.2. Production of TNFa by galactosamine loaded
C3H/HeN mice in vivo.9 Materials: Animals: Male C3H/
HeN mice were purchased from Charles River Japan
(Tokyo, Japan). All mice were used at the age of 7 weeks,
and housed at Sankyo Laboratories (Tokyo, Japan) with free
access to standard rodent chow diet.
1
1630 cmK1. 500 MHz H NMR (CD3OD) d 0.89 (12H, t,
JZ6.8 Hz), 1.30–1.92 (80H, m), 2.01–2.05 (4H, m), 2.20–
2.29 (2H, m), 3.18 (1H, dd, JZ3.9, 9.8 Hz), 3.29 (3H, s),
3.33 (1H, m), 3.39 (3H, s), 3.43 (1H, dd, JZ3.3, 9.8 Hz),
3.46 (1H, m), 3.50–3.67 (9H, m), 3.71–3.85 (6H, m), 4.04–
4.12 (2H, m), 4.48 (1H, d, JZ7.8 Hz), 4.85 (1H, d, JZ
3.9 Hz), 5.31–5.38 (2H, m). ESIMS (negative-ion): m/z
1326 (MKH)K. HRESIMS: calcd for C69H134NO18P2:
1326.9069. Found: 1326.9047.
Reagents: Lipopolysaccharide (LPS, from Escherichia coli
O26:B6) and D-Galactosamine (GalN) were purchased from
Sigma (St Louis, MO). Enzyme-linked immunosorbent
assay (ELISA) kits of murine TNFa were from R&D
Systems (Minneapolis, MN).
4.2. Methods for measurement of biological activity
¨
TNFa production: Naıve C3H/HeN mice (five per group)
were intravenously injected with the test compound solution
(10 ml/kg; dissolved in 0.1% triethylamine /saline solution),
and immediately after, mice were intravenously injected
with a mixture of LPS (0.05 mg/10 ml saline/kg) and GalN
(1 g/10 ml saline/kg). Mice were injected with vehicle
(0.1% triethylamine/saline solution) and saline for negative
control samples, and with vehicle and LPS/GalN for
positive control samples. One hour after injection, venous
blood was collected under ether anesthesia with heparinized
syringes fitted with 23-gauge needles from the abdominal
vena, and was centrifuged at 4 8C for 3 min at 13,230!g to
obtain the plasma. Plasma was stored at K30 8C before
measuring TNFa levels by ELISA. The concentrations of
TNFa of mouse plasma were measured using ELISA
analysis according to the manufacturer’s instructions.
The sources of the materials used in the study are as follows:
lipopolysaccharide (LPS) from E. coli serotype 026:B6 and
12-O-tetradecanoylphorbol acetate (TPA) were from
Sigma, St. Louis, MO; RPMI-1640 medium, fetal bovine
serum (FBS) and newborn calf serum (NBCS) were from
Gibco, Grand Island, NY; and human TNFa ELISA kit and
mouse TNFa ELISA kit were from Genzyme-Techne,
Minneapolis, MN.
4.2.1. Production of TNFa by human whole blood.8
Materials: Lipopolysaccharide (LPS, lot 50K4117, E. coli
026:B6), human tumor necrosis factor alpha (TNFa)
immunoassay kit and 96-well assay plates were purchased
from Sigma, BioSource International, Inc. and Corning Inc.
(Cat. No. 3956), respectively.
Statistical analysis: The percentage of inhibition of TNFa
production was calculated by the following formula:
[1K(concentration of TNFa in the test sampleKconcen-
tration of TNFa in the negative control sample)/(concentra-
tion of TNFa in the positive control sample Kconcentration
of TNFa in the negative control sample)]!100. The
suppressive activities of test compounds are expressed as
the mean of fifty percent inhibitory dose (ID50) of the test
compound, at which the test compound suppresses TNFa
production by 50%. The ID50 was calculated from the
percentage of inhibition using the SAS System for Windows
(version 5).
Whole blood TNFa production: Fresh blood was collected
aseptically in the presence of heparin by venipuncture from
healthy adult volunteers. The subjects did not have any
apparent inflammatory conditions and had taken no drugs
for at least 7 days prior to blood collection. Written
informed consent was obtained from all volunteers before
the experiment. In each well of the plates, 360 mL aliquots
of blood were mixed with 20 mL of LPS solution
(200 ng/mL, final concentration: 10 ng/mL) dissolved in
PBS in the presence (for test sample) or absence (for
positive control sample) of the test compounds solution
(dissolved in DMSO/PBS solution). For the negative control
samples, the same amount of blood was cultured without
either LPS or a test compound solution. After 6 h of
incubation at 37 8C, the plates were centrifuged at 490!g
for 15 min, and the plasma was collected and stored at
K20 8C. The concentrations of TNFa in the plasma were
measured with commercially available immunoassay kits.
References and notes
1. Brade, H.; Opal, S. M.; Vogel, S. N.; Morrison, D. C.

5122
M. Shiozaki et al. / Tetrahedron 61 (2005) 5101–5122
Endotoxin in Health and Diseases; Marcel Dekker Inc.: New
York, Basel, 1999; pp 915–926.
Nishijima, M.; Kuge, S.; Otsuka, T.; Akamatsu, Y. Carbohydr.
Res. 1991, 222, 57–68.
2. (a) Imoto, M.; Yoshimura, H.; Sakaguchi, N.; Kusumoto, S.;
Shiba, T. Tetrahedron Lett. 1985, 26, 1545–1548. (b) Imoto,
M.; Yoshimura, H.; Shimamoto, T.; Sakaguchi, N.; Kusumoto,
S.; Shiba, T. Bull. Chem. Soc. Jpn. 1987, 60, 2205–2214.
(c) Cf. Takahashi, T.; Nakamoto, S.; Ikeda, K.; Achiwa, K.
Tetrahedron Lett. 1986, 27, 1819–1822.
8. Jagger, M. P.; Huo, Z.; Riches, P. G. Clin. Exp. Immunol.
2002, 130, 467–474.
9. Endo, Y.; Shibasaki, M.; Yamaguchi, K.; Kai, K.; Sugawara,
S.; Takada, H.; Kikuchi, H.; Kumagai, K.; Br J. Pharmacol.
1999, 128, 5–12.
10. (a) Liu, W.-C.; Oikawa, M.; Fukase, K.; Suda, Y.; Kusumoto,
S. Bull. Chem. Soc. Jpn. 1999, 72, 1377–1385. (b) Fukase, K.;
Ueno, A.; Fukase, Y.; Oikawa, M.; Suda, Y.; Kusumoto, S.
Bull. Chem. Soc. Jpn. 2003, 76, 485–500. (c) Fukase, K.;
Kirikae, T.; Kirikae, F.; Liu, W.-C.; Oikawa, M.; Suda, Y.;
Kusumoto, S. Bull. Chem. Soc. Jpn. 2001, 74, 2189–2197.
11. (a) Golenbock, D. T.; Hampton, R. Y.; Qureshi, N.; Takayama,
K.; Raetz, C. R. J. Biol. Chem. 1991, 266, 19490–19498 cf.
(b) Oikawa, M.; Shintaku, T.; Yoshizaki, H.; Fukase, K.;
Adachi, S.; Lee, K.; Kusumoto, S. Bull. Chem. Soc. Jpn. 2001,
74, 1455–1461.
3. (a) Qureshi, N.; Honovich, J. P.; Hara, H.; Cotter, R. J.;
Takayama, K. J. Biol. Chem. 1988, 263, 5502–5504.
(b) Qureshi, N.; Takayama, K.; Kurtz, R. Infect. Immun.
1991, 59, 441–444. (c) Qureshi, N.; Takayama, K.; Meyer,
K. C.; Kirkland, T. N.; Bush, C. A.; Chen, L.; Wang, R.;
Cotter, R. J. J. Biol. Chem. 1991, 266, 6532–6538. (d) Christ,
W. J.; McGuinness, P. D.; Asano, O.; Wang, Y.; Mullarkey,
M. A.; Perez, M.; Hawkins, L. D.; Blythe, T. A.; Dubuc, G. R.;
Robidoux, A. L. J. Am. Chem. Soc. 1994, 116, 3637–3638.
(e) Kaltashov, I. A.; Doroshenko, V.; Cotter, R. J.; Takayama,
K.; Qureshi, N. Anal. Chem. 1997, 69, 2317–2322.
12. Saitoh, S.; Akashi, S.; Yamada, T.; Tanimura, N.; Kobayashi,
M.; Konno, K.; Matsumoto, F.; Fukase, K.; Kusumoto, S.;
Nagai, Y.; Kusumoto, Y.; Kosugi, A.; Miyake, K. Int.
Immunol. 2004, 16, 961–969.
4. Rossignol, D. P.; Hawkins, L. D.; Christ, W. J.; Kobayashi, K.;
Kawata, T.; Lynn, M.; Yamatsu, I.; Kishi, Y. Endotoxin in
Health and Diseases; Marcel Dekker Inc.: New York, Basel,
1999; Chapter 47.
13. (a) Watanabe, Y.; Shiozaki, M.; Tanaka, D.; Shimozato, T.;
Kurakata, S. Bull. Chem. Soc. Jpn. 2003, 76, 2341–2352.
(b) Watanabe, Y.; Miura, K.; Shiozaki, M.; Kanai, S.;
Kurakata, S.; Nishijima, M. Carbohydr. Res. 2003, 338,
47–54.
5. Christ, W. J.; Rossignol, D. P.; Kobayashi, S.; Kawata, T. U.S.
Patent 5,935,938, August 10, 1999.
6. Shiozaki, M.; Doi, H.; Tanaka, D.; Shimozato, T.; Kurakata, S.
Bull. Chem. Soc. Jpn., accepted for publication.
7. Shiozaki, M.; Kobayashi, Y.; Ishida, N.; Arai, M.; Hiraoka, T.;