Synthesis of Rubrivivax gelatinosus lipid A and analogues for investigation of the structural basis for immunostimulating and inhibitory activities

Article abstract of DOI:10.1246/bcsj.81.796

To elucidate the structural requirements for the endotoxic and antagonistic activities of lipid A derivatives, we have focused on the effects of the acyl moieties and acidic groups at the 1- and 4′-positions in the present study. We have synthesized new analogues corresponding to Rubrivivax gelatinosus lipid A, which has a characteristic symmetrical distribution of its acyl groups on its two glucosamine residues with shorter acyl groups (decanoyl groups (C₁₀) and lauryl groups (C₁₂)) than Escherichia coli lipid A's. Carboxymethyl (CM) analogues in which one of the phosphates was replaced with a CM group were also synthesized with a different distribution of acyl groups. Biological tests revealed that the acyl group distribution in the lipid A analogue, strongly affected its bioactivity. The synthetic Ru. gelatinosus type lipid A showed potent antagonistic activity against LPS, whereas its 1-O-carboxymethyl analogue showed weak endotoxic activity. These results demonstrate that when lipid A has shorter (C₁₀ and C₁₂) hexa-acyl groups, its bioactivity is more easily affected by small structural differences, such as differences in acidic groups or acyl group distribution, and that they can change bioactivity from endotoxic to agonistic or vice versa at this structural boundary for the bioactivity.

Full text of DOI:10.1246/bcsj.81.796

796 Bull. Chem. Soc. Jpn. Vol. 81, No. 7, 796–819 (2008)
ꢀ 2008 The Chemical Society of Japan
BCSJ Award Article
Synthesis of Rubrivivax gelatinosus Lipid A and Analogues
for Investigation of the Structural Basis for Immunostimulating
and Inhibitory Activities
ꢀ1
Yoshiyuki Fukase,1 Yukari Fujimoto, Yo Adachi,1 Yasuo Suda,2
Shoichi Kusumoto,1;3 and Koichi Fukase
ꢀ1
1Department of Chemistry, Graduate School of Science, Osaka University,
1-1 Machikaneyama Toyonaka, Osaka 560-0043
2Department of Nanostructure and Advanced Materials, Graduate School of Science and Engineering,
Kagoshima University, Kagoshima 890-0065
3Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka 618-8503
Received December 3, 2007; E-mail: yukarif@chem.sci.osaka-u.ac.jp
To elucidate the structural requirements for the endotoxic and antagonistic activities of lipid A derivatives, we have
focused on the effects of the acyl moieties and acidic groups at the 1- and 40-positions in the present study. We have
synthesized new analogues corresponding to Rubrivivax gelatinosus lipid A, which has a characteristic symmetrical dis-
tribution of its acyl groups on its two glucosamine residues with shorter acyl groups (decanoyl groups (C10) and lauryl
groups (C12)) than Escherichia coli lipid A’s. Carboxymethyl (CM) analogues in which one of the phosphates was re-
placed with a CM group were also synthesized with a different distribution of acyl groups. Biological tests revealed that
the acyl group distribution in the lipid A analogue, strongly affected its bioactivity. The synthetic Ru. gelatinosus type
lipid A showed potent antagonistic activity against LPS, whereas its 1-O-carboxymethyl analogue showed weak endo-
toxic activity. These results demonstrate that when lipid A has shorter (C10 and C12) hexa-acyl groups, its bioactivity is
more easily affected by small structural differences, such as differences in acidic groups or acyl group distribution, and
that they can change bioactivity from endotoxic to agonistic or vice versa at this structural boundary for the bioactivity.
The innate immune system is a phylogenetically ancient
defense mechanism conserved between plants and animals.1–5
One of the important roles of innate immunity is the detection
of invading pathogens (bacteria, fungi, viruses, etc.) through in-
nate immune receptors that recognize characteristic structures
that are present in microorganisms, called PAMPs (pathogen-
associated molecular patterns). PAMPs are essential molecules
for pathogens that are not found within the host. In vertebrates,
two diverse families of receptors, i.e., the Toll-like receptor
(TLR) and Nod-like receptor (NLR) families, detect PAMPs
such as the bacterial cell wall peptidoglycan (PGN), lipopoly-
saccharide (LPS) of Gram-negative bacteria, lipoproteins, bac-
terial DNA, viral RNA, etc. to activate the immune system.
Most PAMPs therefore show immunostimulating activity.
LPS is a cell surface glycoconjugate of Gram-negative bac-
teria that is also known as endotoxin,6–12 and is sensed by a
receptor complex consisting of TLR4 and its adaptor protein
MD-2. Via this complex, LPS stimulates immunocompetent
cells such as macrophages and monocytes to produce a variety
of mediators, e.g., cytokines, prostaglandins, the platelet acti-
vating factor, oxygen free radicals, and NO. These mediators
activate and modulate the immune system. If too much LPS
is released during a severe Gram-negative bacterial infection,
the overproduction of these mediators can lead to endotoxin-
related symptoms such as high fever, serious inflammation,
hypotension, and, in serious cases, lethal shock.
LPS consists of a glycolipid component termed lipid A that
is covalently bound to a polysaccharide. It was unequivocally
proved that lipid A is the chemical entity responsible for the
biological activity of LPS by the total synthesis of Escherichia
coli lipid A 1 (synthetic 1 is termed 506) (Figure 1) in
1984.13,14 Lipid A specimens from various bacterial origins
were shown to be closely related structurally and to consist
of: 1) ꢀð1!6Þ disaccharide of D-glucosamines, 2) phosphono
groups at their reducing ends and the 4-position of their non-
reducing glucosamines, and 3) long-chain acyl groups bound
at 2, 20, 3, and 30 positions.
The recognition of LPS and lipid A by the TLR4/MD-2 re-
ceptor complex has been of major interest in the endotoxin re-
search field.1–12 To further our understanding of this issue, we
investigated the precise structural requirements for lipid A bio-
logical activity. It has been previously shown that the acidic
Published on the web July 10, 2008; doi:10.1246/bcsj.81.796

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
797
OH
O
OH
O
OH
O
O
O
O
O
HO
O
O
O
O
O
(HO)2P O
O
(HO)2P O
O
(HO)2P O
HO
HO
O
O
O
O
O
P(OH)
O
P(OH)
O
NH
NH
NH
NH
O
OH
NH
NH
O
O
O
O
O
OH
O
OH
O
O
O
O
P(OH)
O
2
2
2
O
OH
O
O
O
O
OH
OH
O
O
OH
OH
O
O
O
(C10)
(C10)
(C14)
(C14)
(C14)
(C14)
(C14)
(C12)
(C14)
(C14)
(C14)
(C14)
(C14)
(C14)
(C14)
2 : Biosynthetic precursor Ia
3 : RSLA
1 : Lipid A from Escherichia coli
OMe
O
OH
O
OH
O
O
O
O
O
O
O
O
O
O
(HO)2P O
HO
(HO)2P O
HO
(HO)2P O
HO
HO
HO
HO
O
O
O
P(OH)
O
O
P(OH)
O
P(OH)
NH
OMe
NH
NH
NH
NH
O
OH
NH
O
O
O
O
O
2
O
O
2
2
O
O
OH
O
OH
OH
OH
(C10)
(C10)
(C14)
(C14)
(C14)
(C18)
(C14)
(C14)
(C14)
6
5
4 : E5564
OH
O
OH
O
OH
O
O
HO
O
O
O
(HO)2P O
O
O
O
O
O
(HO)2P O
O
(HO)2P O
O
HO
HO
O
O
HO
O
O
P(OH)
O
O
NH
NH
NH
O
O
NH
NH
OH
O
NH
O
O
OH
O
O
P(OH)
P(OH)
O
O
OH
O
2
2
2
O
O
OH
O
OH
OH
OH
O
OH
OH
OH
OH
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C14)
8
(C14)
(C14)
(C14)
(C14)
7
9
Figure 1. Structures of lipid A and its analogues with various acyl groups.
functional groups and acyl groups of lipid A are crucial for its
biological activity.15–18 Additionally, its phosphate groups can
be replaced by other acidic groups (such as carboxylic acid)
without decisively influencing its biological activity.19–21 In
contrast, the structure, number, and chain length of the acyl
groups can dramatically influence its biological activity. The
tetraacyl biosynthetic precursor of lipid A 2 (synthetic 2 is
termed 406) has weaker but clear endotoxic activity in mice,
but quite interestingly it also acts as an antagonist to LPS
and lipid A 1 in human systems.16,17 Rhodobacter sphaeroides
lipid A 3 (RSLA) also shows species-related antagonistic or
agonistic activities in different mammalian hosts.22 The syn-
thetic compounds E5531 and E5564 (Eritoran) 4 exhibit potent
antagonistic activity in human systems.23,24 RSLA, E5531, and
E5564 each have acyl groups containing unsaturated and 2-
keto acyl groups. E5564 is currently under development as a
possible clinical therapeutic for the treatment of sepsis and
septic shock. The N,N0-diacyl analogue 5 does not show any
activity, but the triacyl-type analogues 6 and 7, which lack acyl
groups at the 3- and 30-O-positions, show a weak but definite
ability to inhibit the induction of IL-6 by LPS. Precursor-type
analogues with shorter acyl chains have also been synthe-
sized.25 Analogue 9, which possesses two (R)-3-hydroxytetra-
decanoic acids at the 2- and 20-N-positions and two (R)-3-hy-
droxydecanoic acids at the 3- and 30-O-positions, shows defi-
nite but ca. 10–100 times less potent antagonistic activity than
natural-type 2; whereas analogue 8, which possesses four (R)-
3-hydroxydecanoic acids, does not show this activity. On the
other hand, Boons et al. revealed that an E. coli lipid A ana-
logue had shorter lipids (two C14 and four C12 acids) that
were ca. 100 times more active than E. coli lipid A.26 They al-
so synthesized heptaacylated Salmonella typhimurium lipid A,
which showed much weaker activity than E. coli lipid A, and
using its short-chain analogue they obtained similar results.
In this study, we focused on the structural requirements for
the endotoxic and antagonistic activities of lipid A derivatives,
and in particular, their effects on the human innate immune sys-
tem. We particularly considered the effects of the acyl and acid-
ic groups, and thus prepared and analyzed various structural an-
alogues, including some with different numbers and distribu-
tions of acyl moieties on the lipid A backbone and some with
a carboxymethyl group instead of a phosphate group.
Rubrivivax gelatinosus-type lipid A 10a and 10b (Figure 2)
has shorter acyl groups than E. coli lipid A 1, and a symmet-
rical (3 þ 3) acylation distribution. It was reported that natural
lipid A isolated from Ru. gelatinosus showed endotoxic activ-
ity.27 By contrast, Chromobacterium violaceum lipid A 11 has
acyl groups that are similar to Ru. gelatinosus and shows an-
tagonistic activity.28 The only structural difference between
10 and 11 is the chain lengths of three acyl groups. Since

798 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
OH
OH
O
O
O
O
O
O
O
O
(HO)2P
O
O
(HO)2P
O
O
HO
O
HO
O
O
O
NH
NH
NH
NH
O
O
P(OH)2
O
O
O
P(OH)2
O
O
O
O
O
OH
OH
OH
OH
O
O
O
O
O
O
O
O
R1
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C12)
(C12)
(C12)
(C12)
Lipid A from Rubrivivax gelatinosus
11 : Lipid A from Chromobacterium violaceum
10a : R1CO = C11H23CO (C12)
10b : R1CO = C13H27CO (C14)
Figure 2. Structures of two natural lipid A molecules that each have six acyl groups with symmetrical distributions.
O
O
BnO
O
O
O
O
O
P NEt2
c
Ph
Ph
a
b
d
OCOR1
OCOR1
OH
O
O
O
O
HO
O
92%
74%
94%
NHTroc
NHTroc
BnO
NHTroc
12
13
14
BnO
O
BnO
NH
O
O
O
OCOR1
OH
O
O
OCOR1
OCOR1
g
O
O
CCl3
O
O
O
e, f
74%
P
O
P O
P
O
O
98%
O
O
NHTroc
NHTroc
NHTroc
15
16
17
F3C
HO
O
O
COOH 20
R1COOH =
R2COOH =
O
O
Ph
h, i
j
75%
OCOR1
OCOR1
(C10)
(C12)
13
O
O
HO
O
85%
O
O
NHCOR2
NHCOR2
19
18
21
COOH
(C10)
Scheme 1. Reagents and conditions: (a) R1COOH (20), DCC, DMAP, CH2Cl2, rt, 17 h; (b) BF3 Et2O, Et3SiH, CH3CN, 0 ꢂC, 1.5 h;
ꢁ
(c) 1H-tetrazole, CH2Cl2, rt, 50 min; (d) mCPBA, ꢃ20 ꢂC, 20 min; (e) [Ir(cod)(MePh2P)2]PF6, H2, THF, 2 h; (f) I2, H2O, rt,
30 min; (g) CCl3CN, Cs2CO3, CH2Cl2, rt, 2 h; (h) Zn–Cu couple, AcOH, rt, 3 h; (i) R2COOH (21), DCC, CH2Cl2, rt, 2 h; (j)
TFA, H2O, CH2Cl2, 0 ꢂC, 2.5 h.
we were interested in the structural requirement for the endo-
toxic/antagonistic activity of lipid A with symmetrical (3 þ 3)
acylation distribution, Ru. gelatinosus lipid A 10a was synthe-
sized and analyzed in the present study.
the glycosylation with the trichloroacetimidate 17.30
The glycosyl donor 17 and the glycosyl acceptor 19 were
synthesized as shown in Scheme 1. The hydroxy group at
the 3-position of 1-O-allyl 4,6-O-benzylidene-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-ꢁ-D
-glucopyranoside20
(12) was acylated with (R)-3-(4-trifluoromethylbenzyloxy)-
decanoic acid (20). An unsubstituted benzyl group used for
protecting the hydroxy function on the 3-hydroxyacyl residue
proved to be prone to air-oxidation and gradually transformed
into a corresponding benzoyl group. The p-trifluoromethylben-
zyl group at this position was resistant to oxidation, but was
readily removable by conventional hydrogenolysis.21,30–33
Regioselective reductive opening of the benzylidene of 13
Results
The Synthesis and Biological Activity of Ru. gelatinosus
Lipid A. We have established the efficient synthesis of lipid
A and analogues in our previous studies.20,21,25,29 In the present
study, Ru. gelatinosus lipid A 10a was synthesized using a
similar strategy (Scheme 1). The hydroxy and phosphate
groups were protected with benzyl-type protective groups,
which were removed by catalytic hydrogenation in the last
step. The ꢀ(1!6) disaccharide structures were constructed
by glycosylation of the glycosyl acceptor 19 with the N-Troc
trichloroacetimidate donor 17 (Troc = 2,2,2-trichloroethoxy-
carbonyl). A Lewis acid catalyzed activation was used for
with BF3 OEt2 and Et3SiH gave the 6-O-benzyl-4-OH GlcN
derivative 14.34
ꢁ
The free 4-hydroxy group of 14 was treated with
Watanabe’s reagent and 1-H-tetrazole, and then with m-chlo-

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
799
BnO
O
O
BnO
O
HO
+
NH
CCl3
O
O
O
O
a
OCOR1
OCOR1
OCOR1
OCOR1
O
O
O
O
O
O
HO
O
P
O
HO
O
P
NHCOR2
NHCOR2
72%
NHTroc
NHTroc
17
BnO
19
22
O
BnO
O
O
O
O
O
O
d, e
b, c
OCOR1
OCOR1
OCOR1
OCOR1
O
P
OH
O
O
O
O
O
HO
NHCOR2
O
O
HO
NHCOR2
P
73%
78%
NHCOR2
NHCOR2
23
24
BnO
O
O
O
P
O
O
O
OCOR1
OCOR1
P
O
P
g, h
BnO
BnO
OBn
OBn
O
O
O
OBn
OBn
Rubrivivax gelatinosus lipid A 10a
O P
O
HO
NHCOR2
f
93%
NHCOR2
25
75%
Scheme 2. Reagents and conditions: (a) TMSOTf, CH2Cl2, MS4A, ꢃ20 ꢂC, 1 h; (b) Zn, AcOH, rt, 1.5 h; (c) R2COOH (21),
WSCD HCl, HOBt, CH2Cl2, rt, 21 h; (d) [Ir(cod)(MePh2P)2]PF6, H2, THF, rt, 2 h; (e) I2, H2O, rt, 1 h; (f) LiN(TMS)2, THF,
ꢃ78 ꢂC, 1.5 h; (g) H2 (20 kg cmꢃ2), Pd-black, THF, rt, 44 h; (h) liquid–liquid partition column chromatography using Sephadex
ꢁ
LH-20, CHCl3–MeOH–H2O–iPrOH (8:8:6:1).
roperbenzoic acid (mCPBA) to furnish the phosphate 15 in
Table 1. The Limulus Activity of 10a, 26a–26f, and LPS
94% yield.35 The 1-O-allyl group of 15 was removed via iso-
merization to a 1-propenyl group and subsequently treated
with iodine.36 The resulting 1-OH sugar 16 was then trans-
formed into the glycosyl trichloroacetimidate 17 by treatment
with CCl3CN and Cs2CO3.37
(E. coli 0111:B4), as Tested Using an Endospecy Testꢁ
(Seikagaku Corporation, Tokyo)
ED50/pg mLꢃ1
Ru. gelatinosus lipid A (10a)
CM analogue 26a
10
5000
10000<
—
The glycosyl acceptor 19 was synthesized as follows. The 2-
N-Troc group of 13 was removed using Zn–Cu and acetic acid
and the resulting 2-amino group was then acylated with (R)-3-
(dodecanoyloxy)decanoic acid (21) to give the 2,3-diacyl
derivative 18. Deprotection of the benzylidene group of 18
under the acidic conditions gave the glycosyl acceptor 19.
Glycosylation of the above glycosyl acceptor 19 with the
glycosyl donor 17 gave the desired ꢀ(1!6) disaccharide 22
in 72% yield (Scheme 2). The 20-N-Troc group of 22 was
cleaved and the resulting amino group was acylated with (R)-
3-(dodecanoyloxy)decanoic acid (21) to give the fully acylated
compound 23. The allyl group at the 1-position of 23 was
cleaved via isomerization to a vinyl group with an iridium com-
plex to give 24 in 78% yield. After selective phosphorylation at
the anomeric position with tetrabenzyl pyrophosphate, all the
benzyl-type protecting groups in 25 were removed by catalytic
hydrogenolysis to give the desired Ru. gelatinosus lipid A 10a.
The biological activities of 10a were evaluated in compari-
son to the corresponding LPS (E. coli O111:B4) by measuring
typical endotoxic activity such as Limulus activity and cyto-
kine induction. Cytokine inducing activity was tested in human
peripheral whole-blood cells.38 A mixture of a test sample and
heparinized human peripheral whole-blood collected from an
adult volunteer in RPMI 1640 medium (Flow Laboratories,
Irvine, Scotland) was incubated at 37 ꢂC in 5% CO2 for 24 h.
The levels of cytokines, i.e., interleukin-6 (IL-6) and tumor ne-
crosis factor-ꢁ (TNF-ꢁ), in supernatants of incubated mixtures
were measured using an enzyme-linked immunosorbent assay
(ELISA). Antagonistic activity was examined and compared to
the tetraacyl biosynthetic precursor of lipid A (compound 406)
26b
26c
26d
26e
26f
10000<
50
50
LPS (E. coli 0111:B4)
50
2 using an assay that measured the ability of a compound to
inhibit LPS-induced cytokine production as follows. Samples,
LPS (10 ng mLꢃ1) (E. coli O111:B4; Sigma Chemicals Co.),
and heparinized human peripheral whole blood were mixed
and incubated, and the levels of IL-6 and TNF-ꢁ were mea-
sured (as described above).
The Limulus activity, the hemolymph coagulation activity
on horseshoe crab amoebocyte lysates, was evaluated by
the activation of factor C at various concentrations using an
Endospecy Testꢁ (Seikagaku Corporation, Tokyo) with E. coli
O111:B4 LPS as a positive standard. As clearly seen in
Table 1, Ru. gelatinosus lipid A 10a showed potent Limulus
activity that was comparable to E. coli LPS.
Ru. gelatinosus lipid A 10a showed no cytokine inducing
activity, but a potent ability to antagonize LPS endotoxic ac-
tivity that was comparable to 406 (2) (Figure 3). As mentioned
above, natural Ru. gelatinosus lipid A has immunostimulatory
activity. In contrast, Chromobacterium violaceum lipid A 11,
which has acyl groups similar to Ru. gelatinosus, showed an-
tagonistic activity. Both of these lipid A molecules have short-
er acyl groups than E. coli lipid A, and symmetric (3 þ 3)
acylation distribution. Therefore, our study indicated that these

800 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
A
B
2500
2500
2000
1500
1000
500
2000
1500
1000
500
0
1000 ngmL−1
100 ngmL−1
10 ngmL−1
1 ngmL−1
1000 ngmL−1
100 ngmL−1
10 ngmL−1
0.1 ngmL−1
0
Ru. gelatinosus
lipid A 10a
CM analogue
E. coli LPS 0111:B4
Ru. gelatinosus CM analogue Biosynthetic
lipid A 10a 26a precursor Ia 2
26a
C
D
400
300
250
200
300
200
100
0
1000 ngmL−1
150
100
50
100 ngmL−1
1000 ngmL−1
10 ngmL−1
1 ngmL−1
100 ngmL−1
10 ngmL−1
0.1 ngmL−1
0
-100
-50
Ru. gelatinosus CM analogue
lipid A 10a 26a
E. coli LPS 0111:B4
-100
Ru. gelatinosus CM analogue Biosynthetic
lipid A 10a 26a precursor Ia 2
Figure 3. Cytokine inducing activity and inhibitory activity of Ru. gelatinosus lipid A 10a, its CM analogue 26a, and E. coli LPS
0111:B4 in human peripheral whole-blood cells. A: IL-6 inducing activity, B: inhibitory activity against IL-6 induction by E. coli
LPS 0111:B4 (10 ng mLꢃ1), C: TNF-ꢁ inducing activity, D: Inhibitory activity against TNF-ꢁ induction by E. coli LPS 0111:B4
(10 ng mLꢃ1).
types of lipid A should show antagonistic activity. The reason
why natural Ru. gelatinosus lipid A showed immunostimulato-
ry activity will be discussed later.
having acidic groups ꢀ-glycosidically linked at the 1-position
also showed potent activity.21 We also synthesized two
analogues that had two CM groups at 1- and 40-positions,
E. coli-type (Bis-CM-506) and precursor-type (Bis-CM-406),
both of which showed respective activities.40,41 The acidic
functional groups are concluded to be essential,42 but their strict
type is not necessary for expression of the biological activity.
Although we had already improved the synthetic procedure
for lipid A in many aspects, it still had many reaction steps and
as a consequence considerable time and laborious work was re-
quired for the completion of the synthesis. In order to facilitate
the synthesis, we developed a new synthetic methodology
termed Synthesis based on Affinity Separation (SAS). The ba-
sic principle of SAS is as follows. A tag molecule is covalently
attached to a substrate. The reactions are carried out in solu-
tions, and the desired tagged products are rapidly isolated by
solid-phase extraction using a specific affinity interaction be-
tween the tag and a ligand which is immobilized on a polymer
support. So far, we have successfully used two interactions for
SAS. One was an interaction of a crown ether or a podand
ether tag and polymer-supported ammonium ions43,44 and the
other was the specific molecular recognition between a barbi-
turic acid derivative and its artificial receptor which formed a
tight complex with six hydrogen bonds43b,45 (Figure 5). The
versatility of SAS for glycoconjugate synthesis has already
Synthesis of Ru. gelatinosus Lipid A Analogues by Using
Affinity Separation Method. We were then interested in the
effect of acylation distribution on endotoxic/antagonistic ac-
tivity and planned to synthesize six kinds of Ru. gelatinosus
lipid A analogues 26a–26f having hexaacyl groups (Figure 4).
All of these compounds contained the same acyl groups but
their distributions were different: each compound has two
(R)-3-hydroxydecanoyl groups and two (R)-3-(dodecanoyl-
oxy)decanoyl groups. Analogue 26c had the same acylation
pattern as E. coli lipid A 1, so it was expected that biological
tests of 26c would give additional information on how chain
length effected bioactivity.
1-O-Carboxymethyl (CM) analogues, which had glycosyl
CM groups instead of the glycosyl phosphate moiety in natural
lipid A, were chosen as targets, since they were easier to syn-
thesize than the natural-type because of the chemical instability
of the glycosyl phosphate. We previously synthesized both the
E. coli-type and the precursor-type analogues CM-506 and
CM-406 in which the phosphoryl group at the 1-position was
replaced with a carboxymethyl (CM) group.20,39 The activity
of both CM-506 and CM-406 was indistinguishable from their
corresponding natural-type compounds. The ꢀ-CM analogues

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
801
OH
OH
O
OH
O
O
O
O
HO
O
O
O
O
O
O
O
(HO)2PO
(HO)2PO
HO
HO
(HO)2PO
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
O
O
O
O
O
CO2H
CO2H
O
OH
O
O
OH
O
CO2H
O
O
O
OH
O
O
O
O
O
O
O
OH
OH
O
O
O
O
OH
O
O
O
O
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C12)
(C12)
(C12)
(C12)
(C12)
(C12)
26a : Ru. gelatinosus-type
26b
26c
OH
OH
O
OH
O
O
O
O
HO
O
O
O
O
O
O
O
(HO)2PO
HO
O
(HO)2PO
O
(HO)2PO
HO
O
O
O
O
NH
NH
O
NH
NH
NH
NH
O
O
O
O
CO2H
CO2H
O
O
CO2H
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
OH
O
OH
OH
O
OH
O
O
OH
O
O
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C10)
(C12)
(C12)
(C12)
(C12)
(C12)
26f
(C12)
26d
26e
Figure 4. Lipid A library possessing six acyl groups.
ployed by our previous synthesis and by Boons’ synthesis.25,26
The glycosyl donor 27, whose 2- and 3-positions are pro-
tected with the allyloxycarbonyl (Alloc) and propargyloxycar-
bonyl (Proc) groups respectively, was synthesized as shown in
Scheme 3. The Proc group was stable to neat TFA, but could
be readily cleaved by treatment with Co2(CO)8 and TFA via an
alkyne–Co complex.46 The Proc group could also be cleaved
with Zn–AcOH, Pd0–Et3SiH, or [Ir(cod)(MePh2P)2]PF6 that
was activated with H2 (Ir-complex).46b Since the 1-O-allyl
group could not have been isomerized to a 1-propenyl group
by the Ir-complex in the presence of the N-Alloc group, N-
Fmoc glucosamine allyl glycoside 31 was used as a starting
material. The allyl group was isomerized to a 1-propenyl group
by using the Ir-complex before introduction of the Proc group,
since the latter is readily cleaved with the Ir-complex. The
3-O-Proc derivative 32 formed in 94% yield was treated with
1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-ꢁ]-pyrimidine, poly-
mer-bound, (PTBD) to remove the 2-N-Fmoc group.47 The re-
action was slow with PTBD and 1 day was required for the
complete removal of the Fmoc group, but the solid base was
removed by simple filtration and thus the work-up operation
was quite simple. After the free 2-amino group was again pro-
tected with an Alloc group, reductive opening of the 4,6-O-
benzylidene ring of 33 was effected by the use of the combi-
O
N
H
O
O
N
N
H
O
H
N
O
O
O
O
N
H
N
N
H
H
N
H
O
O
R
N
H
N
O
R = substrate
Figure 5. Host–guest interaction of a polymer-supported
receptor with a barbituric acid tag.
been demonstrated by our total synthesis of E. coli lipid A
based on the latter interaction.31,32
Figure 6 shows the basic synthetic route for constructing the
library. A barbituric acid (BA) tag was attached to the 4-posi-
tion of the glycosyl acceptor via a p-acylaminobenzyl linker
with a glutarylamino spacer. In order to reduce the total num-
ber of reaction steps for the synthesis of the six target com-
pounds, a ꢀ(1!6) disaccharide 40-phosphate 29 was construct-
ed as a common key synthetic intermediate by the coupling of
two monosaccharides, i.e., a glycosyl trichloroacetimidate 27
as a donor and a glycosyl acceptor 28 having the BA-tag.
All the acyl moieties were then introduced step by step to their
respective positions. Acylation of the hydroxy group with the
3-acyloxyfatty acid in the presence of DMAP sometimes
caused ꢀ-elimination of the 3-acyloxy function, especially
when the hydroxy group to be acylated was sterically hindered
by a neighboring long chain N-acyl group. Therefore, acylation
of the 3- or 30-hydroxy group with the 3-acyloxyfatty acid was
carried out prior to 2- and 20-N-acylation. After introducing the
four acyl groups, simultaneous deprotection and cleavage of
the linker by catalytic hydrogenolysis afforded the desired
CM-analogues 26a–26f. The divergent strategy was also em-
nation of Et3SiH and BF3 Et2O. In a small scale experiment
(0.17 mmol of 33), BF3 Et2O was added at once to a solution
ꢁ
ꢁ
of the 4,6-O-benzylidenated compound 33 and Et3SiH in
CH2Cl2 at 0 ꢂC to give the desired 6-O-benzylated product
34 in 93% yield. In a large scale (21.6 mmol of 33), even when
BF3 Et2O was added dropwisely, 30% of 3-O-Alloc derivative
ꢁ
was formed by an undesired reduction of the Proc group. 4-O-
Phosphination of 34 and a subsequent oxidation gave phos-
phate 35 in 91% yield. After removal of the 1-propenyl group
with aqueous I2, treatment with CCl3CN and Cs2CO3 furnish-
ed glycosyl trichloroacetimidate 27.

802 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
HO
BnO
NH
O
O
O
HN
OH
OProc
O
CCl3
O
P
+
O
O
O
O
O
O
O
CO2Bn
O
HN
O
N
N
NHFmoc
NHAlloc
H
H
27
28
BnO
O
O
OProc
O
OH
O
P
O
O
O
O
O
CO2Bn
NHAlloc
NHFmoc
O
HN
O
O
O
HN
O
N
H
N
H
29
BnO
O
O
OCOR
O
OCOR
HO
O
O
P
O
O
O
OCOR
O
OCOR
O
O
O
CO2Bn
O
NHCOR
O
NHCOR
O
O
(HO)2P
O
HO
NHCOR
O
CO2H
HN
HN
NHCOR
O
O
26a-f
N
N
H
H
30a-f
Figure 6. The basic synthesis route for the construction of the lipid A analogue library.
The synthesis of the glycosyl acceptor 28 is shown in
Scheme 4. 4-Azidobenzylglucosamine allyl glycoside 36 was
prepared as previously described.31 The allyl group of 36
was oxidatively cleaved with OsO4 and then with Pb(OAc)4
to give aldehyde 37 in 98% yield. Further oxidation of 37 by
using NaClO2 gave a 1-O-carboxymethyl derivative.48 Benzyl
esterification by slow addition of a phenyldiazomethane solu-
tion gave the desired benzyl ester 38 in 83% yield.49 Treatment
with an excess amount of phenyldiazomethane gave an unde-
sired N-benzylated product. The azido group of 38 was then
reduced using Zn in AcOH, and the resulting amino group
was acylated with glutaric anhydride to afford the carboxylic
acid 39 in 59% yield. The acid 39 was converted to 1-hydroxy-
benzotriazole (HOBt) ester 40, which was then coupled with
the BA-tag moiety 41. The desired BA-tagged product 42 was
obtained in good yield after the affinity separation (outlined as
follows). The reaction mixture in CH2Cl2 was applied to a
short column packed with a resin immobilized artificial recep-
tor of BA. HOBt and small amounts of 39 and 40 were effi-
ciently removed by washing with CH2Cl2, while the desired
42 was retained in the column. Elution of 42 with CH2Cl2–
MeOH (1:1) and concentration gave purified 42. The 3-O-
O
O
O
O
OProc
a, b
Ph
Ph
OH
94%
O
OAllyl
O
O
NHFmoc
NHFmoc
31
c, d
32
O
O
e
Ph
OProc
96%
93%
O
O
NHAlloc
33
BnO
O
BnO
f, g
O
OProc
O
OProc
O
O
91%
P O
O
HO
O
NHAlloc
NHAlloc
34
35
BnO
O
NH
CCl3
O
h, i
OProc
O
MPM group was then removed by treatment with BF3 Et2O
to afford the glycosyl acceptor 28 in 87% yield after affinity
separation.
O
O
ꢁ
73%
P
O
NHAlloc
27
Glycosylation of the BA-tagged 3-O-MPM acceptor 42 with
donor 27 was first attempted by using TMSOTf as a catalyst in
CH2Cl2 at ꢃ20 ꢂC (Scheme 5). Although glycosyl donor 27
disappeared within 1 h, glycosylation of acceptor 42 was in-
complete. Hence, the reaction mixture was once subjected to
the affinity separation. The recovered tagged fraction, which
consisted mainly of 42 and the desired ꢀ-disaccharide 43
was again subjected to glycosylation with 27. Even after this
Scheme 3. Reagents and conditions: (a) [Ir(cod)-
(MePh2P)2]PF6, H2, THF; (b) ProcCl, pyridine, DMAP,
CH2Cl2; (c) PTBD, CH2Cl2; (d) AllocCl, pyridine,
CH2Cl2; (e) Et3SiH, BF3 Et2O, CH2Cl2, 0 ꢂC; (f) N,N-di-
ꢁ
ethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine,
1H-tetrazole, CH2Cl2; (g) mCPBA, ꢃ20 ꢂC; (h) I2, H2O,
THF; (i) CCl3CN, Cs2CO3, CH2Cl2.

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
803
double procedure, the total yield of 43 remained as low as
60%. Glycosylation of a more reactive acceptor 28 with an ad-
ditional free hydroxy group at the 3-position with the same do-
nor 27 using TMSOTf in CH2Cl2 gave 3-O-TMS disaccharide
44 in addition to the desired 29 (80% as a mixture of 29 and
44). Though the unexpectedly introduced TMS group can be
readily cleaved to give 29, further investigation led us to more
favorable conditions for glycosylation: the desired disaccha-
ride 29 was obtained in 96% yield by the use of BF3 Et2O
as a catalyst and THF as a solvent.
ꢁ
The acyl groups were then sequentially introduced to the
respective positions on the disaccharide 29 (Scheme 6 and
Table 2). After acylation of the 3-position of disaccharide
29, the 30-O-Proc group in 45a or 45b was removed by treat-
ment with a stoichiometric amount of an Ir-complex.46b The
30-O-Proc group was also readily cleaved by using Zn–Cu cou-
ple and AcOH. Acylation using diisopropylcarbodiimide
(DIC) and DMAP gave the desired 3,30-di-O-acylated products
46a–46d, which were successfully separated from DMAP,
DIC, and the other by-products by affinity separation. Cleav-
age of the 20-N-Alloc group was carried out by using
Pd(PPh3)4 in the presence of HCO2H and Et3N.50 In contrast,
complete cleavage of the 20-N-Alloc group was not effected by
the use of n-BuNH2 in place of Et3N as an additive. The third
acyl group was introduced to the 20-amino group by using DIC.
Deprotection of the 2-position by treatment with DBU was fol-
lowed by purification using silica-gel short column chromatog-
raphy. Subsequent 2-N-acylation with DIC, affinity separation,
and additional silica-gel chromatography afforded the desired
fully acylated products 30a–30f.
HO
HO
O
OMPM
a, b
O
OMPM
98%
O
O
O
O
CHO
N3
NHFmoc
N3
NHFmoc
36
37
HO
O
OMPM
c, d
e, f
83%
59%
O
O
CO
2Bn
N3
NHFmoc
38
O
HN
O
HO
HN
O
OMPM
O 41
NH2
O
O
O
O
CO2Bn
h
98%
N
H
RO
NHFmoc
39 (R = H)
40 (R = Bt)
g
98%
HO
Table 2 summarizes the reaction time required for all the
acylation steps and the yields of the two-step conversions of
deprotection and acylation. The 3-O-acylation of 29 with ben-
zyloxydecanoic acid 20 and dodecanoyloxydecanoic acid 21
gave 45a and 45b in good yields, respectively. The yields of
the 30-O-acylation of compound 45b, which has a 3-O-acyl-
oxyacyl group, were a little lower than those of 3-O-ben-
zyloxyacylated compound 45a. The 20-N-acylation with ben-
zyloxydecanoic acid 20 afforded 47b, 47d, and 47e in lower
yields than the yields of acylation with dodecanoyloxy-
decanoic acid 21 for the synthesis of 47a, 47c, 47f. Since
the reactivity of the 2-amino group was suppressed by the ster-
ic hindrance of the neighboring 3-O-acyl group, the yields of
the 2-N-acylation reaction were generally not high. Especially,
2-N-acylation of 47d, 47e, and 47f having a 3-O-acyloxyacyl
O
O
O
HN
HN
OR
O
O
O
O
O
CO2Bn
N
N
NHFmoc
H
H
42 (R = MPM)
28 (R = H)
i
87%
Scheme 4. Reagents and conditions: (a) OsO4, NMO, THF/
t-butyl alcohol/water (10:10:1); (b) Pb(OAc)4, benzene/
CH2Cl2 (2:3); (c) NaClO2, NaH2PO4, 2-methyl-2-butene,
THF/t-butyl alcohol/water (2:4:1); (d) phenyldiazo-
methane, Et2O; (e) Zn, AcOH/THF (2:1); (f) glutaric an-
hydride, CH2Cl2; (g) HOBt, DCC, CH2Cl2; (h) Et3N,
DMF, affinity separation; (i) BF3 Et2O, CH2Cl2, 0 ꢂC,
affinity separation.
ꢁ
BnO
NH
CCl3
O
OProc
O
O
P
O
O
O
NHAlloc
HO
BnO
O
O
27
O
O
OProc
O
cat. Lewis Acid
OR
OR
O
O
O
COOBn
O
P
O
O
O
CO2Bn
MS4A
NHFmoc
NHAlloc
NHFmoc
–20 to 0°C
O
O
H
HN
HN
HN
H
N
N
O
NH
O
O
NH
NH
43 (R = MPM)
29 (R = H)
44 (R = TMS)
O
O
O
O
42 (R = MPM)
28 (R = H)
O
Scheme 5. Glycosylation of acceptors possessing BA-tag with donor 27.

804 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
BnO
O
O
O
OProc
O
OCOR1
a, b
c, a, b
O
O
29
P
O
O
O
CO2Bn
3-O-Acylation
3'-O-Acylation
NHAlloc
NHFmoc
O
O
HN
HN
H
NH
N
O
45a (R1= CF3-BnOR)
45b (R1= AcylOR)
O
O
BnO
O
BnO
O
O
OCOR2
O
O
O
OCOR1
O
OCOR1
O
O
O
OCOR2
O
P
d, e, b
P
O
O
O
CO2Bn
O
O
O
CO2Bn
NHCOR3
NHFmoc
2'-N-Acylation
NHAlloc
NHFmoc
O
O
O
O
O
HN
O
HN
H
HN
HN
H
NH
N
NH
O
N
O
O
O
O
47a (R1 = CF3-BnOR, R2 = CF3-BnOR, R3 = AcylOR)
47b (R1 = CF3-BnOR, R2 = AcylOR, R3 = CF3-BnOR)
47c (R1 = CF3-BnOR, R2 = AcylOR, R3 = AcylOR)
47d (R1 = AcylOR, R2 = AcylOR, R3 = CF3-BnOR)
47e (R1 = AcylOR, R2 = CF3-BnOR, R3 = CF3-BnOR)
47f (R1 = AcylOR, R2 = CF3-BnOR, R3 = AcylOR)
46a (R1 = CF3-BnOR, R2 = CF3-BnOR)
46b (R1 = CF3-BnOR, R2 = AcylOR)
46c (R1 = AcylOR, R2 = AcylOR)
46d (R1 = AcylOR, R2 = CF3-BnOR)
BnO
O
O
O
O
OCOR1
O
O
OCOR2
f, g, e, b
P
O
O
O
CO2Bn
NHCOR3
NHCOR4
2-N-Acylation
O
O
HN
HN
H
NH
O
30a (R1 = CF3-BnOR, R2 = CF3-BnOR, R3 = AcylOR, R4 = AcylOR)
30b (R1 = CF3-BnOR, R2 = AcylOR, R3 = CF3-BnOR, R4 = AcylOR)
30c (R1 = CF3-BnOR, R2 = AcylOR, R3 = AcylOR, R4 = CF3-BnOR)
30d (R1 = AcylOR, R2 = AcylOR, R3 = CF3-BnOR, R4 = CF3-BnOR)
30e (R1 = AcylOR, R2 = CF3-BnOR, R3 = CF3-BnOR, R4 = AcylOR)
30f (R1 = AcylOR, R2 = CF3-BnOR, R3 = AcylOR, R4 = CF3-BnOR)
N
O
O
O
O
CF3
O
CF3-BnOR :
AcylOR :
Scheme 6. Reagents and conditions: (a) (R)-3-(4-trifluoromethylbenzyloxy)decanoic acid (20) or (R)-3-(dodecanoyloxy)decanoic
acid (21), DIC, DMAP, CH2Cl2; (b) affinity separation; (c) [Ir(cod)(MePh2P)2]PF6, H2, THF or Zn–Cu, AcOH; (d) Pd(PPh3)4,
HCO2H, Et3N, THF; (e) (R)-3-(4-trifluoromethylbenzyloxy)decanoic acid (20) or (R)-3-(dodecanoyloxy)decanoic acid (21),
DIC, CH2Cl2; (f) DBU, CH2Cl2; (g) silica-gel chromatography.
group gave the fully acylated products always in low yields.
ESI-MS measurements suggested that the undesired by-prod-
ucts 48d and 48f were being formed in the synthesis of 30d
and 30f (positive mode, m=z 2019.49 ½M þ Naꢄþ) (Figure 7).
TLC analysis showed that this side reaction also occurred in
the synthesis of 48a–48c and 48e. Except for the loss of mate-
rial, this undesirable cyclization did not cause serious prob-
lems, since the cyclic by-products which lost the BA-tag were
readily removed from the desired products by the affinity sep-
aration and therefore did not affect the purity.
The biological activities of the six CM-analogues 26a–26f
were evaluated by measuring Limulus activity and cytokine
(IL-6 and TNF-ꢁ) induction, in a manner similar to that men-
tioned above. Compounds 26e and 26f exhibited Limulus ac-
tivity as strong as LPS (Table 1). The Ru. gelatinosus-type an-
alogue compound 26a showed activity, but required concentra-
tions 100 times higher than LPS to activate factor C. In con-
trast, compounds 26b and 26d showed very weak positive
responses, but 26c, which had the same acylation distribution
as E. coli, did not have any activity.
Simultaneous removal of all the benzyl-type protective
groups and the BA-tag moiety using catalytic hydrogenolysis
was investigated. The acylaminobenzyl linker was not cleaved
by the catalytic hydrogenation under neutral conditions. The
acid stability of the CM-analogues allowed us a reaction under
acidic conditions, so that the final deprotection and the cleav-
age of the BA-tag of 30a–30f successfully proceeded by hy-
drogenolysis using Pd(OH)2 in THF–AcOH (3:1) at room tem-
perature for 1 d (Scheme 7). Subsequent purification by liquid–
liquid partition column chromatography afforded the desired
CM-analogues 26a–26f.
The CM-analogue of Ru. gelatinosus lipid A 26a had appa-
rent IL-6 and TNF-ꢁ inducing activities, but it was much less
potent than LPS (Figures 3A, 3C, 8A, and 8C). Compounds
26b–26f did not induce IL-6 or TNF-ꢁ. These results clearly
demonstrate that the distribution of acyl groups also plays a
critical role in determining endotoxic activity under conditions
where the numbers and chain lengths of the acyl groups are
identical.
The inhibitory activities of analogues 26a–26f were tested
on the induction of IL-6 and TNF-ꢁ by LPS (Figure 8B and
8D). Compound 26e had inhibitory activity that was compara-

Y. Fukase et al.
Table 2. Stepwise Acylation Using 20 and 21
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
805
3-O-Acylation
3′-O-Acylation
F.A. Time Yield
46a
2-N-Acylation
F.A. Time Yield
30a
2′-N-Acylation
F.A. Time Yield
47a
F.A. Time Yield
20
9 h
21 18 h
21 14 h
(75%)
(70%)
(49%)
45a
(84%)
20
2 h
47b
(44%)
30b
(39%)
20 18 h
21 14 h
46b
21 10 h
21 11 h
20 13 h
(77%)
47c
(53%)
30c
(35%)
21 19 h
20 18 h
46c
(59%)
47d
(37%)
30d
(14%)
20 12 h
20 25 h
45b
(92%)
21 3.5 h
47e
(42%)
30e
(12%)
20 15 h
21 20 h
46d
(59%)
47f
(63%)
30f
(6%)
21 21 h
20 19 h
O
F3C
(10)
(12)
O
O
COOH
COOH
(10)
20
21
BnO
O
BnO
O
O
O
O
O
OCOR1
O
P
O
O
OCOR2
OCOR3
OCOR1
O
P
O
O
O
O
O
CO2Bn
O
O
O
CO2Bn
O
NHCOR3
NHCOR4
NHCOR2
HN
26a (60%)
O
O
26b(25%)
26c (22%)
26d(67%)
26e (56%)
26f (67%)
HN
HN
H
NH
N
O
O
O
HN
O
30a-f
O
O
CF3
CF3
Scheme 7. Reagents and conditions: H2 (20 kg cmꢃ2),
Pd(OH)2, THF/AcOH (3:1).
O
R1, R3 =
R1, R2 =
R2 =
R3 =
48d :
48f :
O
O
O
while other analogues 26b, 26c, 26d, and 26f were neither
immunostimulatory nor antagonistic. Small structural changes,
i.e., acidic functional groups and acylation distribution, dra-
matically influenced the biological activity, as in the case of
lipid A which has C10 or C12 hexa-acyl groups, which appears
to be a structural boundary for the bioactivity.
Figure 7. Structures of by-products formed during 2-N-
acylation reaction.
ble to biosynthetic precursor 2, but 26a–26d and 26f did not
appear to be inhibitory.
There are two main signal transduction systems for TLR4
signals, which recruit adaptor proteins to TLR4 and induces
cytokines and type I interferon (IFN) by activating the tran-
scription factors, NF-ꢂB and IRF-3, respectively. Seya et al.
reported that compound 26a induced IFN-ꢀ via the IRF-3
pathway, in addition to activating NF-ꢂB, in a similar manner
to E. coli lipid A 1. Both 406 (2) and Ru. gelatinosus lipid A
10a inhibited the production of IFN-ꢀ.51
The reason that changing the acylation distribution of lipid
A analogues 26a–26f effects their bioactivity can be explained
as follows. Recently, Satow et al. reported the crystal struc-
tures of human MD-2 and its complex with antagonist 406
(2).52 Lee et al. reported the 3D structures of the full-length ec-
todomain of the murine TLR4 and the MD-2 complex. They
also determined the structure of the complex of human MD-
2, E5564 (4), and TV3 (a hybrid of the partial structure of hu-
man TLR4 and variable lymphocyte receptor of hagfish).53 In
both MD-2 structures, 2 and 4 bind to the same area in MD-2.
In the complex of human MD-2 with 2, four fatty-acid chains
of 2 are fully confined within a deep hydrophobic cavity that is
sandwiched by two ꢀ-sheets, and phosphate and sugar moie-
ties are located at the cavity ingress (Figure 9A). Molecular
Discussion
As described above, Ru. gelatinosus lipid A 10a showed
potent antagonistic activity against LPS, whereas its 1-O-car-
boxymethyl analogue 26a showed a weak immunostimulatory
activity. Compound 26e showed potent antagonistic activity,

806 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
B
A
25000
20000
15000
10000
5000
20000
16000
12000
8000
4000
1000
100
1
10
100
1000
(ng mL−1
10
0
0
1
LPS
26a
26b
(ng mL−1
)
26c
406
26a
26d
26e
26b
26f
26c
26d
26e
)
26f LPS
C
D
3000
2500
2000
1500
1000
500
2000
1600
1200
800
400
0
1000
100
10
1
10
100
0
1
LPS
26a
26b
26c
26d
(ng mL−1
)
26e
1000
(ng mL−1
26f
406
26a
26b
26c
26d
26e
26f
)
LPS
Figure 8. The cytokine inducing activity and inhibitory activities of 26a–26f, and E. coli LPS 0111:B4, as measured in human
peripheral whole-blood cells. A: IL-6 inducing activity, B: inhibitory activity against IL-6 induction by E. coli LPS 0111:B4
(10 ng mLꢃ1), C: TNF-ꢁ inducing activity, D: inhibitory activity against TNF-ꢁ induction by E. coli LPS 0111:B4 (10 ng mLꢃ1).
A
B
C
Figure 9. Stereo ribbon models of human MD-2 in complexes with 406 (2) (lipid IVa) (A), molecular modeling of human MD-2 in
complex with Ru. gelatinosus lipid A 10a (B), and with 26e (C).
modeling using a united atom AMBERꢂ force field and a
GB/SA continuum solvent model for water, as implemented
in MacroModel (version 7.1), revealed that the Ru. gelatinosus
lipid A 10a and the antagonist 26e could bind to MD-2 in a
manner similar to 406 (2) (Figures 9B and 9C). These results
indicate that the volume of the four C10 and two C12 fatty-
acid chains can fit the hydrophobic pocket of MD-2.
The volume of the acyl groups in 26a–26f should have been
similar, but 26b, 26c, and 26d are inactive in both the human
peripheral blood system and Limulus test and 26f is inactive in
the human peripheral blood system. These results suggest that
the molecular conformation is probably affected by the distri-
bution of the acyl groups. From molecular mechanics calcula-
tions of these compounds, the biologically active compounds
26a, 26e, and 26f had ordered low energy conformations, in
which the acyl chains were aligned in parallel and were closely
packed. On the other hand, the low energy conformations of
the inactive compounds had acyl moieties with disordered
structures (Figure 10). The distribution of the acylation should
therefore affect the tendency of these lipids to aggregate.
Seydel et al. revealed that formation of aggregates is essential
for expression of the endotoxic activity; monomeric lipid A
and LPS prepared by a dialysis procedure showed no activity,
whereas their aggregates at the same concentrations were bio-
logically active.54 Monomeric lipid A and LPS molecules
might be conformationally flexible due to their lack of inter-
molecular hydrophobic interactions and a large entropic loss
should prevent their binding to the LPS receptor system, which

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
807
26a
26e
26c
ꢀ
Figure 10. The lowest energy conformations of 26a, 26e, and 26c, calculated with MacroModel v7.1 (Amber , low mode, GB/SA
water).
consists of LPS binding protein (LBP) in blood, the glycosyl
phosphatidyl inositol (GPI) anchor protein CD14, and the
TLR4/MD-2 complex. LBP binds to oligomeric LPS and
should recognize the particular conformation of lipid A in
the supramolecular assembly; LBP then should transfer lipid
A (LPS) from the aggregates to CD14 and then lipid A
(LPS) should be transferred from CD14 to TLR4/MD2. The
molecular modeling study suggested that the inactive ana-
logues 26b, 26c, and 26d might not form the ordered supramo-
lecular structure, whereas the bioactive compounds should
form the supramolecular assembly.
or antagonistic activity was observed depending on their
anionic charges (carboxylic acid vs. phosphoric acid).58,59 In
addition, we recently found that synthetic tri-acyl type Helico-
bacter pylori lipid A having 1-phosphate shows antagonistic
activity against the induction of inflammatory cytokines such
as IL-6, whereas H. pylori lipid A, in which an ethanolamine
group is linked to the 1-phosphate, shows weak agonistic ac-
tivity.60 The number of anionic charges in all agonists was de-
creased in comparison to their corresponding antagonists. It is
expected that subtle difference in anionic charges decisively
influences the binding manner to TLR4/MD-2 complex at
around the boundary critical structure of lipid A required for
endotoxic or antagonistic activity. The present work showed
the volume of acyl moieties in Ru. gelatinosus lipid A may
corresponds to the boundary structure and hence the differ-
ences in the acidic functional groups affected the bioactivity.
Although the aggregate formation of lipid A and LPS is es-
sential for their biological activity, TLR4/MD-2 should recog-
nize them as single molecules. X-ray crystallographic analysis
indicates that MD-2 binds to the antagonists 406 (2) and E5564
(4) in a 1:1 ratio, and MD-2 forms a stable complex with
TLR4 (i.e., one TLR4/MD-2 binds to one antagonist). It has
been reported that the binding of agonistic lipid A and LPS
induces TLR4 aggregation and initiates intracellular signal-
ing.12,53,55–57 Immunoprecipitation assays using tritium-labeled
lipid A analogues and antiTLR4/MD-2 antibodies revealed
that maximal binding of the antagonistic 406 analogue to hu-
man TLR4/MD-2 was ca. 2-fold higher than that of agonistic
E. coli lipid A 1, suggesting that E. coli lipid A binds to
TLR4/MD-2 in a 1:2 ratio.55 Endotoxic lipid A should be rec-
ognized by two TLR4/MD-2 molecules and consequently in-
duce the dimerization of TLR4/MD-2, whereas binding of an-
tagonistic lipid A to an isolated single TLR4/MD-2 complex
does not induce dimerization of the complex. Although the
mode of the interaction between the TLR4-complex with the
agonistic lipid As and LPS has not been clarified yet, there
must be significant differences between their interactions with
the antagonists and the agonists. Since the four acyl groups of
406 (2) and E5564 (4) almost occupy the hydrophobic pocket
in MD-2, significant structural changes of the pocket seem to
be inevitable when E. coli lipid A 1 binds to MD-2. This struc-
tural change in MD-2 may induce dimerization and activation
of the TLR4–MD-2 complex. However, the difference be-
tween antagonistic Ru. gelatinosus lipid A 10a and agonistic
26a is only an acidic functional group at the 1 position. Similar
results were obtained from our studies of lipid A analogues
that contained acidic amino acid residues; immunostimulatory
Experimental
General Procedures. 1H NMR spectra were measured in the
indicated solvents using a JEOL JNM-LA500, a JEOL JNM-GSX
400, or a Varian UNITYplus 600 spectrometer. The chemical
shifts in CDCl3 and DMSO-d6 are given in ꢃ values using tetra-
methylsilane (TMS) as an internal standard. Mass spectra were
measured using an ESI-TOF mass spectrometer (Applied Biosys-
tems, Marinerꢂ). Specific rotations were measured using a
Perkin-Elmer 241 polarimeter. Elemental analyses were deter-
mined using Yanaco CHN corders MT-3, MT-5, and MT-6. Recy-
cling preparative HPLC was carried out with an LC908 (Japan
Analytical Industry). Silica-gel column chromatography was car-
ried out with Kieselgel 60 (Merck, 0.040–0.063 mm) at medium
pressure (2–4 kg cmꢃ2) using the indicated solvent systems.
Analytical and preparative thin layer chromatographies (TLC)
were performed on precoated Kieselgel 60F254 Plates (Merck,
0.25 mm thickness) and precoated Kieselgel 60F254 Plates (Merck,
0.5 mm thickness), respectively. Anhydrous CH2Cl2 was distilled
from CaH2. Anhydrous CHCl3, THF, Et2O, DMF, CH3CN, tol-
uene, and benzene were purchased from Kanto Chemicals, Tokyo,
Japan. Distilled water, purchased from Otsuka (Tokyo, Japan) or
prepared by a combination of Toray Pure LV-308 (Toray) and
GSL-200 (Advantec, Tokyo, Japan), was used as an eluent for
the liquid–liquid partition column chromatography and as solvent
for the lyophilization. Molecular sieves 4A (MS4A) was activated
by heating at 250 ꢂC in vacuo for 3 h before use. All other com-

808 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
mercially obtained materials were used as received.
1H, ꢁ-CH2 of 3-O-acyl), 2.50 (dd, J ¼ 15:1, 4.4 Hz, 1H, ꢁ-CH2
Allyl 4,6-O-Benzylidene-2-deoxy-2-(2,2,2-trichloroethoxy-
carbonylamino)-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)dec-
anoyl]-ꢀ-D-glucopyranoside (13). To a solution of allyl 4,6-O-
of 3-O-acyl), 1.72–1.49 (m, 2H, ꢁ-CH2 of 3-O-acyl), 1.31–1.26
(m, 10H, CH2 ꢅ 5), 0.87 (t, J ¼ 6:9 Hz, 3H, –CH2–CH3).
Allyl 6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-2,4,3ꢁ5
-
benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
ꢁ
-
benzodioxaphosphepin-3-yl)-2-(2,2,2-trichloroethoxycarbonyl-
amino)-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-
D-glucopyranoside (12) (3.00 g, 6.22 mmol) and (R)-3-(4-trifluoro-
methylbenzyloxy)decanoic acid (20) (2.59 g, 7.46 mmol) in anhy-
drous CH2Cl2 (200 mL) were added DCC (2.31 g, 11.2 mmol) and
DMAP (75.9 mg, 0.622 mmol) at room temperature under Ar at-
mosphere, and the mixture was stirred for 15 h. After additional
stirring for 2 h with the addition of (R)-3-(4-trifluoromethyl-
benzyloxy)decanoic acid (20) (1.14 g, 3.29 mmol), DCC (0.998 g,
4.84 mmol), and DMAP (79.2 mg, 0.648 mmol), the precipitate
was filtrated off and the solution was concentrated under reduced
pressure. The residue was purified by silica-gel flash chromatogra-
phy (300 g, CHCl3:acetone = 70:1) to give 13 (4.62 g, 92%)
as a colorless solid. ESI-MS (positive) m=z 827.3 ½M þ NH4ꢄþ,
832.2 ½M þ Naꢄþ. 1H NMR (400 MHz, CDCl3): ꢃ 7.48 (d,
J ¼ 8:3 Hz, 2H, p-CF3–C6H4–CH2–), 7.39 (dd, J ¼ 8:1, 2.0 Hz,
2H, p-CF3–C6H4–CH2–), 7.30–7.24 (m, 5H, =CH–Ph), 5.90 (m,
1H, –OCH2–CH=CH2), 5.46 (s, 1H, =CH–Ph), 5.42 (t, J ¼
10:0 Hz, 1H, H-3), 5.35 (d, J ¼ 10:0 Hz, 1H, NH), 5.31 (dd,
J ¼ 17:2, 1.4 Hz, 1H, –OCH2–CH=CH2), 5.25 (dd, J ¼ 10:5,
1.2 Hz, 1H, –OCH2–CH=CH2), 4.93 (d, J ¼ 3:7 Hz, 1H, H-1),
4.70 (d, J ¼ 12:1 Hz, 1H, –CO–O–CH2–CCl3), 4.63 (d, J ¼
12:1 Hz, 1H, –CO–O–CH2–CCl3), 4.53 (d, J ¼ 12:2 Hz, 1H, p-
CF3–C6H4–), 4.43 (d, J ¼ 12:4 Hz, 1H, p-CF3–C6H4–CH2–),
4.29 (dd, J ¼ 10:3, 4.7 Hz, 1H, H-6a), 4.22 (ddt, J ¼ 12:7,
5.4, 1.2 Hz, 1H, –OCH2CH=CH2), 4.08 (ddd, J ¼ 10:1, 10.1,
3.8 Hz, 1H, H-2), 4.03 (ddd, J ¼ 12:7, 6.3, 1.2 Hz, 1H,
–OCH2CH=CH2), 3.95 (ddd, J ¼ 9:8, 9.8, 4.7 Hz, 1H, H-5),
3.82 (m, 1H, ꢀ-CH of 3-O-acyl), 3.78 (dd, J ¼ 10:3, 10.3 Hz,
1H, H-6b), 3.71 (t, J ¼ 9:5 Hz, 1H, H-4), 2.65 (dd, J ¼ 15:4,
6.9 Hz, 1H, ꢁ-CH2 of 3-O-acyl), 2.45 (dd, J ¼ 15:4, 5.1 Hz, 1H,
ꢁ-CH2 of 3-O-acyl), 1.33–1.16 (m, 12H, CH2 ꢅ 6), 0.867 (t,
J ¼ 7:3 Hz, 3H, –CH2–CH3). Found: C, 55.31; H, 5.64; N,
1.92%. Calcd for C37H45Cl3F3NO9: C, 54.79; H, 5.59; N, 1.73%.
Allyl 6-O-Benzyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonyl-
ꢀ
-D-
glucopyranoside (15). To a solution of 14 (1.80 g, 2.21 mmol) in
anhydrous CH2Cl2 (30 mL) were added N,N-diethyl-1,5-dihydro-
3H-2,4,3-benzodioxaphosphepin-3-amine (0.801 g, 3.34 mmol)
and 1H-tetrazole (0.465 g, 6.64 mmol) at room temperature under
Ar atmosphere. After the mixture was stirred for 50 min and then
at ꢃ20 ꢂC for 15 min, mCPBA (0.381 g, 2.21 mmol) was added
and stirring was continued for another 20 min. The solution was
quenched by addition of saturated aqueous NaHCO3, and extract-
ed with EtOAc. The organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by silica-gel flash column chromatography (100 g, CHCl3:
acetone = 30:1) to give 4-O-phosphate 15 (2.06 g, 94%) as a col-
22
orless syrup. ½ꢁꢄD ¼ þ34:6 (c 1.00, CHCl3). ESI-MS (positive)
m=z 994.2 ½M þ Hꢄþ, 1016.2 ½M þ Naꢄþ. 1H NMR (400 MHz,
CDCl3): ꢃ 7.50 (d, J ¼ 8:3 Hz, 2H, p-CF3–C6H4–CH2–), 7.43
(d, J ¼ 8:3 Hz, 2H, p-CF3–C6H4–CH2–), 7.39–7.24 (m, 6H,
o-C6H4(CH2O)2P– and Ph–CH2–), 7.17 (ddd, J ¼ 7:5, 7.4,
1.0 Hz, 1H, o-C6H4(CH2O)2P–), 7.12 (d, J ¼ 7:8 Hz, 1H, o-
C6H4(CH2O)2P–), 6.70 (d, J ¼ 7:3 Hz, 1H, o-C6H4(CH2O)2P–),
5.89 (m, 1H, –OCH2–CH=CH2), 5.40 (t, J ¼ 9:8 Hz, 1H, H-3),
5.32–5.27 (m, 2H, NH and –OCH2–CH=CH2), 5.23 (dd,
J ¼ 9:2, 1.0 Hz, 1H, –OCH2–CH=CH2), 5.12–4.94 (m, 5H, o-
C6H4(CH2O)2P– and H-1), 4.76 (dd, J ¼ 18:5, 9.3 Hz, 1H, H-
4), 4.66–4.55 (m, 6H, –CO–O–CH2–CCl3, p-CF3–C6H4–CH2–,
and Ph–CH2–), 4.22 (dd, J ¼ 12:7, 5.3 Hz, 1H, –OCH2CH=CH2),
4.06–3.99 (m, 3H, –OCH2CH=CH2, H-2, and H-5), 3.90 (m, 1H,
ꢀ-CH of 3-O-acyl), 3.80 (dd, J ¼ 11:2, 2.0 Hz, 1H, H-6a), 3.74
(dd, J ¼ 10:7, 4.9 Hz, 1H, H-6b), 2.75 (dd, J ¼ 17:1, 7.8 Hz,
1H, ꢁ-CH2 of 3-O-acyl), 2.56 (dd, J ¼ 17:1, 3.9 Hz, 1H, ꢁ-CH2
of 3-O-acyl), 1.42–1.21 (m, 12H, CH2 ꢅ 6), 0.88 (t, J ¼ 6:8 Hz,
3H, –CH2–CH3).
6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-2,4,3ꢁ5-ben-
zodioxaphosphepin-3-yl)-2-(2,2,2-trichloroethoxycarbonylami-
amino)-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-ꢀ-D-
glucopyranoside (14). To a solution of 13 (1.00 g, 1.23 mmol)
and triethylsilane (0.982 mL, 6.16 mmol) in dry CH3CN (12 mL)
was added diethyl ether–boron trifluoride (1/1) (0.463 mL,
3.69 mmol) dropwise and the mixture was stirred at 0 ꢂC for
1.5 h. The reaction was then quenched with saturated aqueous
NaHCO3 and the mixture was extracted with EtOAc. The organic
layer was washed with saturated aqueous NaHCO3 and brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by silica-gel flash chromatography (50 g, CHCl3:
acetone = 20:1) to give 14 as a colorless syrup (0.742 g, 74%).
no)-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-ꢀ-D-glu-
copyranose (16). To a degassed solution of 15 (980.1 mg, 0.985
mmol) in dry THF (14 mL) was added [Ir(cod)(MePh2P)2]PF6
(83.3 mg, 0.0985 mmol) activated with H2 in THF (10 mL). After
being stirred under Ar at room temperature for 2 h, iodine
(300.3 mg, 1.183 mmol) and water (20 mL) were added and the re-
action mixture was stirred for additional 30 min. The reaction mix-
ture was quenched with aqueous 10% Na2S2O3 (10%, 10 mL).
The mixture was then extracted with EtOAc. The organic layer
was washed with aqueous sat NaHCO3 and brine, dried over
MgSO4, and then concentrated in vacuo. The residue was purified
by silica-gel flash chromatography (40 g, CHCl3:acetone = 10:1)
to give compound 16 as a pale yellow solid (698.7 mg, 74%).
22
½ꢁꢄD ¼ þ38:5 (c 0.757, CHCl3). ESI-MS (positive) m=z 829.3
½M þ NH4ꢄþ, 834.3 ½M þ Naꢄþ. 1H NMR (400 MHz, CDCl3):
ꢃ 7.56 (d, J ¼ 8:3 Hz, 2H, p-CF3–C6H4–CH2–), 7.41 (d, J ¼
8:3 Hz, 2H, p-CF3–C6H4–CH2–), 7.36–7.25 (m, 5H, Ph–CH2–),
5.88 (m, 1H, –OCH2–CH=CH2), 5.31–5.11 (m, 4H, 2-NH, H-3,
and –OCH2–CH=CH2), 4.91 (d, J ¼ 3:9 Hz, 1H, H-1), 4.66 (s,
2H, Ph–CH2–), 4.62–4.54 (m, 4H, –CO–O–CH2–CCl3 and p-
CF3–C6H4–CH2–), 4.53 (d, J ¼ 12:2 Hz, 1H, p-CF3–C6H4–
CH2–), 4.43 (d, J ¼ 12:4 Hz, 1H, p-CF3–C6H4–CH2–), 4.19
(dd, J ¼ 13:0, 5.2 Hz, 1H, –OCH2CH=CH2), 4.03–3.95 (m, 2H,
–OCH2CH=CH2 and H-2), 3.90 (m, 1H, ꢀ-CH of 3-O-acyl),
3.84–3.80 (m, 1H, H-6a), 3.77–3.67 (m, 3H, H-4, H-5, and H-
6b), 2.78 (d, J ¼ 2:9 Hz, 1H, 4-OH), 2.65 (dd, J ¼ 15:0, 7.8 Hz,
22
½ꢁꢄD ¼ þ12:3 (c 0.998, CHCl3). ESI-MS (positive) m=z 976.3
½M þ Naꢄþ. 1H NMR (400 MHz, CDCl3): ꢃ 7.50 (d, J ¼ 8:3 Hz,
2H, p-CF3–C6H4–CH2–), 7.42–7.25 (m, 6H, o-C6H4(CH2O)2P–
and Ph–CH2–), 7.41 (d, J ¼ 8:0 Hz, 2H, p-CF3–C6H4–CH2–),
7.17 (ddd, J ¼ 7:6, 7.6, 1.2 Hz, 1H, o-C6H4(CH2O)2P–), 7.12
(d, J ¼ 7:6 Hz, 1H, o-C6H4(CH2O)2P–), 6.70 (d, J ¼ 7:3 Hz,
1H, o-C6H4(CH2O)2P–), 5.44 (t, J ¼ 10:0 Hz, 1H, H-3), 5.36 (d,
J ¼ 9:5 Hz, 1H, NH), 5.30 (t, J ¼ 3:4 Hz, 1H, H-1), 5.09–4.92
(m, 4H, o-C6H4(CH2O)2P–), 4.71–4.55 (m, 7H, –CO–O–CH2–
CCl3, p-CF3–C6H4–CH2–, Ph–CH2–, and H-4), 4.25 (m, 1H, H-

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
809
5), 3.98 (ddd, J ¼ 10:0, 10.0, 2.9 Hz, 1H, H-2), 3.90 (m, 1H, ꢀ-
CH of 3-O-acyl), 3.79 (dd, J ¼ 10:7, 1.8 Hz, 1H, H-6a), 3.71
(dd, J ¼ 9:8, 6.0 Hz, 1H, H-6b), 3.46 (brs, 1H, C1–OH), 2.75
(dd, J ¼ 17:0, 7.9 Hz, 1H, ꢁ-CH2 of 3-O-acyl), 2.56 (dd,
J ¼ 17:0, 4.0 Hz, 1H, ꢁ-CH2 of 3-O-acyl), 1.36–1.27 (m, 12H,
CH2 ꢅ 6), 0.88 (t, J ¼ 7:0 Hz, 3H, –CH2–CH3). Found: C,
52.59; H, 5.07; N, 1.51%. Calcd for C42H50Cl3F3NO12P: C,
52.81; H, 5.28; N, 1.47%.
0.84 (m, 9H, –CH2–CH3 ꢅ 3). Found: C, 68.75; H, 9.09; N,
2.33%. Calcd for C56H84F3NO10: C, 68.06; H, 8.57; N, 1.42%.
Allyl 2-Deoxy-2-[(R)-3-(dodecanoyloxy)decanoylamino]-3-
O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-ꢀ-D-glucopyr-
anoside (19). To a solution of 18 (3.33 g, 3.37 mmol) in dry
CH2Cl2 (72 mL) was added 90% TFA aqueous solution (3 mL)
at 0 ꢂC. The mixture was stirred under Ar for 2.5 h while warming
gradually up to room temperature. The reaction mixture was
quenched with aqueous sat NaHCO3. The mixture was then ex-
tracted with CHCl3. The organic layer was washed with aqueous
sat NaHCO3 and brine, dried over MgSO4, and then concentrated
in vacuo. The residue was purified by silica-gel flash chromatog-
raphy (160 g, CHCl3:acetone = 5:1 to 3:1) to give compound 19
as a colorless oil (2.28 g, 75%). ESI-MS (positive) m=z 900.6
½M þ Hꢄþ, 922.6 ½M þ Naꢄþ. 1H NMR (400 MHz, CDCl3): ꢃ
7.58 (d, J ¼ 8:1 Hz, 2H, –CH2–C6H4–CF3), 7.42 (d, J ¼ 8:1 Hz,
2H, –C6H4–CF3), 5.94 (d, J ¼ 9:0 Hz, 1H, NH), 5.89 (m, 1H,
–OCH2–CH=CH2), 5.30 (dd, J ¼ 17:3, 1.5 Hz, 1H, –OCH2–
CH=CH2), 5.23 (dd, J ¼ 10:5, 1.2 Hz, 1H, –OCH2–CH=CH2),
5.15–5.05 (m, 2H, H-3 and ꢀ-CH of 2-N-acyl), 4.85 (d,
J ¼ 3:7 Hz, 1H, H-1), 4.57 (s, 2H, –CH2–C6H4–CF3), 4.22 (m,
1H, H-2), 4.18 (ddt, J ¼ 14:7, 5.1, 1.5 Hz, 1H, –OCH2CH=CH2),
3.98 (ddt, J ¼ 12:8, 6.3, 1.2 Hz, 1H, –OCH2CH=CH2), 3.90–3.70
(m, 5H, H-4, H-5, H-6ab, and ꢀ-CH of 3-O-acyl), 2.65 (dd,
J ¼ 14:9, 7.8 Hz, 1H, ꢁ-CH2 of 3-O-acyl), 2.53 (dd, J ¼ 14:9,
4.9 Hz, 1H, ꢁ-CH2 of 3-O-acyl), 2.40 (dd, J ¼ 14:8, 7.0 Hz, 1H,
ꢁ-CH2 of 2-N-acyl’s main chain), 2.35–2.25 (m, 3H, ꢁ-CH2 of
2-N-acyl’s main chain and 2-N-acyl’s side chain), 1.71–1.50 (m,
6H, ꢄ-CH2 of 3-O-acyl, 2-N-acyl’s main chain, and ꢀ-CH2 of
2-N-acyl’s side chain), 1.37–1.16 (m, 36H, CH2 ꢅ 18), 0.90–
0.85 (m, 9H, –CH2–CH3 ꢅ 3).
6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-2,4,3ꢁ5-ben-
zodioxaphosphepin-3-yl)-2-(2,2,2-trichloroethoxycarbonylami-
no)-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-ꢀ-D-glu-
copyranosyl Trichloroacetimidate (17). To a solution of 1-
liberated 16 (123.0 mg, 128.8 mmol) and CCl3CN (64.7 mL,
645 mmol) in dry CH2Cl2 (7 mL) were added Cs2CO3 (24.4 mg,
74.9 mmol) at rt. After being stirred for 1 h, to the reaction mixture
were added CCl3CN (64.7 mL, 645 mmol), Cs2CO3 (32.4 mg,
99.4 mmol), and the reaction mixture was stirred for an additional
45 min. The reaction mixture was quenched with aqueous 10%
Na2S2O3. The mixture was then extracted with EtOAc. The organ-
ic layer was washed with brine, dried over MgSO4, and then con-
centrated in vacuo to give 17 (139.6 mg, 98%) as a pale yellow
solid), which was used for subsequent glycosylation without puri-
fication.
Allyl 4,6-O-Benzylidene-2-deoxy-2-[(R)-3-(dodecanoyloxy)-
decanoylamino]-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)dec-
anoyl]-ꢀ-D-glucopyranoside (18). To a solution of 13 (3.36 g,
4.14 mmol) in AcOH (50 mL) was added Zn–Cu (prepared from
3.5 g of Zn), and the mixture was stirred at rt for 3 h. The insoluble
materials were filtered off, and the filtrate was concentrated in
vacuo. The residual solvent was removed by coevaporation with
toluene (5 mL ꢅ 3). The residue was dissolved in EtOAc, washed
successively with saturated aqueous NaHCO3 and brine, dried
over Na2SO4, and concentrated in vacuo. To a solution of the
residue and (R)-3-(dodecanoyloxy)decanoic acid (21) (2.06 g,
5.55 mmol) in anhydrous CH2Cl2 were added DCC (1.43 g,
6.93 mmol) at room temperature under Ar atmosphere and the
mixture was stirred for 2 h. The insoluble materials were filtered
off, and EtOAc was added to the filtrate. The solution was washed
with aqueous sat NaHCO3 and brine, dried over MgSO4, and then
concentrated in vacuo. The residue was purified by silica-gel flash
chromatography (270 g, toluene:AcOEt = 5:1) to give compound
Allyl 6-O-(6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-
2,4,3ꢁ5-benzodioxaphosphepin-3-yl)-2-(2,2,2-trichloroethoxy-
carbonylamino)-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)dec-
anoyl]-ꢂ-D-glucopyranosyl)-2-deoxy-2-[(R)-3-(dodecanoyloxy)-
decanoylamino]-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)dec-
anoyl]-ꢀ-D-glucopyranoside (22). To a mixture of the imidate
17 (139 mg, 126 mmol), the glycosyl acceptor 19 (94.3 mg,
105 mmol), and MS4A (1 g) in dry CH2Cl2 (7 mL) at ꢃ20 ꢂC
was added TMSOTf (2.64 mL, 14.6 mmol). After being stirred at
the same temperature for 30 min, TMSOTf (2.50 mL, 13.8 mmol)
was added to the solution, and the mixture was stirred further
30 min.
22
18 (3.49 g, 85%) as a colorless solid. ½ꢁꢄD ¼ þ26:0 (c 0.998,
CHCl3). ESI-MS (positive) m=z 988.6 ½M þ Hꢄþ, 1010.6
½M þ Naꢄþ. 1H NMR (400 MHz, CDCl3): ꢃ 7.48 (d, J ¼ 8:1 Hz,
2H, p-CF3–C6H4–CH2–), 7.39 (m, 2H, p-CF3–C6H4–CH2–),
7.31–7.23 (m, 4H, =CH–C6H5), 7.17 (m, 1H, =CH–C6H5),
5.99 (d, J ¼ 9:5 Hz, 1H, NH), 5.90 (m, 1H, –OCH2–CH=CH2),
5.47 (s, 1H, =CH–C6H5), 5.37 (t, J ¼ 10:0 Hz, 1H, H-3), 5.31
(dd, J ¼ 17:1, 1.5 Hz, 1H, –OCH2–CH=CH2), 5.24 (dd, J ¼
10:4, 1.2 Hz, 1H, –OCH2–CH=CH2), 5.09 (m, 1H, ꢀ-CH of
2-N-acyl), 4.87 (d, J ¼ 3:7 Hz, 1H, H-1), 4.53 (d, J ¼ 12:2 Hz,
1H, –CH2–C6H4–CF3), 4.42 (d, J ¼ 12:2 Hz, 1H, –CH2–C6H4–
CF3), 4.36 (ddd, J ¼ 6:8, 6.8, 3.8 Hz, 1H, H-2), 4.29 (dd,
J ¼ 10:2, 4.8 Hz, 1H, H-6a), 4.20 (ddt, J ¼ 12:7, 5.2, 1.5 Hz,
1H, –OCH2CH=CH2), 4.00 (dd, J ¼ 16:6, 6.4, Hz, 1H,
–OCH2CH=CH2), 3.93 (ddd, J ¼ 10:2, 9.8, 5.1 Hz, 1H, H-5),
3.81 (m, 1H, ꢀ-CH of 3-O-acyl), 3.79–3.69 (m, 2H, H-4, H-6b),
2.67 (dd, J ¼ 15:3, 6.8 Hz, 1H, ꢁ-CH2 of 3-O-acyl), 2.49–2.33
(m, 3H, ꢁ-CH2 of 3-O-acyl and 2-N-acyl’s main chain), 2.28
(t, J ¼ 7:4 Hz, 2H, ꢁ-CH2 of 2-N-acyl’s side chain), 1.64–1.46
(m, 6H, ꢄ-CH2 of 3-O-acyl, 2-N-acyl’s main chain, and ꢀ-CH2
of 2-N-acyl’s side chain), 1.37–1.15 (m, 36H, CH2 ꢅ 18), 0.89–
The reaction was quenched with aqueous NaHCO3 (100 mL),
and MS4A was filtered off. The mixture was extracted with
EtOAc. The organic layer was washed with aqueous NaHCO3
and brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica-gel flash chromatography (20 g,
CHCl3:acetone = 20:1) to give 22 as a colorless solid (129 g,
22
72%). ½ꢁꢄD ¼ þ13:5 (c 0.643, CHCl3). ESI-MS (positive) m=z
1836.0 ½M þ Hꢄþ. 1H NMR (500 MHz, CDCl3): ꢃ 7.56 (d,
J ¼ 8:1 Hz, 2H, p-CF3–C6H4–CH2–), 7.52 (d, J ¼ 8:1 Hz, 2H,
p-CF3–C6H4–CH2–), 7.44 (d, J ¼ 7:9 Hz, 2H, p-CF3–C6H4–
CH2–), 7.41 (d, J ¼ 7:9 Hz, 2H, p-CF3–C6H4–CH2–), 7.40–7.26
(m, 6H, o-C6H4(CH2O)2P– and Ph–CH2–), 7.19 (dd, J ¼ 7:4,
7.4 Hz, 1H, o-C6H4(CH2O)2P–), 7.12 (d, J ¼ 7:3 Hz, 1H, o-
C6H4(CH2O)2P–), 6.75 (d, J ¼ 7:5 Hz, 1H, o-C6H4(CH2O)2P–),
5.91 (d, J ¼ 9:5 Hz, 1H, 2-NH), 5.87 (m, 1H, –OCH2–CH=CH2),
5.42 (t, J ¼ 9:9 Hz, 1H, H-30), 5.29 (dd, J ¼ 17:2, 1.5 Hz, 1H,
–OCH2–CH=CH2), 5.21 (m, 2H, –OCH2–CH=CH2 and 20-NH),
5.12 (dd, J ¼ 10:5, 9.3 Hz, 1H, H-3), 5.07 (m, 1H, ꢀ-CH of 2-
N-acyl), 5.06–4.90 (m, 4H, o-C6H4(CH2O)2P–), 4.83–4.79 (m,

810 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
2H, H-1 and H-10), 4.67 (m, 1H, H-40), 4.66–4.53 (m, 8H,
–CO–O–CH2–CCl3, p-CF3–C6H4–CH2– ꢅ 2, and Ph–CH2–),
4.21 (ddd, J ¼ 10:8, 9.4, 3.7 Hz, 1H, H-2), 4.16 (ddt, J ¼ 12:9,
5.3, 1.4 Hz, 1H, –OCH2CH=CH2), 4.07 (d, J ¼ 9:3 Hz, 1H,
H-6a), 3.94 (ddt, J ¼ 12:8, 6.3, 1.1 Hz, 1H, –OCH2CH=CH2),
3.90–3.86 (m, 2H, ꢀ-CH of 3-O-acyl and 30-O-acyl), 3.84–3.76
(m, 3H, H-5, H-60a, and H-60b), 3.74–3.70 (m, 2H, H-6b and
H-50), 3.64 (ddd, J ¼ 9:2, 9.2, 4.3 Hz, 1H, H-4), 3.50 (dd,
J ¼ 18:4, 8.3 Hz, 1H, H-20), 2.84 (brs, 1H, C4-OH), 2.74 (dd,
J ¼ 16:8, 7.5 Hz, 1H, ꢁ-CH2 of 30-O-acyl), 2.66–2.61 (m, 2H,
ꢁ-CH2 of 3-O-acyl and 30-O-acyl), 2.50 (dd, J ¼ 15:3, 4.6 Hz,
1H, ꢁ-CH2 of 3-O-acyl), 2.38 (dd, J ¼ 14:7, 6.8 Hz, 1H, ꢁ-CH2
of 2-N-acyl’s main chain), 2.29 (dd, J ¼ 14:8, 5.3 Hz, 1H, ꢁ-
CH2 of 2-N-acyl’s main chain), 2.28–2.25 (m, 2H, ꢁ-CH2 of 2-
N-acyl’s side chain), 1.60–1.50 (m, 6H, ꢄ-CH2 of 2-N-acyl’s main
chain, 3-O-acyl, and 30-O-acyl), 1.38–1.25 (m, 48H, CH2 ꢅ 24),
0.89–0.86 (m, 12H, –CH2–CH3 ꢅ 4).
OH), 2.75–2.60 (m, 3H,
2.47 (dd, J ¼ 15:7, 4.8 Hz, 1H,
(m, 2H,
-CH2 of 2-N-acyl’s main chain and 20-N-acyl’s main
chain), 2.30–2.21 (m, 6H, -CH2 of 2-N-acyl’s main chain,
2-N-acyl’s side chain, 20-N-acyl’s main chain, and 2-N-acyl’s
side chain), 1.60–1.51 (m, 8H, -CH2 of 2-N-acyl’s main chain,
ꢁ
-CH2 of 3-O-acyl and 30-O-acyl),
-CH2 of 3-O-acyl), 2.38–2.33
ꢁ
ꢁ
ꢁ
ꢄ
2-N0-acyl’s main chain, 3-O-acyl, and 30-O-acyl), 1.38–1.25 (m,
76H, CH2 ꢅ 38), 0.89–0.86 (m, 18H, –CH2–CH3 ꢅ 6).
6-O-(6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-2,4,3ꢁ5
-
benzodioxaphosphepin-3-yl)-2-[(R)-3-(dodecanoyloxy)decanoyl-
amino]-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-ꢂ-D-
glucopyranosyl)-2-deoxy-2-[(R)-3-(dodecanoyloxy)decanoyl-
amino]-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-ꢀ-D-
glucopyranose (24). To a solution of 23 (70.4 mg, 34.9 mmol)
in dry THF (4 mL) was added [Ir(cod)(MePh2P)2]PF6 (9.3 mg,
11 mmol) activated with H2 in THF (4 mL). After being stirred un-
der Ar at room temperature for 2 h, iodine (9.5 mg, 37 mmol) and
water (4 mL) were added and the reaction mixture was stirred for
an additional 1 h. The reaction mixture was quenched with aque-
ous 10% Na2S2O3. The mixture was then extracted with EtOAc.
The organic layer was washed with aqueous sat NaHCO3 and
brine, dried over MgSO4, and then concentrated in vacuo. The
residue was purified by silica-gel flash chromatography (5 g,
Allyl 6-O-(6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-
2,4,3ꢁ5-benzodioxaphosphepin-3-yl)-2-[(R)-3-(dodecanoyloxy)-
decanoylamino]-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)dec-
anoyl]-
decanoylamino]-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)dec-
anoyl]- -glucopyranoside (23). To a solution of 22
ꢂ-D-glucopyranosyl)-2-deoxy-2-[(R)-3-(dodecanoyloxy)-
ꢀ-D
(104.9 mg, 57.1 mmol) in AcOH (3 mL) was added Zn powder
(400 mg), and the mixture was stirred at rt for 1.5 h. The insolu-
ble materials were filtered off, and the filtrate was concentrated
in vacuo. The residual solvent was removed by coevaporation
with toluene (5 mL ꢅ 3). The residue was dissolved in EtOAc,
washed successively with saturated aqueous NaHCO3 and brine,
dried over Na2SO4, and concentrated in vacuo. To a solution of
the residue and (R)-3-(dodecanoyloxy)decanoic acid (21)
(29.5 mg, 79.6 mmol) in anhydrous CH2Cl2 were added HOBt
CHCl3:acetone = 5:1) to give compound 24 as a pale yellow solid
2þ
(53.8 mg, 78%). ESI-MS (positive) m=z 988.0 ½M þ 2Hꢄ
,
1975.2 ½M þ Hꢄþ. 1H NMR (500 MHz, CDCl3): ꢃ 7.57 (d, J ¼
8:1 Hz, 2H, p-CF3–C6H4–CH2–), 7.52 (d, J ¼ 8:1 Hz, 2H, p-
CF3–C6H4–CH2–), 7.44 (d, J ¼ 8:4 Hz, 2H, p-CF3–C6H4–CH2–),
7.41 (d, J ¼ 8:1 Hz, 2H, p-CF3–C6H4–CH2–), 7.38–7.31 (m, 4H,
Ph–CH2–), 7.29–7.25 (m, 3H, o-C6H4(CH2O)2P– and Ph–CH2–),
7.19 (dd, J ¼ 7:7, 7.7 Hz, 1H, o-C6H4(CH2O)2P–), 7.12 (d,
J ¼ 7:5 Hz, 1H, o-C6H4(CH2O)2P–), 6.76 (d, J ¼ 7:8 Hz, 1H, o-
C6H4(CH2O)2P–), 6.03 (d, J ¼ 7:9 Hz, 1H, 20-NH), 5.92 (d,
J ¼ 9:1 Hz, 1H, 2-NH), 5.45 (dd, J ¼ 10:4, 9.2 Hz, 1H, H-30),
5.19 (d, J ¼ 7:9 Hz, 1H, H-10), 5.15 (brs, 1H, H-1), 5.13–5.06
(m, 2H, H-3 and ꢀ-CH of 20-N-acyl), 5.03–4.88 (m, 5H, ꢀ-CH
of 2-N-acyl and o-C6H4(CH2O)2P–), 4.67–4.52 (m, 8H, p-CF3–
C6H4–CH2– ꢅ 2, Ph–CH2–, H-40, and C1-OH), 4.14 (dd, J ¼
9:4, 9.4 Hz, 1H, H-2), 4.04–3.98 (m, 2H, H-5 and H-60a), 3.91–
3.84 (m, 2H, ꢀ-CH of 3-O-acyl and 30-O-acyl), 3.83 (m, 1H, H-
60b), 3.73–3.70 (m, 3H, H-6a, H-6b, and H-50), 3.50 (ddd,
J ¼ 8:6, 7.9, 7.9 Hz, 1H, H-20), 3.42 (ddd, J ¼ 9:4, 9.4, 4.1 Hz,
1H, H-4), 2.91 (d, J ¼ 4:4 Hz, 1H, C4-OH), 2.74–2.62 (m, 3H,
ꢁ-CH2 of 3-O-acyl and 30-O-acyl), 2.50 (dd, J ¼ 15:1, 4.9 Hz,
1H, ꢁ-CH2 of 3-O-acyl), 2.40–2.34 (m, 2H, ꢁ-CH2 of 2-N-acyl’s
main chain and 20-N-acyl’s main chain), 2.32–2.21 (m, 6H, ꢁ-CH2
of 2-N-acyl’s main chain, 2-N-acyl’s side chain, 20-N-acyl’s main
chain, and 2-N-acyl’s side chain), 1.58–1.53 (m, 8H, ꢄ-CH2 of 2-
N-acyl’s main chain, 2-N0-acyl’s main chain, 3-O-acyl, and 30-O-
acyl), 1.38–1.25 (m, 76H, CH2 ꢅ 38), 0.89–0.86 (m, 18H, –CH2–
CH3 ꢅ 6).
(6.54 mg, 48.4 mmol) and WSCD HCl (21.0 mg, 110 mmol) at
ꢁ
room temperature under Ar atmosphere, and the mixture was
stirred for 21 h. Aqueous sat NaHCO3 was added to the mixture,
and the mixture was extracted with EtOAc. The organic layer
was washed with aqueous sat NaHCO3 and brine, dried over
MgSO4, and then concentrated in vacuo. The residue was puri-
fied by silica-gel flash chromatography (9 g, CHCl3:acetone =
10:1) to give compound 23 (78.7 g, 73%) as a colorless oil.
½
ꢁ 2D2
ꢄ
¼ þ16:4 (c 0.700, CHCl3). ESI-MS (positive) m=z
2014.4 ½M þ Hꢄþ, 2035.4 ½M þ Naꢄþ. 1H NMR (400 MHz,
CDCl3):
7.55 (d, J ¼ 8:3 Hz, 2H, p-CF3–C6H4–CH2–), 7.51
ꢃ
(d, J ¼ 8:0 Hz, 2H, p-CF3–C6H4–CH2–), 7.43 (d, J ¼ 8:3 Hz,
2H, p-CF3–C6H4–CH2–), 7.40 (d, J ¼ 8:0 Hz, 2H, p-CF3–
C6H4–CH2–), 7.37–7.24 (m, 6H, o-C6H4(CH2O)2P– and Ph–
CH2–), 7.18 (ddd, J ¼ 7:6, 7.6, 1.2 Hz, 1H, o-C6H4(CH2O)2P–),
7.11 (d, J ¼ 7:3 Hz, 1H, o-C6H4(CH2O)2P–), 6.73 (d, J ¼
7:5 Hz, 1H, o-C6H4(CH2O)2P–), 6.01 (d, J ¼ 7:8 Hz, 1H, 20-
NH), 5.92 (d, J ¼ 9:3 Hz, 1H, 2-NH), 5.86 (m, 1H, –OCH2–
CH=CH2), 5.41 (dd, J ¼ 10:4, 9.2 Hz, 1H, H-30), 5.27 (ddd,
J ¼ 17:3, 2.9, 1.5 Hz, 1H, –OCH2–CH=CH2), 5.19 (dd, J ¼
10:8, 1.5 Hz, 1H, –OCH2–CH=CH2), 5.15 (dd, J ¼ 10:3,
6-O-{6-O-Benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-2,4,3ꢁ5
-
benzodioxaphosphepin-3-yl)-2-[(R)-3-(dodecanoyloxy)decanoyl-
amino]-3-O-[(R)-3-(4-trifluoromethylbenzyloxy)decanoyl]-ꢂ-D-
glucopyranosyl}-1-O-bis(benzyloxy)phosphoryl-2-deoxy-2-[(R)-
3-(dodecanoyloxy)decanoylamino]-3-O-[(R)-3-(4-trifluorometh-
ylbenzyloxy)decanoyl]-ꢀ-D-glucopyranose (25). To a mixture
of 24 (33.1 mg, 16.8 mmol) and tetrabenzyl diphosphate (13.5
mg, 25.1 mmol) in dry THF (4 mL) was added 1.08 M (1 M =
1 mol dmꢃ3) LiN(TMS)2 (22.0 mL, 23.8 mmol) at ꢃ78 ꢂC and the
mixture was stirred at ꢃ78 ꢂC for 40 min. After addition of tetra-
benzyl diphosphate (12.1 mg, 22.5 mmol) in dry THF (4 mL) was
8.8 Hz, 1H, H-3), 5.10–5.03 (m, 2H,
ꢀ-CH of 2-N-acyl and
20-N-acyl), 4.99–4.91 (m, 5H, o-C6H4(CH2O)2P– and H-10),
4.81 (d, J ¼ 3:7 Hz, 1H, H-1), 4.67–4.50 (m, 7H, p-CF3–
C6H4–CH2– ꢅ 2, Ph–CH2–, and H-40), 4.21 (ddd, J ¼ 9:3,
9.3, 3.7 Hz, 1H, H-2), 4.15 (dd, J ¼ 12:9, 5.3 Hz, 1H,
–OCH2CH=CH2), 4.02 (dd, J ¼ 10:7, 1.9 Hz, 1H, H-6a), 3.94
(dd, J ¼ 12:9, 5.3 Hz, 1H, –OCH2CH=CH2), 3.91–3.84 (m,
2H,
ꢀ
-CH of 3-O-acyl and 30-O-acyl), 3.83 (m, 1H, H-60a),
3.74–3.61 (m, 7H, H-4, H-5, H-6b, H-20, H-50, H-60b, and C4-

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
811
added 1.08 M LiN(TMS)2 (10.0 mL, 10.8 mmol), the reaction mix-
ture was further stirred for 50 min. After addition of saturated
aqueous NaHCO3, the mixture was extracted with EtOAc. The
organic layer was washed with saturated aqueous NaHCO3 and
brine, dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by ULTRA
PACKꢂ ꢅ11 ꢅ 150 mm (YAMAZEN Co., Tokyo, CHCl3:
acetone:Et3N = 10:1:0.002) to give 25 as a yellowish oil
(28.2 mg, 75%). ESI-MS (positive) m=z 2234.4 ½M þ Hꢄþ.
1H NMR (500 MHz, CDCl3): ꢃ 7.57 (d, J ¼ 8:1 Hz, 2H, p-CF3–
C6H4–CH2–), 7.49 (d, J ¼ 8:1 Hz, 2H, p-CF3–C6H4–CH2–),
7.44–7.41 (m, 4H, p-CF3–C6H4–CH2–), 7.41–7.31 (m, 15H,
Ph–CH2– and (Ph–CH2O)2P–), 7.28–7.26 (m, 1H, o-
C6H4(CH2O)2P–), 7.18 (ddd, J ¼ 7:5, 7.5, 1.2 Hz, 1H, o-
C6H4(CH2O)2P–), 7.10 (d, J ¼ 7:0 Hz, 1H, o-C6H4(CH2O)2P–),
6.74 (d, J ¼ 7:0 Hz, 1H, o-C6H4(CH2O)2P–), 6.52 (d, J ¼
8:1 Hz, 20-NH), 5.93 (d, J ¼ 8:7 Hz, 1H, 2-NH), 5.66 (dd,
J ¼ 5:0, 3.4 Hz, 1H, H-1), 5.33 (dd, J ¼ 10:7, 10.5 Hz, 1H,
H-30), 5.11 (dd, J ¼ 9:5, 9.5 Hz, 1H, H-3), 5.09 (m, 1H, ꢀ-CH of
20-N-acyl), 5.05–4.99 (m, 8H, o-C6H4(CH2O)2P– and (Ph–
CH2O)2P–), 4.95–4.88 (m, 2H, ꢀ-CH of 2-N-acyl and H-10),
4.66–4.50 (m, 7H, p-CF3–C6H4–CH2– ꢅ 2, Ph–CH2–, and H-
40), 4.22 (m, 1H, H-2), 3.98–3.96 (m, 2H, H-5 and H-60a), 3.93–
3.85 (m, 2H, ꢀ-CH of 3-O-acyl and 30-O-acyl), 3.82–3.79 (m,
2H, H-6a and H-60b), 3.72–3.68 (m, 2H, H-6b and H-20), 3.67–
3.61 (m, 2H, H-4 and H-50), 2.72 (dd, J ¼ 16:8, 7.5 Hz, 1H, ꢁ-
CH2 of 30-O-acyl), 2.66–2.61 (m, 2H, ꢁ-CH2 of 3-O-acyl and
30-O-acyl), 2.55 (dd, J ¼ 15:6, 4.7 Hz, 1H, ꢁ-CH2 of 3-O-acyl),
2.38 (dd, J ¼ 15:6, 6.1 Hz, 1H, ꢁ-CH2 of 20-N-acyl’s main chain),
2.30–2.21 (m, 6H, ꢁ-CH2 of 2-N-acyl’s main chain and side
chain, 20-N-acyl’s main chain and side chain), 2.11 (m, 1H,
ꢁ-CH2 of 2-N-acyl’s main chain), 1.58–1.50 (m, 8H, ꢄ-CH2 of
2-N-acyl’s main chain, 2-N0-acyl’s main chain, 3-O-acyl, and
30-O-acyl), 1.36–1.22 (m, 76H, CH2 ꢅ 38), 0.89–0.86 (m, 18H,
–CH2–CH3 ꢅ 6).
alyst with H2 three times (each 30 s), the mixture was stirred under
Ar atmosphere at room temperature for 1.5 h. The reaction mix-
ture was concentrated in vacuo. The residue was dissolved in an-
hydrous CH2Cl2 (380 mL), and then DMAP (50.0 mg, 409 mmol),
pyridine (20.0 mL, 247 mmol), and 2-propynyl chloroformate
(7.20 mL, 74.2 mmol) were added to the solution at 0 ꢂC under
Ar atmosphere. After stirring at room temperature for 1 h, the
reaction was quenched by addition of water. The mixture was ex-
tracted with CH2Cl2 and the organic layer was washed with 0.5 M
HCl and brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica-gel flash column chromatography
(400 g, CHCl3:acetone = 40:1) to give 32 as colorless powder
22
(14.1 g, 94%). ½ꢁꢄD ¼ þ52:0 (c 1.00, CHCl3). ESI-MS (positive)
m=z 634.22 ½M þ Naꢄþ, 1246.23 ½2M þ Naꢄþ. 1H NMR
(500 MHz, CDCl3): ꢃ 7.77 (d, J ¼ 7:6 Hz, 2H, (C6H4)2–CH–
CH2–OCO), 7.58 (dd, J ¼ 8:8, 7.6 Hz, 2H, (C6H4)2–CH–CH2–
OCO), 7.49 (dd, J ¼ 5:2, 2.0 Hz, 2H, C6H5–CH=), 7.41 (dd,
J ¼ 7:3, 7.3 Hz, 2H, (C6H4)2–CH–CH2–OCO), 7.37 (dd, J ¼
5:2, 2.0 Hz, 3H, C6H5–CH=), 7.32 (d, J ¼ 8:8 Hz, 2H,
(C6H4)2–CH–CH2–OCO), 6.12 (dd, J ¼ 12:0, 1.6 Hz, 1H, –O–
CH=CH–CH3), 5.56 (s, 1H, C6H5–CH=), 5.23 (dd, J ¼ 12:0,
6.9 Hz, 1H, –O–CH=CH–CH3), 5.20 (d, J ¼ 10:8 Hz, 1H, 2-
NH), 5.17 (dd, J ¼ 10:1, 10.1 Hz, 1H, H-3), 4.91 (d, J ¼
3:5 Hz, 1H, H-1), 4.76 (d, J ¼ 2:3 Hz, 1H, –OCH2–CꢆCH of
Proc), 4.64 (d, J ¼ 2:3 Hz, 1H, –OCH2–CꢆCH of Proc), 4.38
(dd, J ¼ 10:5, 10.5 Hz, 2H, (C6H4)2–CH–CH2–OCO), 4.29 (dd,
J ¼ 10:3, 4.8 Hz, 1H, H-6a), 4.22 (dd, J ¼ 10:5, 10.5 Hz, 1H,
(C6H4)2–CH–CH2–OCO), 4.14 (ddd, J ¼ 10:1, 10.1, 3.5 Hz, 1H,
H-2), 3.93 (ddd, J ¼ 9:9, 9.9, 4.8 Hz, 1H, H-5), 3.80–3.73 (m, 2H,
H-4 and H-6b), 2.33 (s, 1H, –OCH2–CꢆCH of Proc), 1.57 (dd,
J ¼ 6:9, 1.6 Hz, 3H, –O–CH=CH–CH3). Anal. Calcd for
C35H33NO9: C, 68.73; H, 5.44; N, 2.29%. Found: C, 68.65; H,
5.58; N, 2.29%.
1-Propenyl 2-Allyloxycarbonylamino-4,6-O-benzylidene-2-
deoxy-3-O-(2-propynyloxycarbonyl)-ꢀ-D-glucopyranoside (33).
To a solution of 32 (116 mg, 190 mmol) in CH2Cl2 (1.5 mL) was
added 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-ꢁ]pyrimidine poly-
mer-bound (PTBD) (30.0 mg, 240 mmol) at room temperature and
the mixture was shaken for 1 d. PTBD was removed by filtration
and the filtrate was concentrated in vacuo to give 2-N-deprotected
product as a pale yellow solid: Yield 75.2 mg (quant.). To a solu-
tion of the 2-N-free product (74.0 mg, 190 mmol) in anhydrous
CH2Cl2 (1.5 mL) were added allyl chloroformate (30.0 mL,
283 mmol) and pyridine (25.0 mL, 309 mmol) at 0 ꢂC under Ar
atmosphere. After stirring for 1 h, the reaction was quenched by
addition of water and extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica-gel column chromatog-
raphy (12 g, CHCl3:acetone = 40:1) to give 33 as a colorless solid
6-O-{2-Deoxy-2-[(R)-3-(dodecanoyloxy)decanoylamino]-3-O-
[(R)-3-hydrodecanoyl]-ꢂ-D-glucopyranosyl}-2-deoxy-2-[(R)-3-
(dodecanoyloxy)decanoylamino]-3-O-[(R)-3-hydrodecanoyl]-ꢀ-
D-glucopyranose 1,40-Bisphosphate (10a). To a solution of 25
(37.0 mg, 16.6 mmol) in dry THF (4 mL) was added Pd-black
(42 mg) at rt and the mixture was stirred at rt under H2 (20 atm)
for 44 h. After addition of 10% Et3N–THF solution (46.3 mL),
Pd-black was filtered off with a membrane filter. The organic layer
was concentrated under reduced pressure. The residue was lyophi-
lized with water to give crude 10a. The compound 10a was puri-
fied by liquid–liquid partition column chromatography (5 g of
Sephadexꢁ LH-20, CHCl3:MeOH:iPrOH:H2O = 8:8:1:6), where-
in organic and aqueous layers were used for stationary and mobile
phases, respectively, to give 10a (23.9 mg, 93%) as a white pow-
der. ESI-MS (negative) m=z 771.5 ½M ꢃ 2Hꢄ2ꢃ, 1543.9 ½M ꢃ Hꢄꢃ.
1H NMR (500 MHz, CDCl3:MeOH-d4 = 1:1): ꢃ 5.40–5.00 (m,
4H), 4.80–4.4 (m, 2H), 4.4–4.18 (m, 3H), 4.18–3.78 (m, 4H),
3.6–3.4 (m, 1H), 3.4–3.06 (m, 7H), 2.59–2.26 (m, 12H), 1.62–
1.38 (m, 8H, ꢄ-CH2 of 3-O-acyl, 30-O-acyl, 2-N-acyl’s main
chain, and 20-N-acyl’s main chain), 1.38–1.0 (m, 66H, –CH2– ꢅ
33), 0.89 (t, J ¼ 6:3 Hz, 18H, –CH3 ꢅ 6).
21
(86.3 mg, 96%). ½ꢁꢄD ¼ þ79:2 (c 0.97, CHCl3). ESI-MS (posi-
tive) m=z 474.20 ½M þ Hꢄþ, 496.17 ½M þ Naꢄþ. 1H NMR
(400 MHz, CDCl3): ꢃ 7.46 (dd, J ¼ 4:0, 2.4 Hz, 2H, C6H5–
CH=), 7.35 (dd, J ¼ 4:0, 2.4 Hz, 3H, C6H5–CH=), 6.12 (dd, J ¼
12:0, 1.6 Hz, 1H, –O–CH=CH–CH3), 5.90 (dddd, J ¼ 16:0, 10.8,
10.8, 5.6 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.52 (s, 1H, C6H5–
CH=), 5.30 (dd, J ¼ 16:0, 1.4 Hz, 1H, –OCH2–CH=CH2 of
Alloc), 5.23–5.11 (m, 4H, 2-NH, H-3, –O–CH=CH–CH3, and
–OCH2–CH=CH2 of Alloc), 5.08 (d, J ¼ 3:6 Hz, 1H, H-1),
4.72 (d, J ¼ 2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 4.68 (d,
J ¼ 2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 4.61–4.54 (m, 2H,
–OCH2–CH=CH2 of Alloc), 4.28 (dd, J ¼ 10:0, 4.4 Hz, 1H,
H-6a), 4.13 (ddd, J ¼ 10:4, 10.4, 3.6 Hz, 1H, H-2), 3.91 (ddd,
1-Propenyl 4,6-O-Benzylidene-2-deoxy-2-(9-fluorenylme-
thoxycarbonylamino)-3-O-(2-propynyloxycarbonyl)-
copyranoside (32). To a degassed solution of 31 (13.1 g,
ꢀ-D-glu-
24.7 mmol) in anhydrous THF (300 mL) was added (1,5-cyclo-
octadiene)[bis(methyldiphenylphosphine)]iridium(I) hexafluoro-
phosphate (500 mg, 591 mmol). After activation of the iridium cat-

812 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
J ¼ 9:6, 9.6, 4.4 Hz, 1H, H-5), 3.77 (dd, J ¼ 10:0, 4.4 Hz, 1H,
H-6b), 3.75 (dd, J ¼ 9:6, 9.6 Hz, 1H, H-4), 2.46 (t, J ¼ 2:4 Hz,
1H, –OCH2–CꢆCH of Proc), 1.57 (dd, J ¼ 6:8, 1.6 Hz, 3H,
–O–CH=CH–CH3). Anal. Calcd for C24H27NO9: C, 60.88; H,
5.75; N, 2.96%. Found: C, 60.76; H, 5.89; N, 3.02%.
1H, H-2), 3.99 (ddd, J ¼ 9:9, 9.9, 4.8 Hz, 1H, H-5), 3.83 (d,
J ¼ 10:3 Hz, 1H, H-6a), 3.77 (dd, J ¼ 10:3, 4.8 Hz, 1H, H-6b),
2.43 (t, J ¼ 2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 1.55 (dd,
J ¼ 6:9, 1.6 Hz, 3H, –O–CH=CH–CH3). Anal. Calcd for
C32H36NO12P: C, 58.45; H, 5.52; N, 2.13%. Found: C, 58.45;
H, 5.64; N, 2.11%.
1-Propenyl 2-Allyloxycarbonylamino-6-O-benzyl-2-deoxy-
3-O-(2-propynyloxycarbonyl)-ꢀ-D-glucopyranoside (34). To
a solution of 33 (82.3 mg, 174 mmol) in anhydrous CH2Cl2
(1.5 mL) were added triethylsilane (260 mL, 1.63 mmol) and boron
trifluoride diethyl etherate (40.0 mL, 316 mmol) at 0 ꢂC under Ar
atmosphere. After stirring for 2 h, the reaction was quenched by
addition of saturated aqueous NaHCO3 and extracted with EtOAc.
The organic layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica-gel col-
umn chromatography (11 g, CHCl3:acetone = 40:1) to give 34 as
2-Allyloxycarbonylamino-6-O-benzyl-2-deoxy-4-O-(1,5-di-
hydro-3-oxo-3H-2,4,3ꢁ5-benzodioxaphosphepin-3-yl)-3-O-(2-
propynyloxycarbonyl)-D-glucopyranosyl Trichloroacetimidate
(27). To a solution of 35 (4.69 g, 7.13 mmol) in THF (150 mL)
were added water (100 mL) and iodine (1.82 g, 7.17 mmol) at
room temperature. After the mixture was stirred for 30 min, aque-
ous 10% Na2S2O3 was added to quench the reaction. The mixture
was extracted with EtOAc and the organic layer was washed with
saturated aqueous NaHCO3 and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica-gel flash
column chromatography (250 g, CHCl3:acetone = 5:1 to 3:1) to
give 1-OH product as a colorless foamy solid (3.16 g, 73%).
ESI-MS (positive) m=z 618.31 ½M þ Hꢄþ, 640.29 ½M þ Naꢄþ.
1H NMR (500 MHz, CDCl3) selected data for ꢁ-isomer: ꢃ 7.35–
7.27 (m, 7H, C6H4–CH2– and o-C6H4(CH2O)2P–), 7.21 (dd,
J ¼ 8:2, 4.6 Hz, 2H, o-C6H4(CH2O)2P–), 5.91–5.84 (m, 1H,
–OCH2–CH=CH2 of Alloc), 5.28 (d, J ¼ 17:2 Hz, 1H, –OCH2–
CH=CH2 of Alloc), 5.23–5.06 (m, 6H, H-3, o-C6H4(CH2O)2P–,
and –OCH2–CH=CH2 of Alloc), 4.96 (d, J ¼ 9:6 Hz, 1H, 2-
NH), 4.73 (d, J ¼ 2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 4.66
(d, J ¼ 3:8 Hz, 1H, H-1), 4.65–4.55 (m, 6H, H-4, C6H4–CH2–,
–OCH2–CꢆCH of Proc, and –OCH2–CH=CH2 of Alloc), 4.22
(ddd, J ¼ 9:9, 9.9, 4.8 Hz, 1H, H-5), 4.09 (ddd, J ¼ 9:6, 9.6,
3.8 Hz, 1H, H-2), 3.83 (dd, J ¼ 10:8, 4.8 Hz, 1H, H-6a), 3.74
(dd, J ¼ 10:8, 9.9 Hz, 1H, H-6b), 3.40 (brs, 1H, C1-OH), 2.48
(brs, 1H, –OCH2–CꢆCH of Proc). Anal. Calcd for C29H32NO12P:
C, 56.40; H, 5.22; N, 2.27%. Found: C, 56.46; H, 5.23; N, 2.21%.
To a solution of the 1-OH product (2.66 g, 4.31 mmol) in anhy-
drous CH2Cl2 (50 mL) were added trichloroacetonitrile (9.32 mL,
43.1 mmol) and Cs2CO3 (700 mg, 2.15 mmol). After stirring for
1 h, the reaction mixture was quenched by addition of saturated
aqueous NaHCO3, and extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and concentrated in
vacuo to give 27 (3.25 g, 99%) as a pale yellow solid, which
was used for the subsequent glycosylation without further purifi-
cation.
21
a colorless solid (76.8 mg, 93%). ½ꢁꢄD ¼ þ40:0 (c 0.64, CHCl3).
ESI-MS (positive) m=z 498.21 ½M þ Naꢄþ. 1H NMR (400 MHz,
CDCl3): ꢃ 7.37–7.27 (m, 5H, C6H5–CH2–), 6.14 (dd, J ¼ 12:0,
1.6 Hz, 1H, –O–CH=CH–CH3), 5.89 (dddd, J ¼ 17:3, 10.8,
10.8, 5.6 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.28 (dd, J ¼
17:3, 1.4 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.22–5.16 (m,
3H, 2-NH, –O–CH=CH–CH3, and –OCH2–CH=CH2 of Alloc),
5.06 (d, J ¼ 3:2 Hz, 1H, H-1), 4.96 (dd, J ¼ 10:8, 10.8 Hz, 1H,
H-3), 4.73 (d, J ¼ 2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 4.69
(d, J ¼ 2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 4.62–4.51 (m, 2H,
–OCH2–CH=CH2 of Alloc), 4.59 (d, J ¼ 12:1 Hz, 2H, C6H5–
CH2–), 4.02 (ddd, J ¼ 10:8, 10.8, 3.2 Hz, 1H, H-2), 3.88 (ddd,
J ¼ 10:8, 9.5, 3.2 Hz, 1H, H-4), 3.82–3.78 (m, 2H, H-5 and H-
6a), 3.67 (dd, J ¼ 10:1, 3.2 Hz, 1H, H-6b), 2.72 (d, J ¼ 3:2 Hz,
1H, C4-OH), 2.51 (t, J ¼ 2:4 Hz, 1H, –OCH2–CꢆCH of Proc),
1.55 (dd, J ¼ 6:8, 1.6 Hz, 3H, –O–CH=CH–CH3). Anal. Calcd
for C24H29NO9: C, 60.62; H, 6.15; N, 2.95%. Found: C, 60.71;
H, 6.19; N, 2.99%.
1-Propenyl 2-Allyloxycarbonylamino-6-O-benzyl-2-deoxy-
4-O-(1,5-dihydro-3-oxo-3H-2,4,3ꢁ5-benzodioxaphosphepin-3-
yl)-3-O-(2-propynyloxycarbonyl)-ꢀ-D-glucopyranoside
(35).
To a solution of 34 (4.03 g, 8.48 mmol) in anhydrous CH2Cl2
(100 mL) were added N,N-diethyl-1,5-dihydro-3H-2,4,3-benzo-
dioxaphosphepin-3-amine (2.10 g, 8.78 mmol) and 1H-tetrazole
(2.97 g, 42.4 mmol) at room temperature under Ar atmosphere.
After the mixture was stirred for 30 min and then at ꢃ20 ꢂC for
10 min, mCPBA (2.10 g, 8.52 mmol) was added and stirring was
continued for another 20 min. The solution was quenched by addi-
tion of saturated aqueous NaHCO3, and extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica-
gel flash column chromatography (300 g, CHCl3:acetone = 20:1)
Formylmethyl 4-O-(4-Azidophenylmethyl)-2-deoxy-2-(9-fluo-
renylmethoxycarbonylamino)-3-O-(4-methoxyphenylmethyl)-
ꢀ-D-glucopyranoside (37).
To a solution of 36 (4.58 g,
6.61 mmol) in THF–t-BuOH–water (10:10:1) (84 mL) were added
NMO (3.00 g, 25.6 mmol) and OsO4 in water (25 g Lꢃ1, 10.0 mL,
984 mmol) at room temperature. After stirring for 4 h, the mixture
was added to 10% aqueous Na2S2O3 and extracted with EtOAc.
The organic layer was washed successively with 10% aqueous
Na2S2O3 and brine, dried over MgSO4, and concentrated in vacuo
to give the crude diol (4.88 g), which was subjected to the follow-
ing oxidation without further purification. To a suspension of
crude diol thus obtained in anhydrous benzene–CH2Cl2 (2:3)
(100 mL) was added Pb(OAc)4 (90% purity, 4.40 g, 9.92 mmol)
at room temperature under Ar atmosphere. After stirring for 4 h,
the mixture was filtered through a short silica-gel column (30 g)
using EtOAc as an eluent. The filtrate was concentrated in vacuo
and then the residue was purified by silica-gel flash column chro-
matography (200 g, CHCl3:acetone = 5:1) to give 37 (4.49 g,
98%) as a pale brown foamy solid. ESI-MS (positive) m=z
717.31 ½M þ Naꢄþ. 1H NMR (400 MHz, CDCl3): ꢃ 9.65 (s, 1H,
21
to give 35 as a colorless foamy solid (5.01 g, 91%). ½ꢁꢄD ¼ þ37:0
(c 1.00, CHCl3). ESI-MS (positive) m=z 658.22 ½M þ Hꢄþ, 680.20
½M þ Naꢄþ. 1H NMR (500 MHz, CDCl3): ꢃ 7.37–7.32 (m, 5H,
C6H4–CH2– and o-C6H4(CH2O)2P–), 7.28 (d, J ¼ 6:9 Hz, 2H,
C6H4–CH2–), 7.26–7.19 (m, 2H, o-C6H4(CH2O)2P–), 6.15 (d,
J ¼ 12:2 Hz, 1H, –O–CH=CH–CH3), 5.90 (dddd, J ¼ 17:2,
10.3, 5.9, 5.9 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.36 (d, J ¼
17:2 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.25 (d, J ¼ 10:3 Hz,
1H, –OCH2–CH=CH2 of Alloc), 5.23–5.07 (m, 6H, H-3, o-
C6H4(CH2O)2P–, and –O–CH=CH–CH3), 5.03 (d, J ¼ 9:6 Hz,
1H, 2-NH), 4.93 (d, J ¼ 3:5 Hz, 1H, H-1), 4.73 (d, J ¼ 2:5 Hz,
1H, –OCH2–CꢆCH of Proc), 4.69–4.66 (m, 2H, H-4 and
–OCH2–CꢆCH of Proc), 4.64–4.62 (m, 2H, –OCH2–CH=CH2
of Alloc), 4.58 (d, J ¼ 11:6 Hz, 1H, C6H4–CH2–), 4.56 (d,
J ¼ 11:6 Hz, 1H, C6H4–CH2–), 4.09 (ddd, J ¼ 10:3, 9.6, 3.5 Hz,

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
813
–OCH2–CHO), 7.76 (d, J ¼ 7:3 Hz, 2H, (C6H4)2–CH–CH2–
OCO), 7.59 (dd, J ¼ 7:6, 7.3 Hz, 2H, (C6H4)2–CH–CH2–OCO),
7.41 (dd, J ¼ 7:3, 7.3 Hz, 2H, (C6H4)2–CH–CH2–OCO), 7.35
(d, J ¼ 8:3 Hz, 2H, p-N3–C6H4–CH2–), 7.32 (d, J ¼ 7:6 Hz, 2H,
(C6H4)2–CH–CH2–OCO), 7.16 (d, J ¼ 8:8 Hz, 2H, p-CH3O–
C6H4–CH2–), 7.01 (d, J ¼ 8:3 Hz, 2H, p-N3–C6H4–CH2–),
6.75 (d, J ¼ 8:8 Hz, 2H, p-CH3O–C6H4–CH2–), 4.85 (d, J ¼
10:3 Hz, 1H, 2-NH), 4.83 (d, J ¼ 3:3 Hz, 1H, H-1), 4.80 (d,
J ¼ 11:2 Hz, 1H, p-N3–C6H4–CH2–), 4.70 (d, J ¼ 11:5 Hz, 1H,
p-N3–C6H4–CH2–), 4.66 (d, J ¼ 11:3 Hz, 1H, p-CH3O–C6H4–
CH2–), 4.62 (d, J ¼ 11:3 Hz, 1H, p-CH3O–C6H4–CH2–), 4.44
(dd, J ¼ 10:7, 6.3 Hz, 2H, (C6H4)2–CH–CH2–OCO), 4.21 (dd,
J ¼ 6:3, 6.3 Hz, 1H, (C6H4)2–CH–CH2–OCO), 3.94 (ddd, J ¼
10:3, 10.3, 3.3 Hz, 1H, H-2), 3.85–3.78 (m, 2H, H-3, H-6a, and
–OCH2–CHO), 3.75–3.65 (m, 5H, H-5, H-6b, and p-CH3O–
C6H4–CH2–), 3.60 (dd, J ¼ 8:7, 8.7 Hz, 1H, H-4).
were removed by filtration, the filtrate was concentrated in vacuo.
The residual AcOH was removed by co-evaporation with toluene
three times. The residue was dissolved in EtOAc and washed with
saturated aqueous NaHCO3 and brine. The organic layer was dried
over MgSO4 and concentrated in vacuo. To a solution of the res-
idue in CH2Cl2 (30 mL) was added glutaric anhydride (520 mg,
4.56 mmol) at room temperature, and the mixture was stirred
for 1 d. The reaction mixture was concentrated in vacuo. The
residue was purified by silica-gel flash column chromatography
(150 g, CHCl3:acetone = 5:1 to CHCl3:MeOH = 5:1) to give
39 (1.95 g, 59%) as a colorless solid. ESI-MS (negative) m=z
887.353 ½M ꢃ Hꢄꢃ. 1H NMR (400 MHz, CDCl3): ꢃ 7.77 (d,
J ¼ 7:3 Hz, 2H, (C6H4)2–CH–CH2–OCO), 7.63 (dd, J ¼ 7:3,
7.0 Hz, 2H, –OCH2–COOCH2–C6H5), 7.58 (dd, J ¼ 7:8,
7.3 Hz, 2H, (C6H4)2–CH–CH2–OCO), 7.32 (dd, J ¼ 7:3, 7.3 Hz,
2H, (C6H4)2–CH–CH2–OCO), 7.30–7.25 (m, 3H, –OCH2–
COOCH2–C6H5), 7.29 (d, J ¼ 8:3 Hz, 2H, p-RCONH–C6H4–
CH2–), 7.23 (d, J ¼ 8:3 Hz, 2H, p-RCONH–C6H4–CH2–), 7.16
(d, J ¼ 8:8 Hz, 2H, p-CH3O–C6H4–CH2–), 6.74 (d, J ¼ 8:8 Hz,
2H, p-CH3O–C6H4–CH2–), 5.22 (d, J ¼ 9:2 Hz, 1H, 2-NH),
5.18 (d, J ¼ 2:5 Hz, 2H, –OCH2–COOCH2–C6H5), 4.86 (d,
J ¼ 3:4 Hz, 1H, H-1), 4.79 (d, J ¼ 11:5 Hz, 1H, p-RCONH–
C6H4–CH2–), 4.73 (d, J ¼ 11:5 Hz, 1H, p-RCONH–C6H4–CH2–),
4.64 (d, J ¼ 13:8 Hz, 1H, p-CH3O–C6H4–CH2–), 4.59 (d, J ¼
13:8 Hz, 1H, p-CH3O–C6H4–CH2–), 4.40 (dd, J ¼ 12:8, 7.2 Hz,
2H, (C6H4)2–CH–CH2–OCO), 4.23–4.20 (m, 3H, (C6H4)2–CH–
CH2–OCO and –OCH2–COOCH2–C6H5), 3.98 (ddd, J ¼ 10:5,
9.2, 3.4 Hz, 1H, H-2), 3.80–3.75 (m, 2H, H-3 and H-6a), 3.73–
3.63 (m, 5H, H-5, H-6b, and p-CH3O–C6H4–CH2–), 3.60 (dd,
J ¼ 8:8, 8.8 Hz, 1H, H-4), 2.45 (dd, J ¼ 5:9, 5.9 Hz, 2H, CO–
CH2–CH2–CH2–CO), 2.42 (dd, J ¼ 5:9, 5.9 Hz, 2H, CO–CH2–
CH2–CH2–CO), 2.00 (dd, J ¼ 5:9, 5.9 Hz, 2H, CO–CH2–CH2–
CH2–CO). Anal. Calcd for C50H52N2O13: C, 67.56; H, 5.90; N,
3.15%. Found: C, 67.59; H, 5.86; N, 3.27%.
Benzyloxycarbonylmethyl
4-O-(4-Azidophenylmethyl)-2-
deoxy-2-(9-fluorenylmethoxycarbonylamino)-3-O-(4-methoxy-
phenylmethyl)-ꢀ-D-glucopyranoside (38). To a solution of 37
(8.15 g, 11.7 mmol), NaH2PO4 (2.20 g, 18.3 mmol), and 2-meth-
yl-2-butene (6.22 mL, 58.7 mmol) in THF–t-BuOH–water (2:4:1)
(280 mL) was added NaClO2 (80% purity, 4.0 g, 35.4 mmol) at
room temperature and the mixture was stirred for 9 h. The reaction
mixture was acidified by addition of 1 M HCl and extracted with
CHCl3. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo to give crude carboxylic acid
product. To a suspension of the crude carboxylic product in Et2O
(100 mL) was added solution of phenyldiazomethane in Et2O
(0.24 M, 60 mL, 14.4 mmol) at room temperature and the mixture
was stirred for 1 h. After another solution of phenyldiazomethane
(=(diazo)phenylmethane) in Et2O (0.24 M, 60 mL, 14.4 mmol)
was added, the mixture was stirred for an additional 1 h and then
concentrated in vacuo. The residue was purified by silica-gel flash
column chromatography (450 g, CHCl3:acetone = 40:1 to 3:1)
21
to give 38 (7.79 g, 83%) as a pale yellow solid. ½ꢁꢄD ¼ þ21:6
Benzyloxycarbonylmethyl 4-O-{4-[4-(Benzotriazolyloxycar-
boxyl)butyrylamino]phenylmethyl}-2-deoxy-2-(9-fluorenylme-
thoxycarbonylamino)-3-O-(4-methoxyphenylmethyl)-ꢀ-D-glu-
(c 0.99, CHCl3). ESI-MS (positive) m=z 801.27 ½M þ Hꢄþ,
þ
1
823.32 ½M þ Naꢄ . H NMR (400 MHz, CDCl3): ꢃ 7.75 (d, J ¼
7:2 Hz, 2H, (C6H4)2–CH–CH2–OCO), 7.63 (dd, J ¼ 7:2, 7.0 Hz,
2H, –OCH2–COOCH2–C6H5), 7.59 (dd, J ¼ 7:8, 7.2 Hz, 2H,
(C6H4)2–CH–CH2–OCO), 7.37 (d, J ¼ 8:3 Hz, 2H, p-N3–C6H4–
CH2–), 7.32 (dd, J ¼ 7:2, 7.2 Hz, 2H, (C6H4)2–CH–CH2–OCO),
7.30–7.25 (m, 3H, –OCH2–COOCH2–C6H5), 7.16 (d, J ¼
8:8 Hz, 2H, p-CH3O–C6H4–CH2–), 6.98 (d, J ¼ 8:3 Hz, 2H, p-
N3–C6H4–CH2–), 6.74 (d, J ¼ 8:8 Hz, 2H, p-CH3O–C6H4–
CH2–), 5.45 (d, J ¼ 9:2 Hz, 1H, 2-NH), 5.18 (d, J ¼ 2:4 Hz,
2H, –OCH2–COOCH2–C6H5), 4.86 (d, J ¼ 3:6 Hz, 1H, H-1),
4.81 (d, J ¼ 11:3 Hz, 1H, p-N3–C6H4–CH2–), 4.70 (d, J ¼
11:5 Hz, 1H, p-N3–C6H4–CH2–), 4.66 (d, J ¼ 13:8 Hz, 1H, p-
CH3O–C6H4–CH2–), 4.62 (d, J ¼ 13:8 Hz, 1H, p-CH3O–C6H4–
CH2–), 4.40 (dd, J ¼ 12:8, 7.2 Hz, 2H, (C6H4)2–CH–CH2–
OCO), 4.23–4.20 (m, 3H, (C6H4)2–CH–CH2–OCO and –OCH2–
COOCH2–C6H5), 3.98 (ddd, J ¼ 10:5, 9.2, 3.6 Hz, 1H, H-2),
3.81–3.75 (m, 4H, H-3, H-5, and H-6a,b), 3.70 (s, 3H, p-CH3O–
C6H4–CH2–), 3.60 (dd, J ¼ 8:8, 8.8 Hz, 1H, H-4), 1.79 (brs,
1H, C6-OH). Anal. Calcd for C45H44N4O10: C, 67.49; H, 5.54;
N, 7.00%. Found: C, 67.42; H, 5.53; N, 6.87%.
copyranoside (40). To a mixture of 39 (1.00 g, 1.12 mmol) and
HOBt (182 mg, 1.35 mmol) in anhydrous CH2Cl2 (20 mL) was
added DCC (340 mg, 1.65 mmol), and the mixture was stirred at
room temperature for 5 h. After the insoluble materials were
removed by filtration, the filtrate was concentrated in vacuo to
give 40 (1.10 g, 98%) as a pale yellow solid, which was used
for the subsequent coupling reaction without further purification.
General Procedure for Affinity Separation. After comple-
tion of the reaction, the reaction mixture was directly applied to
the resin column (7.0 g: 1:5 cm ꢅ 7 cm; 13 g: 2:5 cm ꢅ 10 cm,
CH2Cl2) unless otherwise noted. After untagged compounds were
washed off with toluene–CH2Cl2 (1:1) then CH2Cl2, the tagged
compound was eluted with CH2Cl2–MeOH (1:1). Evaporation
of the solvents afforded the desired product having the BA-tag.
Benzyloxycarbonylmethyl
2-Deoxy-4-O-(4-{4-[4-(1-ethyl-
2,4,6-trioxo-3,5-diazacyclohexylmethyl)phenylmethylamino-
carbonyl]butyrylamino}phenylmethyl)-2-(9-fluorenylmethoxy-
carbonylamino)-3-O-(4-methoxyphenylmethyl)-ꢀ-D-glucopyr-
anoside (42). To a solution of 41 (637 mg, 2.32 mmol) and acti-
vated ester 40 (2.80 g, 2.78 mmol) in anhydrous DMF (40 mL) was
added Et3N (420 mL, 3.01 mmol) at room temperature under Ar at-
mosphere and the mixture was stirred for 1.5 h. EtOAc was added
to the mixture and the organic layer was washed with 10% aque-
ous citric acid and brine, dried over MgSO4, and concentrated in
vacuo. The residue was dissolved in CH2Cl2 and then subjected to
Benzyloxycarbonylmethyl 4-O-[4-(4-Carboxylbutyrylami-
no)phenylmethyl]-2-deoxy-2-(9-fluorenylmethoxycarbonylami-
no)-3-O-(4-methoxyphenylmethyl)-ꢀ-D-glucopyranoside (39).
To a suspension of 38 (3.05 g, 3.81 mmol) in AcOH–THF (2:1)
(60 mL) was added zinc powder (2.50 g), and the mixture was stir-
red at room temperature for 2.5 h. After the insoluble materials

814 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
affinity separation (13 g ꢅ 4) to give a mixture of 42 and 41. The
mixture thus obtained was dissolved in anhydrous DMF (40 mL)
and activated ester 40 (2.80 g, 2.78 mmol) and Et3N (420 mL,
3.01 mmol) were added at room temperature under Ar atmosphere.
After stirring for 1.5 h, EtOAc was added to the mixture and the
mixture was washed with 10% aqueous citric acid and brine, dried
over MgSO4, and concentrated in vacuo. The residue was dis-
solved in CH2Cl2 and then subjected to affinity separation
(13 g ꢅ 4) to give 42 (2.61 g, 98%) as a colorless foamy solid.
NHCO), 5.95 (brs, 1H, 2-NH), 5.16 (d, J ¼ 2:5 Hz, 2H,
–OCH2–COOCH2–C6H5), 4.87 (d, J ¼ 3:3 Hz, 1H, H-1), 4.79
(d, J ¼ 10:8 Hz, 1H, p-RCONH–C6H4–CH2–), 4.65 (d, J ¼
10:8 Hz, 1H, p-RCONH–C6H4–CH2–), 4.36 (dd, J ¼ 12:8,
7.8 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 4.26 (d, J ¼ 5:8 Hz, 2H,
BA–CH2–C6H4–CH2NHCO–), 4.21–4.19 (m, 3H, (C6H4)2–CH–
CH2–OCO– and –OCH2–COOCH2–C6H5), 3.92 (ddd, J ¼ 10:5,
10.5, 3.3 Hz, 1H, H-2), 3.84 (ddd, J ¼ 11:3, 9.6, 3.8 Hz, 1H,
H-5), 3.76–3.70 (m, 3H, H-3 and H-6a,b), 3.50 (dd, J ¼ 9:6,
9.6 Hz, 1H, H-4), 3.17 (s, 2H, BA–CH2–C6H4–CH2NHCO–),
2.29 (dd, J ¼ 7:3, 7.3 Hz, 2H, CO–CH2–CH2–CH2–CO), 2.24
(dd, J ¼ 7:3, 7.3 Hz, 2H, CO–CH2–CH2–CH2–CO), 2.14 (q, J ¼
7:3 Hz, 2H, –CH2–CH3), 1.89 (dd, J ¼ 7:3, 7.3 Hz, 2H, CO–CH2–
CH2–CH2–CO), 0.86 (t, J ¼ 7:3 Hz, 3H, –CH2–CH3). Anal. Calcd
for C56H59N5O14: C, 65.55; H, 5.80; N, 6.83%. Found: C, 65.43;
H, 5.96; N, 6.78%.
21
½ꢁꢄD ¼ þ26:7 (c 1.03, CHCl3). ESI-MS (positive) m=z 1168.41
½M þ Naꢄþ. 1H NMR (400 MHz, DMSO-d6): ꢃ 8.27 (brs, 2H,
CONHCO ꢅ 2), 7.74 (d, J ¼ 7:6 Hz, 2H, (C6H4)2–CH–CH2–
OCO–), 7.61 (dd, J ¼ 8:0, 6.6 Hz, 2H, –OCH2–COOCH2–C6H5),
7.48 (dd, J ¼ 8:3, 7.6 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 7.37
(dd, J ¼ 7:6, 7.6 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 7.32 (d,
J ¼ 7:3 Hz, 2H, p-RCONH–C6H4–CH2–), 7.30–7.25 (m, 3H,
–OCH2–COOCH2–C6H5), 7.23 (d, J ¼ 7:3 Hz, 2H, p-RCONH–
C6H4–CH2–), 7.19 (d, J ¼ 7:8 Hz, 2H, BA–CH2–C6H4–
CH2NHCO–), 7.16 (d, J ¼ 8:6 Hz, 2H, p-CH3O–C6H4–CH2–),
7.04 (d, J ¼ 7:8 Hz, 2H, BA–CH2–C6H4–CH2NHCO–), 6.74 (d,
J ¼ 8:6 Hz, 2H, p-CH3O–C6H4–CH2–), 6.53 (brs, 1H, –CH2–
NHCO), 5.38 (d, J ¼ 9:3 Hz, 1H, 2-NH), 5.17 (d, J ¼ 2:1 Hz,
2H, –OCH2–COOCH2–C6H5), 4.86 (d, J ¼ 3:8 Hz, 1H, H-1),
4.79 (d, J ¼ 10:8 Hz, 1H, p-RCONH–C6H4–CH2–), 4.71 (d, J ¼
10:8 Hz, 1H, p-CH3O–C6H4–CH2–), 4.64 (d, J ¼ 10:8 Hz, 1H,
p-CH3O–C6H4–CH2–), 4.59 (d, J ¼ 11:3 Hz, 1H, p-RCONH–
C6H4–CH2–), 4.39 (dd, J ¼ 12:8, 7.8 Hz, 2H, (C6H4)2–CH–
CH2–OCO–), 4.26 (d, J ¼ 5:6 Hz, 2H, BA–CH2–C6H4–
CH2NHCO–), 4.20–4.19 (m, 3H, (C6H4)2–CH–CH2–OCO– and
–OCH2–COOCH2–C6H5), 3.96 (ddd, J ¼ 11:3, 9.3, 3.8 Hz, 1H,
H-2), 3.76 (dd, J ¼ 11:3, 9.6 Hz, 1H, H-3), 3.71–3.66 (m, 3H,
H-5 and H-6a,b), 3.65 (s, 3H, p-CH3O–C6H4–CH2–), 3.57 (dd,
J ¼ 9:6, 9.6 Hz, 1H, H-4), 3.20 (s, 2H, BA–CH2–C6H4–
CH2NHCO–), 2.34 (dd, J ¼ 7:2, 7.2 Hz, 2H, CO–CH2–CH2–
CH2–CO), 2.24 (dd, J ¼ 7:2, 7.2 Hz, 2H, CO–CH2–CH2–CH2–
CO), 2.15 (q, J ¼ 7:2 Hz, 2H, –CH2–CH3), 1.94 (dd, J ¼ 7:2,
7.2 Hz, 2H, CO–CH2–CH2–CH2–CO), 0.88 (t, J ¼ 7:2 Hz, 3H,
–CH2–CH3). Anal. Calcd for C64H67N5O15: C, 67.06; H, 5.89;
N, 6.11%. Found: C, 67.09; H, 5.87; N, 6.01%.
Benzyloxycarbonylmethyl 6-O-[2-Allyloxycarbonylamino-
6-O-benzyl-2-deoxy-4-O-(1,5-dihydro-3-oxo-3H-2,4,3ꢁ5-ben-
zodioxaphosphepin-3-yl)-3-O-(2-propynyloxycarbonyl)-ꢂ-D-
glucopyranosyl]-2-deoxy-4-O-(4-{4-[4-(1-ethyl-2,4,6-trioxo-3,5-
diazacyclohexylmethyl)phenylmethylaminocarbonyl]butyryl-
amino}phenylmethyl)-2-(9-fluorenylmethoxycarbonylamino)-
ꢀ-D-glucopyranoside (29). To a mixture of donor 27 (505 mg,
663 mmol), acceptor 28 (454 mg, 442 mmol) and MS4A (1.0 g) in
anhydrous THF (15.0 mL) was added diethyl ether–boron trifluo-
ride (1/1) (30.0 mL, 237 mmol) at 0 ꢂC under Ar atmosphere. After
stirring for 1.5 h, another donor 27 (170 mg, 223 mmol) was added,
and the mixture was stirred for additional 1 h. The reaction was
quenched by addition of saturated aqueous NaHCO3. After re-
moval of insoluble materials by filtration, the filtrate was extracted
with EtOAc. The organic layer was washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was dissolved
in CH2Cl2 and then subjected to affinity separation (13 g ꢅ 1)
to give 29 (689 mg, 96%) as a colorless solid. ESI-MS (positive)
2þ
m=z 1657.54 ½M þ Naꢄþ, 835.29 ½M þ 2Naꢄ
.
1H NMR (400
MHz, DMSO-d6): ꢃ 8.27 (d, J ¼ 5:6 Hz, 2H, CONHCO ꢅ 2),
7.87 (d, J ¼ 7:2 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 7.75 (d, J ¼
6:4 Hz, 2H, –OCH2–COOCH2–C6H5), 7.53 (dd, J ¼ 8:3, 7.2 Hz,
2H, (C6H4)2–CH–CH2–OCO–), 7.40 (dd, J ¼ 7:2, 7.2 Hz, 2H,
(C6H4)2–CH–CH2–OCO–), 7.36–7.33 (m, 9H, (C6H4)2–CH–
CH2–OCO–, p-RCONH–C6H4–CH2–, and C6H4–CH2–), 7.32–
7.27 (m, 7H, BA–CH2–C6H4–CH2NHCO–, o-C6H4(CH2O)2P–,
and –OCH2–COOCH2–C6H5), 7.26 (d, J ¼ 7:2 Hz, 2H, o-
C6H4(CH2O)2P–), 7.12 (d, J ¼ 8:0 Hz, 2H, BA–CH2–C6H4–
CH2NHCO–), 6.96 (d, J ¼ 7:6 Hz, 2H, p-RCONH–C6H4–CH2–),
5.80 (dddd, J ¼ 15:8, 10.5, 10.5, 5.8 Hz, 1H, –OCH2–CH=CH2 of
Alloc), 5.29 (d, J ¼ 15:8 Hz, 1H, –OCH2–CH=CH2 of Alloc),
5.20 (d, J ¼ 10:5 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.18–
5.11 (m, 9H, 2-NH, 20-NH, H-30, o-C6H4(CH2O)2P, and
–OCH2–COOCH2–C6H5), 4.86 (d, J ¼ 2:8 Hz, 1H, H-1), 4.80
(d, J ¼ 11:2 Hz, 1H, p-RCONH–C6H4–CH2–), 4.68 (d, J ¼
2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 4.61–4.59 (m, 7H, H-10,
H-40, C6H4–CH2–, p-RCONH–C6H4–CH2–, –OCH2–CꢆCH of
Proc, and –OCH2–CH=CH2 of Alloc), 4.56 (d, J ¼ 12:5 Hz,
1H, C6H4–CH2–), 4.38 (d, J ¼ 7:2 Hz, 2H, (C6H4)2–CH–CH2–
OCO–), 4.26–4.22 (m, 5H, (C6H4)2–CH–CH2–OCO, –OCH2–
COOCH2–C6H5, and BA–CH2–C6H4–CH2NHCO–), 4.01 (dd,
J ¼ 8:9, 2.5 Hz, 1H, H-60a), 3.85 (brd, J ¼ 8:8 Hz, 1H, H-3), 3.80
(brd, J ¼ 9:5 Hz, 1H, H-2), 3.78–3.70 (m, 3H, H-4, H-6a, and H-
50), 3.68–3.65 (m, 2H, H-20 and H-60b), 3.45–3.40 (m, 2H, H-5
and H-6b), 3.08 (s, 2H, BA–CH2–C6H4–CH2NHCO–), 2.46
(t, J ¼ 2:5 Hz, 1H, –OCH2–CꢆCH of Proc), 2.36 (dd, J ¼ 6:8,
Benzyloxycarbonylmethyl
2-Deoxy-4-O-(4-{4-[4-(1-ethyl-
2,4,6-trioxo-3,5-diazacyclohexylmethyl)phenylmethylamino-
carbonyl]butyrylamino}phenylmethyl)-2-(9-fluorenylmethoxy-
carbonylamino)-ꢀ-D-glucopyranoside (28). To a solution of 42
(130 mg, 113 mmol) in anhydrous CH2Cl2 (5.0 mL) was added di-
ethyl ether–boron trifluoride (1/1) (15.0 mL, 118 mmol) at 0 ꢂC un-
der Ar atmosphere. After stirring at 0 ꢂC for 3 h, the reaction was
quenched by addition of saturated aqueous NaHCO3 and extracted
with EtOAc. The organic layer was washed with saturated aque-
ous NaHCO3 and brine, dried over MgSO4, and concentrated in
vacuo. The residue was dissolved in CH2Cl2 and then subjected
to affinity separation (13 g) to give 28 (101 mg, 87%) as a pale
21
yellow foamy solid. ½ꢁꢄD ¼ þ28:1 (c 0.96, CHCl3). ESI-MS
þ
1
(positive) m=z 1048.47 ½M þ Naꢄ . H NMR (400 MHz, DMSO-
d6): ꢃ 8.42 (brs, 2H, CONHCO ꢅ 2), 7.74 (d, J ¼ 7:6 Hz, 2H,
(C6H4)2–CH–CH2–OCO–), 7.62 (dd, J ¼ 8:0, 6.6 Hz, 2H,
–OCH2–COOCH2–C6H5), 7.48 (dd, J ¼ 8:7, 7.6 Hz, 2H,
(C6H4)2–CH–CH2–OCO–), 7.37 (dd, J ¼ 7:6, 7.6 Hz, 2H,
(C6H4)2–CH–CH2–OCO–), 7.32 (d, J ¼ 8:3 Hz, 2H, p-RCONH–
C6H4–CH2–), 7.30–7.25 (m, 3H, –OCH2–COOCH2–C6H5),
7.23 (d, J ¼ 8:3 Hz, 2H, p-RCONH–C6H4–CH2–), 7.19 (d, J ¼
7:8 Hz, 2H, BA–CH2–C6H4–CH2NHCO–), 7.00 (d, J ¼ 7:8 Hz,
2H, BA–CH2–C6H4–CH2–NHCO–), 6.72 (brs, 1H, –CH2–

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
815
6.8 Hz, 2H, CO–CH2–CH2–CH2–CO), 2.18 (dd, J ¼ 6:8, 6.8 Hz,
2H, CO–CH2–CH2–CH2–CO), 1.98 (q, J ¼ 7:6 Hz, 2H, –CH2–
CH3), 1.84 (dd, J ¼ 6:8, 6.8 Hz, 2H, CO–CH2–CH2–CH2–CO),
0.78 (t, J ¼ 7:6 Hz, 3H, –CH2–CH3). Anal. Calcd for
C85H89N6O25P: C, 62.80; H, 5.52; N, 5.17%. Found: C, 62.78;
H, 5.51; N, 5.29%.
times (each 30 s), the mixture was stirred under Ar atmosphere
at room temperature for 1.5 h. The mixture was concentrated in
vacuo to give crude 30-O-deprotected product. To a solution of
the crude 30-O-free product and (R)-3-(4-trifluoromethylbenzyl-
oxy)decanoic acid (20) (50.0 mg, 144 mmol) in anhydrous CH2Cl2
(4.0 mL) were added DIC (40.0 mL, 255 mmol) and DMAP
(1.0 mg, 8.2 mmol) at room temperature under Ar atmosphere.
After stirring for 8 h, the reaction mixture was directly subjected
to affinity separation (13 g ꢅ 1) to give 46a as a pale yellow solid
3-O-Acylated Compounds 45a and 45b. 45a: To a solution
of 29 (434 mg, 267 mmol) and (R)-3-(4-trifluoromethylbenzyloxy)-
decanoic acid (20) (150 mg, 433 mmol) in anhydrous CH2Cl2
(10 mL) were added DMAP (3.3 mg, 27 mmol) and DIC
(125 mL, 798 mmol) at room temperature under Ar atmosphere
and the mixture was stirred for 2 h. To the reaction mixture was
added toluene (10 mL) and the mixture was subjected to affinity
separation (13 g ꢅ 3). After untagged compounds were eluted
with toluene–CH2Cl2 (1:1) and CH2Cl2, 45a was obtained as a
pale yellow solid (435 mg, 84%) by elution with CH2Cl2–MeOH
24
(117 mg, 75%). ½ꢁꢄD ¼ þ18:3 (c 0.67, CHCl3). ESI-MS (posi-
tive) m=z 2222.34 ½M þ Naꢄþ. 1H NMR (400 MHz, DMSO-d6,
40 ꢂC): ꢃ 9.79 (s, 1H, NH), 8.22 (t, J ¼ 3:8 Hz, 2H, NH), 7.84
(d, J ¼ 7:6 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 7.66 (dd, J ¼
7:3, 6.9 Hz, 2H, –OCH2–COOCH2–C6H5), 7.58 (dd, J ¼ 8:1,
7.6 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 7.54 (d, J ¼ 8:0 Hz, 4H,
p-CF3–C6H4–CH2–), 7.52 (d, J ¼ 8:1 Hz, 2H, (C6H4)2–CH–
CH2–OCO–), 7.41–7.31 (m, 14H, (C6H4)2–CH–CH2–OCO–,
p-RCONH–C6H4–CH2–, p-CF3–C6H4–CH2–, and C6H5–CH2–),
7.30–7.25 (m, 6H, BA–CH2–C6H4–CH2NHCO–, o-C6H4-
(CH2O)2P–, and –OCH2–COOCH2–C6H5), 7.14–7.10 (m, 4H,
o-C6H4(CH2O)2P– and BA–CH2–C6H4–CH2NHCO–), 6.95 (d,
J ¼ 8:1 Hz, 2H, p-RCONH–C6H4–CH2–), 5.95–5.85 (m, 1H,
–OCH2–CH=CH2 of Alloc), 5.44 (d, J ¼ 6:5 Hz, 1H, NH), 5.38
(dd, J ¼ 10:1, 10.1 Hz, 1H, H-30), 5.31 (d, J ¼ 14:5 Hz, 1H,
–OCH2–CH=CH2 of Alloc), 5.23 (d, J ¼ 10:5 Hz, 1H, –OCH2–
CH=CH2 of Alloc), 5.18–5.08 (m, 8H, NH, H-3, o-
C6H4(CH2O)2P, and –OCH2–COOCH2–C6H5), 4.95 (brs, 1H,
H-1), 4.60 (dd, J ¼ 10:1, 10.1 Hz, 2H, p-RCONH–C6H4–CH2–),
4.53–4.47 (m, 9H, H-10, H-40, C6H5–CH2–, p-CF3–C6H4–CH2–,
and –OCH2–CH=CH2 of Alloc), 4.39 (d, J ¼ 7:2 Hz, 2H,
(C6H4)2–CH–CH2–OCO–), 4.21 (dd, J ¼ 7:2, 7.2 Hz, 1H,
(C6H4)2–CH–CH2–OCO–), 4.15–4.07 (m, 4H, –OCH2–
COOCH2–C6H5 and BA–CH2–C6H4–CH2NHCO–), 3.95–3.87
(m, 2H, H-2 and H-60a), 3.81–3.74 (m, 4H, H-4, H-6a, H-50,
and ꢀ-CH of 3-O-acyl), 3.68–3.58 (m, 5H, H-5, H-6b, H-20, H-
60b, and ꢀ-CH of 30-O-acyl), 3.06 (d, J ¼ 3:7 Hz, 2H, BA–
CH2–C6H4–CH2NHCO–), 2.65 (dd, J ¼ 15:0, 5.3 Hz, 1H, ꢁ-
CH2 of 3-O-acyl), 2.53–2.45 (m, 2H, ꢁ-CH2 of 3-O-acyl and 30-
O-acyl), 2.41 (dd, J ¼ 15:1, 5.6 Hz, 1H, ꢁ-CH2 of 30-O-acyl),
2.28 (dd, J ¼ 7:1, 7.1 Hz, 2H, –CO–CH2–CH2–CH2–CO–), 2.17
(dd, J ¼ 7:1, 7.1 Hz, 2H, –CO–CH2–CH2–CH2–CO–), 1.96 (q,
J ¼ 7:3 Hz, 2H, –CH2–CH3 of BA), 1.81 (dd, J ¼ 7:1, 7.1 Hz,
2H, –CO–CH2–CH2–CH2–CO–), 1.55–1.48 (m, 4H, ꢄ-CH2 of
3-O-acyl and 30-O-acyl), 1.31–0.99 (m, 20H, CH2 ꢅ 10), 0.86–
0.79 (m, 6H, –CH2–CH3 of 3-O-acyl and 30-O-acyl), 0.75
(t, J ¼ 7:1 Hz, 3H, –CH2–CH3 of BA).
23
(1:1) and evaporation of the solvents. ½ꢁꢄD ¼ þ22:9 (c 0.94,
CHCl3). ESI-MS (positive) m=z 1975.42 ½M þ Naꢄþ, 999.35
2þ
1
½M þ 2Naꢄ . H NMR (400 MHz, DMSO-d6, 40 ꢂC): ꢃ 7.84 (d,
J ¼ 7:2 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 7.67 (dd, J ¼ 8:9,
7.2 Hz, 2H, (C6H4)2–CH–CH2–OCO–), 7.63 (dd, J ¼ 7:3,
6.9 Hz, 2H, –OCH2–COOCH2–C6H5), 7.55 (d, J ¼ 8:0 Hz, 2H,
p-CF3–C6H4–CH2–), 7.52 (dd, J ¼ 7:2, 7.2 Hz, 2H, (C6H4)2–
CH–CH2–OCO–), 7.41–7.31 (m, 12H, (C6H4)2–CH–CH2–OCO–,
p-RCONH–C6H4–CH2–, p-CF3–C6H4–CH2–, and C6H5–CH2–),
7.30–7.25 (m, 6H, BA–CH2–C6H4–CH2NHCO–, o-C6H4-
(CH2O)2P–, and –OCH2–COOCH2–C6H5), 7.15–7.10 (m, 4H,
o-C6H4(CH2O)2P– and BA–CH2–C6H4–CH2NHCO–), 6.95 (d,
J ¼ 8:0 Hz, 2H, p-RCONH–C6H4–CH2–), 5.95–5.83 (m, 1H,
–OCH2–CH=CH2 of Alloc), 5.43 (d, J ¼ 6:5 Hz, 1H, NH), 5.28
(d, J ¼ 15:4 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.21 (d, J ¼
10:5 Hz, 1H, –OCH2–CH=CH2 of Alloc), 5.17–5.07 (m, 9H,
NH, H-3, H-30, o-C6H4(CH2O)2P, and –OCH2–COOCH2–C6H5),
4.95 (brs, 1H, H-1), 4.79 (dd, J ¼ 10:3, 10.3 Hz, 2H, p-RCONH–
C6H4–CH2–), 4.67 (d, J ¼ 2:8 Hz, 1H, –OCH2–CꢆCH of Proc),
4.61–4.52 (m, 8H, H-10, H-40, C6H5–CH2–, p-CF3–C6H4–CH2–,
–OCH2–CꢆCH of Proc, and –OCH2–CH=CH2 of Alloc), 4.48
(d, J ¼ 12:5 Hz, 1H, C6H5–CH2–), 4.39 (d, J ¼ 7:2 Hz, 2H,
(C6H4)2–CH–CH2–OCO–), 4.21 (dd, J ¼ 7:2, 7.2 Hz, 1H,
(C6H4)2–CH–CH2–OCO–), 4.18–4.10 (m, 4H, –OCH2–
COOCH2–C6H5 and BA–CH2–C6H4–CH2NHCO–), 3.93–3.86
(m, 2H, H-2 and H-60a), 3.78 (brs, 1H, ꢀ-CH of 3-O-acyl),
3.76–3.72 (m, 3H, H-4, H-6a, and H-50), 3.65–3.58 (m, 4H,
H-5, H-6b, H-20, and H-60b), 3.07 (s, 2H, BA–CH2–C6H4–
CH2NHCO–), 2.51–2.45 (m, 3H, –OCH2–CꢆCH of Proc and ꢁ-
CH2 of 3-O-acyl), 2.29 (dd, J ¼ 6:8, 6.8 Hz, 2H, –CO–CH2–
CH2–CH2–CO–), 2.18 (dd, J ¼ 6:8, 6.8 Hz, 2H, –CO–CH2–
CH2–CH2–CO–), 1.96 (q, J ¼ 7:6 Hz, 2H, –CH2–CH3 of BA),
1.81 (dd, J ¼ 6:8, 6.8 Hz, 2H, –CO–CH2–CH2–CH2–CO–),
1.60–1.47 (m, 2H, ꢄ-CH2 of 3-O-acyl), 1.31–0.99 (m, 10H,
CH2 ꢅ 5), 0.79 (t, J ¼ 7:6 Hz, 3H, –CH2–CH3 of 3-O-acyl),
0.74 (t, J ¼ 7:2 Hz, 3H, –CH2–CH3 of BA).
46b: In a manner similar to the synthesis of 46a, 45a (288 mg,
147 mmol) was deprotected and acylated with (R)-3-(dodecanoyl-
oxy)decanoic acid (21) to yield 46b as a pale yellow solid
(252 mg, 77%). ESI-MS (positive) m=z 2249.29 ½M þ Naꢄþ.
46c:
To a solution of 45b (235 mg, 119 mmol) in AcOH
(5.0 mL) was added Zn–Cu couple (200 mg) at room temperature,
and the mixture was stirred for 2 h. After insoluble materials were
filtered off, the filtrate was concentrated in vacuo, and the residual
AcOH was removed by co-evaporation with toluene three times.
The residue was dissolved in EtOAc, washed successively with
saturated aqueous NaHCO3 and brine, dried over MgSO4, and
concentrated in vacuo. To a solution of the residue and (R)-3-(do-
decanoyloxy)decanoic acid (21) (120 mg, 346 mmol) in anhydrous
CH2Cl2 (5.0 mL) were added DMAP (1.5 mg, 12 mmol) and DIC
(110 mL, 703 mmol) at room temperature under Ar atmosphere
and the mixture was stirred for 11 h. To the reaction mixture
45b: In a manner similar to the synthesis of 45a, 29 (221 mg,
136 mmol) was acylated with (R)-3-(dodecanoyloxy)decanoic acid
(21) to yield 45b as a pale yellow solid (247 mg, 93%). ESI-MS
(positive) m=z 1977.87 ½M þ Hꢄþ, 1999.81 ½M þ Naꢄþ, 1000.41
2þ
½M þ H þ Naꢄ
.
3-,30-O-Diacylated Compounds 46a, 46b, 46c, and 46d. 46a:
To a degassed solution of 45a (140 mg, 71.6 mmol) in anhydrous
THF (4.0 mL) was added (1,5-cyclooctadiene)[bis(methyldi-
phenylphosphine)]iridium(I) hexafluorophosphate (65.0 mg, 76.9
mmol). After activation of the iridium catalyst with hydrogen three

816 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
was added toluene (5.0 mL) and the mixture was subjected to
affinity separation (13.0 g ꢅ 3) to give 46c as a pale yellow solid
47b: In a manner similar to the synthesis of 47a, 46b (111 mg,
49.8 mmol) was deprotected and acylated with (R)-3-(4-trifluoro-
methylbenzyloxy)decanoic acid (20) to yield 47b as a pale yellow
solid (54.5 mg, 44%). ESI-MS (positive) m=z 2468.98 ½M þ Hꢄþ,
2489.97 ½M þ Naꢄþ.
(155 mg, 59%). ESI-MS (positive) m=z 2224.80 ½M þ Hꢄþ,
2þ
2246.33 ½M þ Naꢄþ, 1123.62 ½M þ H þ Naꢄ
,
1134.97
2þ
½M þ 2Naꢄ
.
46d: In a manner similar to the synthesis of 46c, 45b (98.2 mg,
49.6 mmol) was deprotected and acylated with (R)-3-(4-trifluoro-
methylbenzyloxy)decanoic acid (20) to yield 46d as a pale yellow
solid (59.6 mg, 59%). ESI-MS (positive) m=z 2270.29 ½M þ Naꢄþ,
47c: In a manner similar to the synthesis of 47a, 46b (110 mg,
49.7 mmol) was deprotected and acylated with (R)-3-(dodecanoyl-
oxy)decanoic acid (21) to yield 47c as a pale yellow solid
(65.9 mg, 53%). ESI-MS (positive) m=z 2492.93 ½M þ Hꢄþ,
2514.23 ½M þ Naꢄþ.
2þ
1136.00 ½M þ H þ Naꢄ
.
20-N-,3-,30-O-Triacylated Compounds 47a, 47b, 47c, 47d,
47e, and 47f. 47a: To a solution of 46a (50.0 mg, 22.7 mmol)
in anhydrous THF (2.0 mL) was added Et3N (31.7 mL, 226 mmol),
HCO2H (8.6 mL, 220 mmol), and tetrakis(triphenylphosphine)-
palladium(0) (5.2 mg, 4.5 mmol) at room temperature under Ar
atmosphere. After the mixture was stirred for 1 h, EtOAc was add-
ed. The organic layer was washed with 1 M HCl, saturated aque-
ous NaHCO3, and brine. The EtOAc layer was dried over MgSO4
and concentrated in vacuo to give 20-N-deprotected product.
To a solution of the crude 2-N-free product and (R)-3-(do-
decanoyloxy)decanoic acid (21) (42.1 mg, 114 mmol) in anhydrous
CH2Cl2 (4.0 mL) were added DIC (36.0 mL, 230 mmol) at room
temperature under Ar atmosphere. After stirring for 18 h, toluene
(4.0 mL) was added and the reaction mixture was subjected to
affinity separation (13 g ꢅ 1) to give 47a as a pale yellow solid
47d: In a manner similar to the synthesis of 47a, 46c (59.6 mg,
26.5 mmol) was deprotected and acylated with (R)-3-(4-trifluoro-
methylbenzyloxy)decanoic acid (20) to yield 47d as a pale
yellow solid (24.5 mg, 37%). ESI-MS (positive) m=z 1244.15
2þ
½M þ 2Hꢄ
.
47e: In a manner similar to the synthesis of 47a, 46d (78.0 mg,
35.1 mmol) was deprotected and acylated with (R)-3-(4-trifluoro-
methylbenzyloxy)decanoic acid (20) to yield 47e as a pale
yellow solid (41.2 mg, 42%). ESI-MS (positive) m=z 2469.18
2þ
½M þ Hꢄþ, 2489.74 ½M þ Naꢄþ, 1245.40 ½M þ H þ Naꢄ
,
2þ
1256.35 ½M þ 2Naꢄ
.
47f: In a manner similar to the synthesis of 47a, 46d (78.0 mg,
35.1 mmol) was deprotected and acylated with (R)-3-(dodecanoyl-
oxy)decanoic acid (21) to yield 47f as a pale yellow solid
(67.4 mg, 63%). ESI-MS (positive) m=z 2515.20 ½M þ Naꢄþ,
(38.1 mg, 70%). ESI-MS (positive) m=z 2268.61 ½M þ Hꢄþ,
1256.95 ½M þ H þ Naꢄ2þ, 1268.40 ½M þ 2Naꢄ
.
2þ
2þ
2490.30 ½M þ Naꢄþ, 1245.33 ½M þ 2Naꢄ . H NMR (400 MHz,
2-,20-N-,3-,30-O-Tetraacylated Compounds 30a, 30b, 30c,
1
DMSO-d6, 40 ꢂC): ꢃ 8.22 (s, 1H, NH), 7.83 (d, J ¼ 7:3 Hz,
2H, (C6H4)2–CH–CH2–OCO–), 7.65 (dd, J ¼ 8:3, 7.3 Hz, 2H,
–OCH2–COOCH2–C6H5), 7.56 (dd, J ¼ 8:3, 7.3 Hz, 2H,
(C6H4)2–CH–CH2–OCO–), 7.50 (d, J ¼ 7:3 Hz, 4H, p-CF3–
C6H4–CH2–), 7.39 (dd, J ¼ 7:3, 7.3 Hz, 2H, (C6H4)2–CH–CH2–
OCO–), 7.35–7.30 (m, 14H, (C6H4)2–CH–CH2–OCO–, p-
RCONH–C6H4–CH2–, p-CF3–C6H4–CH2–, and C6H5–CH2–),
7.28–7.25 (m, 6H, BA–CH2–C6H4–CH2NHCO–, o-C6H4-
(CH2O)2P–, and –OCH2–COOCH2–C6H5), 7.21 (dd, J ¼ 8:2,
4.8 Hz, 2H, o-C6H4(CH2O)2P), 7.14 (d, J ¼ 7:8 Hz, 2H, BA–
CH2–C6H4–CH2NHCO–), 7.08 (d, J ¼ 7:8 Hz, 2H, p-RCONH–
C6H4–CH2–), 6.57 (d, J ¼ 8:8 Hz, 1H, 20-NH), 6.11 (d, J ¼
8:5 Hz, 1H, 2-NH), 5.45 (d, J ¼ 6:5 Hz, 1H, NH), 5.45 (dd,
J ¼ 10:2, 10.2 Hz, 1H, H-30), 5.33 (dd, J ¼ 10:5, 10.5 Hz,
1H, H-3), 5.19–5.01 (m, 6H, o-C6H4(CH2O)2P and –OCH2–
COOCH2–C6H5), 4.89 (d, J ¼ 3:3 Hz, 1H, H-1), 4.81 (d, J ¼
11:2 Hz, 1H, p-RCONH–C6H4–CH2–), 4.61–4.54 (m, 11H, H-10,
H-40, C6H5–CH2–, p-CF3–C6H4–CH2–, p-RCONH–C6H4–CH2–,
and o-C6H4(CH2O)2P), 4.38 (d, J ¼ 7:3 Hz, 2H, (C6H4)2–CH–
CH2–OCO–), 4.30–4.20 (m, 5H, (C6H4)2–CH–CH2–OCO–,
–OCH2–COOCH2–C6H5, and BA–CH2–C6H4–CH2NHCO–),
4.01 (dd, J ¼ 13:2, 2.8 Hz, 1H, H-60a), 3.84–3.80 (m, 4H, H-2,
ꢀ-CH of 3-O-acyl and 30-O-acyl, and ꢀ-CH of 20-N-acyl), 3.75–
3.72 (m, 3H, H-4, H-6a, and H-50), 3.68–3.65 (m, 2H, H-20 and
H-60b), 3.45–3.41 (m, 2H, H-5 and H-6b), 3.08 (s, 2H, BA–
CH2–C6H4–CH2NHCO–), 2.65 (dd, J ¼ 15:0, 5.5 Hz, 1H, ꢁ-
CH2 of 3-O-acyl), 2.53–2.44 (m, 2H, ꢁ-CH2 of 3-O-acyl and 30-
O-acyl), 2.40 (dd, J ¼ 15:0, 5.6 Hz, 1H, ꢁ-CH2 of 30-O-acyl),
2.30 (dd, J ¼ 6:3, 6.3 Hz, 2H, –CO–CH2–CH2–CH2–CO–), 2.28
(dd, J ¼ 14:5, 7.0 Hz, 2H, ꢁ-CH2 of 20-N-acyl’s side chain),
2.21 (dd, J ¼ 16:5, 8.5 Hz, 2H, ꢁ-CH2 of 20-N-acyl’s main chain),
2.17 (dd, J ¼ 6:3, 6.3 Hz, 2H, –CO–CH2–CH2–CH2–CO–), 1.97
(q, J ¼ 7:3 Hz, 2H, –CH2–CH3 of BA), 1.92 (dd, J ¼ 6:3,
6.3 Hz, 2H, –CO–CH2–CH2–CH2–CO–), 1.55–1.01 (m, 54H,
CH2 ꢅ 27), 0.88–0.72 (m, 15H, –CH2–CH3 ꢅ 5).
30d, 30e, and 30f.
30a:
To a solution of 47a (38.0 mg,
15.4 mmol) in CH2Cl2 (2.0 mL) was added DBU (2.5 mL,
16.7 mmol) at room temperature and the mixture was stirred for
1.5 h. The reaction mixture was directly subjected to silica-gel col-
umn chromatography (5.0 g, CHCl3:MeOH = 10:1) to give 2-N-
deprotected product as a colorless solid: Yield 19.0 mg (63%).
To a solution of the 2-N-free product and (R)-3-(dodecanoyloxy)-
decanoic acid (21) (28.0 mg, 75.6 mmol) in anhydrous CH2Cl2
(2.0 mL) were added DIC (25.0 mL, 160 mmol) at room tempera-
ture under Ar atmosphere. After stirring for 14 h, to the reaction
mixture was added toluene (2.0 mL) and the mixture was
subjected to affinity separation (13 g ꢅ 1) to give 30a as a pale
yellow solid (18.7 mg, 49%). ESI-MS (positive) m=z 2620.88
2þ
½M þ Naꢄþ, 1321.05 ½M þ 2Naꢄ
.
1H NMR (400 MHz, DMSO-
d6, 40 ꢂC): ꢃ 9.81 (s, 1H, NH), 8.25 (s, 1H, NH), 7.65 (dd, J ¼
8:5, 7.3 Hz, 2H, –OCH2–COOCH2–C6H5), 7.55 (d, J ¼ 7:3 Hz,
4H, p-CF3–C6H4–CH2–), 7.35–7.29 (m, 12H, p-RCONH–C6H4–
CH2–, p-CF3–C6H4–CH2–, and C6H5–CH2–), 7.26–7.20 (m, 7H,
BA–CH2–C6H4–CH2NHCO–, o-C6H4(CH2O)2P–, and –OCH2–
COOCH2–C6H5), 7.16 (d, J ¼ 7:8 Hz, 2H, BA–CH2–C6H4–
CH2NHCO–), 7.02 (d, J ¼ 7:8 Hz, 2H, p-RCONH–C6H4–CH2–),
6.39 (d, J ¼ 8:3 Hz, 1H, 20-NH), 6.20 (d, J ¼ 7:3 Hz, 1H, 2-NH),
5.60 (d, J ¼ 6:5 Hz, 1H, NH), 5.48 (dd, J ¼ 10:5, 9.3 Hz, 1H,
H-30), 5.36 (dd, J ¼ 10:5, 10.5 Hz, 1H, H-3), 5.23–5.01 (m, 6H,
o-C6H4(CH2O)2P and –OCH2–COOCH2–C6H5), 4.85 (d, J ¼
3:5 Hz, 1H, H-1), 4.75 (d, J ¼ 10:5 Hz, 1H, p-RCONH–C6H4–
CH2–), 4.62–4.50 (m, 11H, H-10, H-40, C6H5–CH2–, p-CF3–
C6H4–CH2–, p-RCONH–C6H4–CH2–, and o-C6H4(CH2O)2P),
4.33–4.20 (m, 4H, –OCH2–COOCH2–C6H5 and BA–CH2–
C6H4–CH2NHCO–), 4.01 (dd, J ¼ 13:2, 1.8 Hz, 1H, H-60a),
3.88–3.79 (m, 4H, H-2, ꢀ-CH of 3-O-acyl and 30-O-acyl, and ꢀ-
CH of 20-N-acyl), 3.75–3.72 (m, 5H, H-4, H-6a, H-50, H-60b, and
ꢀ-CH of 2-N-acyl), 3.68–3.64 (m, 2H, H-20 and H-60b), 3.45–3.40
(m, 2H, H-5 and H-6b), 3.12 (d, J ¼ 3:1 Hz, 2H, BA–CH2–C6H4–
CH2NHCO–), 2.65 (dd, J ¼ 15:1, 5.5 Hz, 1H, ꢁ-CH2 of 3-O-

Y. Fukase et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008)
817
acyl), 2.53–2.43 (m, 2H, ꢁ-CH2 of 3-O-acyl and 30-O-acyl),
2.40 (dd, J ¼ 15:0, 5.6 Hz, 1H, ꢁ-CH2 of 30-O-acyl), 2.31
(dd, J ¼ 7:0, 7.0 Hz, 2H, –CO–CH2–CH2–CH2–CO–), 2.28–
2.20 (m, 8H, ꢁ-CH2 of 20-N-acyl’s main and side chains, and
ꢁ-CH2 of 20-N-acyl’s main and side chains), 2.16 (dd, J ¼ 7:0,
7.0 Hz, 2H, –CO–CH2–CH2–CH2–CO–), 2.08 (q, J ¼ 6:9 Hz,
2H, –CH2–CH3 of BA), 1.97 (dd, J ¼ 7:0, 7.0 Hz, 2H, –CO–
CH2–CH2–CH2–CO–), 1.61–1.43 (m, 12H, ꢄ-CH2 of 2-N-acyl,
20-N-acyl, 3-O-acyl, and 30-O-acyl), 1.35–1.08 (m, 76H,
–CH2 ꢅ 38), 0.88 (t, J ¼ 6:9 Hz, 21H, –CH2–CH3 ꢅ 7).
1H, ꢁ-CH2 of 2-N-acyl’s main chain), 2.28–2.21 (m, 10H, ꢁ-
CH2 of 3-O-acyl, 30-O-acyl, 2-N-acyl’s main and side chain,
and 20-N-acyl’s main and side chain), 1.60–1.52 (m, 8H, ꢄ-CH2
of 3-O-acyl, 30-O-acyl, 2-N-acyl’s main chain, and 20-N-acyl’s
main chain), 1.31–1.16 (m, 66H, –CH2– ꢅ 33), 0.85 (t, J ¼
6:6 Hz, 18H, –CH3 ꢅ 6).
26b:
In a manner similar to the synthesis of 26a, 30b
(18.0 mg, 6.93 mmol) was hydrogenolytically deprotected to give
26b as a colorless solid (2.7 mg, 25%). ESI-MS (negative) m=z
2ꢃ
1521.24 ½M ꢃ Hꢄꢃ, 760.26 ½M ꢃ 2Hꢄ
.
30b:
In a manner similar to the synthesis of 30a, 47b
26c: In a manner similar to the synthesis of 26a, 30c (17.0 mg,
6.54 mmol) was hydrogenolytically deprotected to give 26c as a
colorless solid (2.1 mg, 22%). ESI-MS (negative) m=z 1521.54
.
26d: In a manner similar to the synthesis of 26a, 30d (3.3 mg,
(55.0 mg, 22.3 mmol) was deprotected and acylated with (R)-3-
(dodecanoyloxy)decanoic acid (21) to yield 16b as a pale yellow
solid: Yield 19.7 mg (39%); ESI-MS (positive) m=z 2622.53
½M þ Naꢄþ.
½M ꢃ Hꢄꢃ, 760.39 ½M ꢃ 2Hꢄ
2ꢃ
30c: In a manner similar to the synthesis of 30a, 47c (66.0 mg,
26.8 mmol) was deprotected and acylated with (R)-3-(4-trifluoro-
methylbenzyloxy)decanoic acid (20) to yield 30c as a pale yellow
1.27 mmol) was hydrogenolytically deprotected to give 26d as a
colorless solid (1.3 mg, 67%). ESI-MS (negative) m=z 761.23
2ꢃ
½M ꢃ 2Hꢄ
.
26e: In a manner similar to the synthesis of 26a, 30e (5.2 mg,
solid (17.9 mg, 35%). ESI-MS (positive) m=z 2620.30 ½M þ Naꢄþ,
2þ
1299.57 ½M þ 2Hꢄ
.
In a manner similar to the synthesis of 30a, 47d
2.00 mmol) was hydrogenolytically deprotected to give 26e as a
colorless solid (1.7 mg, 56%). ESI-MS (negative) m=z 1522.11
30d:
2ꢃ
(24.7 mg, 9.91 mmol) was deprotected and acylated with (R)-3-
(4-trifluoromethylbenzyloxy)decanoic acid (20) to yield 30d as a
pale yellow solid (3.3 mg, 14%). ESI-MS (positive) m=z 2621.46
½M ꢃ Hꢄꢃ, 760.46 ½M ꢃ 2Hꢄ
.
26f: In a manner similar to the synthesis of 26a, 30f (3.8 mg,
1.46 mmol) was hydrogenolytically deprotected to give 26f as a
colorless solid (1.5 mg, 67%). ESI-MS (negative) m=z 1521.93
2þ
½M þ Naꢄþ, 1311.14 ½M þ H þ Naꢄ2þ, 1321.56 ½M þ 2Naꢄ
.
2ꢃ
30e: In a manner similar to the synthesis of 30a, 47e (42.7 mg,
17.3 mmol) was deprotected and acylated with (R)-3-(dodecanoyl-
oxy)decanoic acid (21) to yield 30e as a pale yellow solid (5.2 mg,
12%). ESI-MS (positive) m=z 2620.55 ½M þ Naꢄþ, 1311.06
½M ꢃ Hꢄꢃ, 760.44 ½M ꢃ 2Hꢄ
.
Limulus Assay. Limulus activity of synthetic samples were
measured by means of the Endospecy Testꢁ (Seikagaku Kogyo,
Tokyo, Japan) using an LPS specimen from E. coli O111:B1
(Sigma-Aldrich Chemical Co.) as a reference standard. A solution
of a test sample in 1% DMSO in distilled water (30 mL) was mixed
with the reagent in the Endospecy ES-50M set (30 mL) and incu-
bated in duplicate in a 96-well plastic plate (Toxipet plate 96F,
Seikagaku Kogyo) at 37 ꢂC for 30 min. Sodium nitrate (75 mL,
0.04% in 0.48 mol dmꢃ3 hydrochloric acid), 75 mL of 0.3% ammo-
nium sulfate, and 75 mL of 0.07% N-1-naphthylethylenediamine
dichloride were added successively. The absorbance at 414 nm
of each well was measured using a micro plate reader.
½M þ H þ Naꢄ2þ, 1321.42 ½M þ 2Naꢄ
.
2þ
30f: In a manner similar to the synthesis of 30a, 47f (68.4 mg,
27.4 mmol) was deprotected and acylated with (R)-3-(4-trifluoro-
methylbenzyloxy)decanoic acid (20) to yield 30f as a pale
yellow solid (3.0 mg, 6.0%). ESI-MS (positive) m=z 1312.33
2þ
½M þ H þ Naꢄ2þ, 1321.77 ½M þ 2Naꢄ
.
CM-Analogues 26a, 26b, 26c, 26d, 26e, and 26f. 2-Deoxy-6-
O-[2-deoxy-2-((R)-3-(dodecanoyloxy)decanoylamino)-3-O-((R)-
3-hydroxydecanoyl)-4-O-phosphono-ꢂ-D-glucopyranosyl]-2-
((R)-3-(dodecanoyloxy)decanoylamino)-3-O-((R)-3-hydroxy-
decanoyl)-ꢀ-D-glucopyranosyloxyacetic Acid (26a): To a solu-
tion of 30b (18.0 mg, 6.93 mmol) in THF–AcOH (3:1) (2.4 mL)
was added Pd(OH)2 (25.0 mg). The mixture was stirred under
19 kg cmꢃ2 of hydrogen at room temperature for 1 d. After remov-
al of the Pd catalyst by filtration, the solvent was evaporated in
vacuo. The crude product was purified by liquid–liquid partition
column chromatography (5.0 g of Sephadexꢁ LH-20, CHCl3:
MeOH:water:i-PrOH = 10:10:10:1.3). The organic layer was the
stationary phase, and the aqueous layer was the mobile phase in
this chromatography. After removal of the solvent in vacuo, the
residue was lyophilized from sterilized water to afford 26a as a
Cytokine Assay. Heparinized human whole blood diluted with
RPMI 1640 (Biken, Osaka, Japan) (v/v, 1/4) was incubated at
37 ꢂC for 24 h in humidified air containing 5% (v/v) CO2 in a
96-well culture plate (Becton Dickson) with or without various
doses of test specimens for interleukin-6 (IL-6) assay. The
amounts of cytokine induced were measured from the culture su-
pernatants using the appropriate ELISA kit systems (IL-6 and
TNF-ꢁ, ELISA Development Kit human IL-6 and ELISA Devel-
opment Kit human TNF-ꢁ, Genzyme TECHNE Co., Minneapolis,
MN, USA). These assays were performed according to the manu-
facturer’s instructions, and the cytokine amount was determined
from a standard curve prepared for each assay. Assays were
repeated three times. Similar results were obtained in repeated
experiments.
colorless solid (4.0 mg, 60%). ESI-MS (negative) m=z 1521.77
2ꢃ
½M ꢃ Hꢄꢃ, 760.35 ½M ꢃ 2Hꢄ
.
1H NMR (600 MHz, CDCl3:
MeOH-d4 = 1:1) ꢃ 5.20–5.04 (m, 2H, ꢀ-CH of 2-N-acyl and 20-
N-acyl), 5.19 (dd, J ¼ 8:7, 8.7 Hz, 1H, H-3), 5.14 (dd, J ¼ 8:9,
8.9 Hz, 1H, H-30), 4.74 (d, J ¼ 3:1 Hz, 1H, H-1), 4.66 (d,
J ¼ 7:4 Hz, 1H, H-10), 4.20–4.16 (m, 2H, H-20 and H-40), 4.03
(d, J ¼ 12:3 Hz, 1H, –OCH2–COOH), 4.01–3.96 (m, 3H, H-6a,
H-60a, and ꢀ-CH of 30-O-acyl), 3.83 (d, J ¼ 12:3 Hz, 1H,
–OCH2–COOH), 3.81–3.68 (m, 5H, H-20, H-5, H-6b, H-60b, and
ꢀ-CH of 3-O-acyl), 3.56 (dd, J ¼ 8:7, 8.7 Hz, 1H, H-4), 3.46
(dd, J ¼ 6:9, 6.9 Hz, 1H, H-50), 2.45 (dd, J ¼ 12:8, 6.3 Hz, 1H,
ꢁ-CH2 of 20-N-acyl’s main chain), 2.38 (dd, J ¼ 12:9, 3.8 Hz,
This work was supported in part by Grants-in-Aid for
Scientific research (Nos. 14380288, 15310149, 17035050,
17310128, and 18032046) from the Japan Society for the
Promotion of Science, the Grant-in-Aid for Creative Scientific
Research ‘‘In vivo Molecular Science for Discovery of New
Biofunctions and Pharmaceutical Drugs’’ No. 13NP0401 from
the Ministry of Education, Culture, Sports, Science and
Technology of Japan; and grants from Suntory Institute for
Bioorganic Research (SUNBOR Grant), the Houansha Foun-

818 Bull. Chem. Soc. Jpn. Vol. 81, No. 7 (2008) BCSJ AWARD ARTICLE
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Ms. K. Aoyama at Hyogo medical college for their assistance
in the cytokine induction assay.
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