Synthesis and biological activities of biscarboxymethyl lipid A analogues

Article abstract of DOI:10.3987/com-06-s(o)51

Biscarboxymethyl analogues of Escherichia coli lipid A and the biosynthetic precursor-type lipid A were synthesized in order to elucidate the role of the acidic functional groups in the biological activity. Both analogues showed respective activities simi

Full text of DOI:10.3987/com-06-s(o)51

HETEROCYCLES, Vol. 69, 2006
395
HETEROCYCLES, Vol. 69, 2006, pp. 395 - 415. © The Japan Institute of Heterocyclic Chemistry
Received, 8th September, 2006, Accepted, 30th October, 2006, Published online, 31st October, 2006. COM-06-S(O)51
SYNTHESIS AND BIOLOGICAL ACTIVITIES OF
BISCARBOXYMETHYL LIPID A ANALOGUES
Mikayo Kataoka, Masaya Hashimoto, Yasuo Suda, Shoichi Kusumoto, and
Koichi Fukase*
Department of Chemistry, Graduate School of Science, Osaka University, 1-1
Machikaneyama, Toyonaka, Osaka 560-0043. koichi@chem.sci.osaka-u.ac.jp
Abstract – Biscarboxymethyl analogues of Escherichia coli lipid A and the
biosynthetic precursor-type lipid A were synthesized in order to elucidate the role
of the acidic functional groups in the biological activity. Both analogues showed
respective activities similar to those of their natural counterparts, i. e., the
hexaacyl E. coli lipid A analogue showed potent immunostimulating activity,
whereas the tetraacylated biosynthetic precursor-type lipid A analogue showed
the antagonistic activity. The present study clearly indicates that the acidic
functional groups but not phosphate groups are essential for the expression of the
biological activities.
INTRODUCTION
Lipopolysaccharide (LPS), also termed endotoxin, is a cell surface component of Gram-negative bacteria
and known as the strong stimulator of mammal immune system. LPS induces various inflammatory
mediators such as cytokines [tumor necrosis factor α (TNF-α), interleukin (IL) 1β, 6, 8, 12, 15, 18, and
etc], prostaglandins, and nitrogen oxide. These mediators stimulate immune system and also cause
clinical manifestations of bacterial infections such as fever, inflammation, hypotension, and, in severe
cases, lethal shock.1 LPS consists of a glycolipid component termed lipid A and a polysaccharide part. It
was unequivocally proved that lipid A is the chemical entity responsible for the biological activity of
LPS by our total synthesis of Escherichia coli lipid A (1) (a synthetic 1 is termed 506) (Fig. 1).2,3 E. coli
lipid A consists of β(1-6) disaccharide of glucosamines, 1,4’-diphosphoryl group, long-chain acyl groups
bound at 2, 2’, 3, and 3’ positions. A biosynthetic precursor of LPS (2) (a synthetic 2 is termed 406),
which has four acyl groups, shows antagonistic activity in humans but displays endotoxic activity in
rodents.3-6

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HETEROCYCLES, Vol. 69, 2006
LPS is sensed by a receptor complex consisting of toll like receptor 4 (TLR4)7-11 and an adaptor protein
MD-212-15. Golenbock et al. demonstrated that the above species-specific responses to 2 are mediated by
TLR4.11 Miyake et al. reported that MD-2 is required for LPS signaling and is also responsible for
species-specific activity of 2.12-14 Miyake et al. also demonstrated by using our synthetic lipid A and its
radio-labeled analogue that TLR4-MD-2 directly associates to LPS.15,16
It has been shown that both phosphoryl groups at the 1-, and 4’-positions are important for the biological
activities of lipid A. For example, both 1- and 4’-monophosphoryl lipid A have considerably lower
activity than the natural type bisphosphorylated lipid A, and the dephosphorylated lipid A does not show
any activity. To clarify the relationship between the biological activity and the role of the acidic
functional groups in lipid A, we synthesized both the E. coli-type and the precursor-type analogues (3)
(CM-506) and (4) (CM-406) in which the phosphoryl group at the 1-position is replaced with a
carboxymethyl (CM) group. Both the E. coli-type (3) and the biosynthetic precursor-type (4) showed
indistinguishable activity with the corresponding natural-type compounds.17,18 We also synthesized lipid
A analogues having acidic groups β-glycosidically linked at the 1-position.19 The β-CM analogues also
showed potent activity. The acidic functional group at 1-position are concluded to be essential but their
strict spatial arrangement is not required for expression of the biological activity. In the present study, we
synthesized two new analogues, E. coli-type (5) (Bis-CM-506) and precursor-type (6) (Bis-CM-406)
having two carboxymethyl groups at 1 and 4’-positions in order to investigate the role of the acidic
functional group at the 4’-position.
OH
OH
O
4'
O
O
O
O
O
O
Y O
HO
Y O
HO
O
1
O
O
NH
O
O
O
HN
NH
O
X
O
O
HN
O
X
O
O
O
O
O
OH
OH
OH
O
OH
OH
OH
O
1 (X, Y = -P(O)(OH)2): Escherichia coli Lipid A (506)
3 (X = -CH2COOH, Y = -P(O)(OH)2): CM analog (CM-506)
5 (X, Y = -CH2COOH): Bis-CM analog (Bis-CM-506)
2 (X, Y = -P(O)(OH)2): Biosynthetic precursor (406)
4 (X = -CH2COOH, Y = -P(O)(OH)2): CM analog (CM-406)
6 (X, Y = -CH2COOH): Bis-CM analog (Bis-CM-406)
Figure 1 Structures of lipid A and analogues.
RESULTS AND DISCUSSION

HETEROCYCLES, Vol. 69, 2006
397
We have established the efficient synthesis of lipid A and analogues in our previous studies.17-20 In the
present study, both Bis-CM-506 (5) and Bis-CM-406 (6) were synthesized via the similar strategy
(Scheme 1). Hydroxy and carboxy groups were protected with benzyl groups, which were removed by
catalytic hydrogenation at the last step. The β(1-6) disaccharide structures were constructed by glycosy-
lation of the glycosyl acceptor (12) with the N-Troc trichloroacetimidate donors. Compound (8) was used
as a common starting material for the glycosyl acceptor and the donors. The carboxymethyl group in 12
was formed by oxidative cleavage of the ally group. Both glycosyl donors (16) and (22) were derived
from the key intermediate (14), which was synthesized from 8 via 4-O-carboxymethylation.
BnO
O
O
HO
HO
O
O
O
Ph
OCOR1
OCOR1
OCOR1
BnO
OBn
O
OAllyl
NHTroc
O
OAllyl
NHTroc
NHCOR1
14
O
O
8
12
BnO
BnO
O
O
OCOR1
OCOR2
O C CCl3
NH
O C CCl3
NH
BnO
BnO
O
O
NHTroc
NHTroc
O
O
22
OBn
O
O
16
R1COOH:
C11H23
OH
+
12
12
16
22
Bis-CM-406 (6)
Bis-CM-506 (5)
O
C13H27
O
+
R2COOH:
C11H23
OH
Scheme 1 Synthesis strategy of Bis-CM analogues.
The glycosyl acceptor (12) was synthesized as shown in Scheme 2. The hydroxy group at 3-postion of
1-O-allyl 4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside17 (7)
was acylated with (R)-3-benzyloxytetradecanoic acid20. The 2-N-Troc group was removed by zinc pow-
der and acetic acid and the resulting 2-amino group was then acylated with (R)-3-benzyloxytetradecanoic
acid to give 2,3-diacyl derivative (9). Allyl glycoside (9) was treated with OsO4 and the resulting diol
was oxidatively cleaved with Pb(OAc)4 to give 1-O-formylmethyl glycoside (10), which was further oxi-
dized with NaClO2.17 The resulting carboxyl group was protected with benzyl group by using phenyldia-
zomethane to give 11. Deprotection of benzylidene group of 11 under the acidic conditions gave the
glycosyl acceptor (12).

398
HETEROCYCLES, Vol. 69, 2006
O
O
O
O
O
i) Zn, AcOH/MeOH/THF
(1:5:1), sonication
O
R1COOH
O
O
Ph
Ph
Ph
OCOR1
OCOR1
OH
ii) R1COOH, WSCI·HCl
HOAt, CHCl3
DCC, DMAP
CH2Cl2
OAllyl
NHTroc
O
OAllyl
NHTroc
OAllyl
NHCOR1
86%
78%
9
7
8
i) OsO4, NMO,
THF/t-BuOH/ H2O
(10:10:1)
i) NaClO2, NaHPO4
2-methyl-2-butene
t-BuOH/ H2O (4:1)
O
O
O
O
O
Ph
O
Ph
OCOR1
OCOR1
O
OBn
ii) Pb(OAc)4, benzene
O
ii) PhCHN2, CH2Cl2, 0°C
86%
O
NHCOR1
84%
NHCOR1
10
11
O
HO
TFA, CH2Cl2, H2O
90%
O
OBn
O
OCOR1
OBn
R1COOH:
C11H23
OH
HO
O
NHCOR1
O
12
Scheme 2 Synthesis of glycosyl acceptor (12).
One of the key step in the synthesis of Bis-CM lipid A analogues was the introduction of the car-
boxymethyl group at 4’-position. Since the 4-OH group of glucosamine residue is the most sterically
hindered position, we planned to introduce the carboxymethyl group before the formation of the disac-
charide (Scheme 3). Regioselective reductive opening of the benzylidene (8) with BF3•OEt2 and Et3SiH
gave the 6-O-benzyl-4-OH GlcN derivative (13).21 We then investigated the reaction conditions for the
introduction of carboxymethyl group to the 4-OH group. Among the reaction conditions tested, reaction
of compound (16) with benzyl iodoacetate and silver oxide gave 4-CM derivative (14) in a good yield.
Other basic conditions using NaH or BuLi decomposed the starting material. The 1-O-allyl group of 14
was removed via isomerization to 1-propenyl group and subsequent treatment with iodine.22 The result-
ing 1-OH sugar (15) was then transformed to the glycosyl donor (16).
O
BnO
BnO
O
O
O
Ph
BF3·OEt2, Et3SiH
CH2Cl2, 0 °C
Ag2O, ICH2COOBn
OCOR1
OCOR1
OCOR1
BnO
MS4A, toluene
rt, 10 d
O
OAllyl
NHTroc
HO
OAllyl
NHTroc
O
OAllyl
NHTroc
O
79%
85%
8
13
14
BnO
BnO
CCl3CN, Cs2CO3
O
i) [Ir(cod)(PMePh2)2]PF6, H2, THF
ii) I2, H2O
O
OCOR1
OCOR1
O C CCl3
NH
OH
MS4A, CH2Cl2
quant.
BnO
BnO
O
O
99%
NHTroc
O
O
NHTroc
16
15
Scheme 3 Synthesis of glycosyl donor (16).

HETEROCYCLES, Vol. 69, 2006
399
HO
BnO
O
O
TMSOTf, CH2Cl2
OCOR1
OCOR1
+
O C CCl3
NH
OBn
BnO
-15°C
71%
HO
O
O
NHCOR1
NHTroc
O
O
16
12
O
O
BnO
BnO
O
i) Zn-Cu, AcOH
ii) R1COOH
WSCI·HCl
O
O
O
OCOR1
OCOR1
OCOR1
OCOR1
OBn
OBn
BnO
BnO
HO
NHTroc
O
HO
NHCOR1
O
O
O
NHCOR1
NHCOR1
O
HOAt, CHCl3, rt
O
O
O
17
45%
18
Pd-black, H2, THF
quant.
Bis-CM-406 (6)
Scheme 4 Synthesis of Bis-CM-406 (6).
Synthesis of Bis-CM-406 was then carried out as described in Scheme 4. Glycosylation of the above
glycosyl acceptor (12) with the glycosyl donor (16) gave the desired β(1-6) disaccharide (17) in 71%
yield.23 The 2'-N-Troc group of 17 was cleaved and the resulting amino group was acylated with
(R)-3-benzyloxytetradecanoic acid to give the protected Bis-CM-406 (18). The final catalytic hydrogena-
tion gave the desired Bis-CM-406 (6) (m/z = 1360.0 [(M-H)-]).
BnO
BnO
BnO
O
O
O
O
O
DDQ (1 eq.)
H2O, CH2Cl2
BnO
OCOR1
BnO
O
OAllyl
NHTroc
O
OAllyl
NHTroc
BnO
O
O
O
O
OAllyl
NHTroc
O
48%
O
14
HO
BnO
C11H23
14
C11H23
19
BnO
BnO
O
O
i) [Ir(cod)(PMePh2)2]PF6, H2, THF
OCOR2
OCOR2
tetradecanoic acid
OH
BnO
BnO
ii) I2, H2O
DCC, DMAP, CH2Cl2
O
OAllyl
O
O
NHTroc
84%
O
NHTroc
20
21
98%
BnO
O
CCl3CN, Cs2CO3
O
OCOR2
C13H27
R2COOH:
O
O
O C CCl3
NH
MS4A, CH2Cl2
quant.
BnO
O
C11H23
OH
NHTroc
O
22
Scheme 5 Synthesis of glycosyl donor (22).

400
HETEROCYCLES, Vol. 69, 2006
Synthesis of Bis-CM-506 (5) is shown in Scheme 5 and 6. In our previous study, we found that the ben-
zyl group on 3-benzyloxyalkanoyl moiety was more prone to oxidation than usual O-benzyl group.19
We hence attempted to covert bezyloxytetradecanoate (14) to tetradecanoyloxytetradecanoate (20) via
selective cleavage of the benzyl group on benzyloxyalkanoyl moiety by using
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). Benzyl group on 3-benzyloxytetradecanoyl moiety in 14
was thus cleaved by DDQ to give 19 in 48% yield with 17% recovery of 14 and other undesired
6-hydroxyl products. The hydroxy group of resulting 19 was then acylated with tetradecanoic acid to
give 20. Deprotection of 1-O-allyl group of 20 and subsequent formation to 1-O-trichloroacetoimidate
afforded glycosyl donor (22).
Glycosylation of the acceptor (12) with 22 gave the disaccharide (23) in 93% yield. The 2’-N-Troc group
of 23 was cleaved by Zn-Cu and acetic acid and 3-(decanoyloxy)tetradecanoic acid was then introduced
to the 2’-amino group. The fully protected Bis-CM-506 (24) was thus obtained in 62% yield. Finally,
catalytic hydrogenation of 24 removed all the benzyl groups to give the desired Bis-CM-506 (5) (m/z =
1753.1 [(M-H)-]).
HO
BnO
O
O
TMSOTf, CH2Cl2
OCOR1
OCOR2
+
O C CCl3
NH
OBn
BnO
-15 °C
93%
HO
O
O
NHCOR1
NHTroc
O
O
22
12
O
O
BnO
BnO
O
i) Zn-Cu, AcOH
ii) R3COOH
WSCI·HCl
O
O
O
OCOR2
OCOR1
OCOR1
OCOR2
OBn
OBn
BnO
BnO
HO
NHTroc
O
HO
NHCOR3
O
O
O
NHCOR1
NHCOR1
O
HOAt, CHCl3, rt
O
O
O
23
62%
24
O
Pd-black, H2, THF
55%
C11H23
R3COOH:
O
O
Bis-CM-506 (5)
C11H23
OH
Scheme 6 Synthesis of Bis-CM-506 (5).
Biological activities of the hexaacyl Bis-CM-506 (5), CM-506 (3), and E. coli lipid A 506 (1) were
evaluated by measurement of cytokine inducing activity (IL-6, TNF-α) in human peripheral whole-blood
cells (Figure 2).24 The levels of induced cytokines were measured by means of the enzyme-linked im-
munosorbent assay (ELISA). Bis-CM-506 (5) showed potent but slightly weaker activity than both natu-
ral type lipid A (1) and CM-506 (3).
Antagonistic activity of the tetraacyl Bis-CM-406 (6) and the biosynthetic precursor 406 (2) was exam-
ined by inhibition to cytokine induction by LPS. A mixture of a test sample, LPS (10 ng ml-1) (E. coli

HETEROCYCLES, Vol. 69, 2006
401
0111:B4; Sigma Chemicals Co.), and human peripheral whole-blood was incubated and the levels of cy-
tokines were measured by ELISA. Bis-CM-406 (6) showed definite antagonistic activity but was 10-fold
less active than 406 (2) (Figure 3).
10 ng/mL
3000
2000
1 ng/mL
)
L
0.1 ng/mL
)
m
L
/
2000
g
m
/
p
(
g
0.01 ng/mL
p
α
(
1000
-
6
F
-
N
L
I
T
1000
0
0
Bis-CM-506(5) 506(1) CM-506(3)
Bis-CM-506(5) 506(1) CM-506(3)
Figure 2 IL-6 and TNF-α inducing activity.
1000 ng/mL
100 ng/mL
10 ng/mL
1 ng/mL
10000
5000
0
750
)
)
L
L
m
/
m
/
g
g
500
p
p
(
(
α
6
-
-
F
L
I
N
T
250
0
Bis-CM-406 (6) 406 (2)
+LPS +LPS
control
LPS
Bis-CM-406 (6) 406 (2)
+LPS +LPS
control
LPS
(10 ng/mL) (10 ng/mL) (10 ng/mL)
(10 ng/mL) (10 ng/mL) (10 ng/mL)
Figure 3 Inhibitory activity of Bis-CM-406 and 406.
As described, we synthesized two new lipid A analogues E. coli-type (5) (Bis-CM-506) and precur-
sor-type (6) (Bis-CM-406) having two carboxy groups in place of two phosphates in natural compounds.
Both analogues showed potent but slightly weaker activities than natural types. These results clearly in-
dicated that the acidic functional groups but phosphate groups are essential for expression of the
biological activities. The receptor complex should recognize negative charges on two acidic functional
groups at 1 and 4’-positions. The precise biological activities and biophysical properties of two Bis-CM
analogues have been reported separately.25

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EXPERIMENTAL
General methods
NMR spectra were measured with a JEOL JNM-GX400 and JEOL JMM-GX270 at 30 °C unless other-
wise specified, and analyzed using Alice program (version 2.0). The proton chemical shifts in CDCl3 are
given in δ values from tetramethylsilane as an internal standard, and the chemical shifts in other solvents
or conditions are given in δ values from the residual proton signal of the solvent. Mass spectra were ob-
tained with ESI-TOF mass spectrometer (Applied Biosystems, MarinerTM). Specific optical rotations
were measured on a Perkin-Elmer 241 polarimeter. Silica-gel chromatography was performed using Ki-
eselgel 60 (Merck, 0.040-0.063 mm) under medium pressure (2-4 kg/cm2) using the indicated solvent
systems. Analytical and preparative thin-layer chromatographies (TLC) were performed on Kieselgel
60F254 Plates (Merck, 0.25 mm thickness) and precoated Kieselgel 60F254 Plates (Merck, 0.5 mm thick-
ness), respectively. Recycling preparative HPLC was carried out with Japan Analytical Industry LC908
by using CHCl3 as an eluent. Nonaqueous reactions were carried out under argon atmosphere unless oth-
erwise noted. Anhydrous dichloromethane (CH2Cl2) was prepared by distillation from calcium hydride
and phosphorus pentoxide. Anhydrous acetonitrile, tetrahydrofuran (THF), and toluene were purchased
from Kanto Chemicals Co. Distilled water was purchased from Otsuka (Tokyo, Japan) or prepared by a
combination of Toray Pure LV-308 (Toray) and GSL-200 (Advantec, Tokyo, Japan). Molecular sieves
4A was activated in vacuo at 250 °C for 3 h before use. All other commercially obtained materials were
used as received.
Allyl
4,6-O-benzylidene-3-O-((R)-3-benzyloxytetradecanoyl)-2-deoxy-2-(2,2,2-trichloroethoxy-
carbonylamino)-α-D-glucopyranoside (8). To a solution of allyl 4,6-O-benzylidene-2-deoxy-
2-(2,2,2-trichloroethoxycarboylamino)-α-D-glucopyranoside (7) (6.47 g, 13.4 mmol) and
(R)-3-benzyloxytetradecanoic acid (5.38 g, 16.1 mmol) in dry CH2Cl2(100 mL) were added DMAP (318
mg, 2.60 mmol) and DCC (4.02 g, 19.5 mmol) at room temperature under N2 atmosphere. After being
stirred for 2 h, the insoluble materials were removed by filtration and the filtrate was diluted with CHCl3.
The organic solution was washed with aqueous 10% citric acid, saturated aqueous NaHCO3, and brine,
dried over MgSO4, and concentrated in vacuo. The filtrate was concentrated in vacuo and the residue was
purified by silica-gel column chromatography (500 g, toluene/AcOEt = 50/1) to give 8 as a white solid
1
(9.20 g, 86%). MALDI-TOF (positive) m/z 822.3 [(M+Na)+]. H NMR (400 MHz, CDCl3) δ 7.42-7.21
(m, 10H, OCH2Ph, PhCH), 5.95-5.85 (m, Jtrans = 17.1 Hz, Jcis = 10.4 Hz, Jvic = 5.3 Hz, 1H, OCH2CH=CH2),
5.47 (s, 1H, PhCH), 5.47-5.39 (dd, J3,2 = 10.1 Hz, J3,4 = 9.9 Hz, 1H, H-3), 5.37-5.34 (d, JN,2 = 10.1 Hz, 1H,
NH), 5.34-5.29 (dd, Jtrans = 17.2 Hz, Jgem = 1.5 Hz, 1H, OCH2CH=CH2), 5.26-5.23 (dd, Jcis = 10.4 Hz, Jgem
= 1.5 Hz, 1H, OCH2CH=CH2), 4.94-4.93 (d, J1,2 = 3.7 Hz, 1H, H-1), 4.74-4.71 (d, Jjem = 12.1 Hz, 1H,
OCH2Ph), 4.60-4.57 (d, Jjem = 12.1 Hz, 1H, OCH2Ph), 4.52-4.49 (d, Jjem = 12.1 Hz, 1H, CH2 of Troc),

HETEROCYCLES, Vol. 69, 2006
403
4.41-4.38 (d, Jjem = 12.1 Hz, 1H, CH2 of Troc), 4.30-4.27 (m, J6a,5 = 4.7 Hz, J6a,6b = 10.2 Hz, 1H, H-6a),
4.24-4.19 (dd, Jjem = 12.7 Hz, Jvic = 5.3 Hz, 1H, OCH2CH=CH2), 4.10-4.00 (m, J2,1 = 3.7 Hz, J2,3 = 10.1,
J2,N = 10.1 Hz, Jjem = 12.8 Hz, Jvic = 6.4 Hz, 2H, OCH2CH=CH2, H-2), 3.98-3.92 (ddd, J5, 4 = 9.8 Hz, J5,6a =
4.7 Hz, J5,6b = 9.9 Hz, 1H, H-5), 3.83-3.68 (m, 3H, C3H of acyl, H-6b, H-4), 2.70-2.64 (dd, Jjem = 15.3 Hz,
Jvic = 6.4 Hz, 1H, C2H2 of acyl), 2.45-2.40 (dd, Jjem = 15.3 Hz, Jvic = 6.0 Hz, 1H, C2H2 of acyl), 1.53-1.41
(m, 2H, C4H2 of acyl), 1.32-1.18 (m, 18H, C5- 13H2 of acyl), 0.90-0.86 (t, Jvic = 7.2 Hz, 3H, C14H3 of acyl).
25
Anal. Calcd for C87H119N2O17P: C, 60.11; H, 6.81; N, 1.75%. Found: C, 60.61; H, 6.70; N, 1.74. [α]D
=
+35.8 (c 1.00, CHCl3).
Allyl
4,6-O-benzylidene-3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-benzyloxytetradecanoyl-
amino)-2-deoxy-α-D-glucopyranoside (9). To a solution of 8 (2.00 g, 2.51 mmol) in
AcOH/MeOH/THF = 1/5/1 (21 mL) was added zinc powder (2.07 g). The suspension was sonicated for
30 sec. and stirred at room temperature for 20 min. After additional sonication for 30 min and being
stirred for 2h, the mixture was filtered through cerite pad. To the filtrate was added saturated aqueous
NaHCO3 (20 mL) and then the mixture was extracted with AcOEt. Combined extracts were washed with
saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo to give crude amine.
To the solution of the amine, (R)-3-benzyloxytetradecanoic acid (78.8 mg, 2.36 mmol) and HOAt (440
mg, 3.23 mmol) in dry CHCl3 (22 mL) was added WSCI•HCl (620 mg, 3.23 mmol) at room temperature
under N2 atmosphere. After being stirred over night, the mixture was quenched with water (30 mL) and
extracted with CHCl3. Combined organic layer was washed with water, saturated aqueous NaHCO3 and
brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica-gel column
chromatography (80 g, toluene/AcOEt = 50/1) to give 9 as a white solid (1.84 g, 78%). MALDI-TOF
(positive), m/z 962.7 [(M+Na)+]. 1H NMR (400 MHz, CDCl3) δ 7.42-7.20 (m, 15H, OCH2Ph x 2, PhCH),
5.37-5.34 (d, JN, 2 = 10.1 Hz, 1H, NH), 5.78-5.68 (m, 1H, OCH2CH=CH2), 5.46 (s, 1H, PhCH), 5.41-5.36
(dd, J3,2 = 10.1 Hz, J3,4 = 9.9 Hz, 1H, H-3), 5.22-5.17 (dd, Jtrans = 17.2 Hz, Jgem = 1.5 Hz, 1H,
OCH2CH=CH2), 5.14-5.10 (dd, Jcis = 10.4 Hz, Jgem = 1.4 Hz, 1H, OCH2CH=CH2), 4.80-4.79 (d, J1,2 = 3.7
Hz, 1H, H-1), 4.56-4.48 (m, Jjem = 11.6 Hz, 3H, OCH2Ph, OCH2Ph), 4.43-4.36 (m, J2,1 = 3.7 Hz, J2,3 = 10.5,
J2,N = 9.6 Hz, Jjem = 11.6 Hz, 2H, H-2, OCH2Ph), 4.06-4.00 (dd, Jjem = 12.7 Hz, Jvic = 5.5 Hz, 1H,
OCH2CH=CH2), 3.94-3.88 (ddd, J5, 4 = 9.8 Hz, J5, 6a = 4.6 Hz, J5, 6b = 9.8 Hz, 1H, H-5), 3.84-3.68 (m, 5H,
OCH2CH=CH2, C3H of acyl x 2, H-6b, H-4), 2.70-2.64 (m, Jjem = 15.3 Hz, Jvic = 6.3 Hz, 1H, C2H2 of acyl),
2.44-2.39 (dd, Jjem = 15.3 Hz, Jvic = 6.1 Hz, 1H, C2H2 of acyl), 2.37-3.27 (m, 2H, C2H2 of acyl), 1.61-1.20
(m, 40H, C4-13H2 of acyl x 2), 0.90-0.86 (m, 3H, C14H3 of acyl x 2). Anal. Calcd for C87H119N2O17P: C,
25
74.09; H, 9.11; N, 1.49%. Found: C, 73.81; H, 9.06; N, 1.50. [α]D = +36.3 (c 1.00, CHCl3).
Formylmethyl
4,6-O-benzylidene-3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-benzyloxytetra-
decanoylamino)-2-deoxy-α-D-glucopyranoside (10). To a solution of 9 (126 mg, 0.134 mmol) in

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HETEROCYCLES, Vol. 69, 2006
THF/t-BuOH/H2O = 10/10/1 (20.1 mL) were added 4-methylmorpholine N-oxide (64.7 mg, 0.536 mmol)
and OsO4 (1 M, 26.9 mL, 26.9 mmol) at room temperature. After being stirred for 8 h, the reaction was
quenched with 10% aqueous Na2S2O3. The mixture wasextracted with AcOEt, washed with 10% aque-
ous Na2S2O3, and concentrated in vacuo to give the crude diol. To the solution of the diol in anhydrous
benzene (1.2 mL) was added Pb(OAc)4 (93%, 76.8 mg, 0.161 mmol) at room temperature under N2 at-
mosphere. After being stirred for 1 h, the mixture was filtrated through cerite pad and concentrated in
vacuo. The residue was purified by silica gel column chromatography (10 g, CHCl3/acetone = 20/1) to
give 10 as a white solid (109 mg, 86%). 1H NMR (400 MHz, CDCl3) δ 9.41 (s, 1H, CHO), 7.37-7.23 (m,
15H, OCH2Ph x 2, CHPh), 6.58-6.55 (d, JN, 2 = 9.5 Hz, NH), 5.45-5.35 (m, 2H, CHPh, H-3), 4.73-4.72 (d,
J1,2 = 3.5 Hz, 1H, H-1), 4.58-4.37 (m, 5H, OCH2Ph x 2, H-2), 4.24-4.20 (dd, J6a,5 = 4.9 Hz, J6a,6b = 10.2 Hz,
1H, H-6a), 3.94-3.67 (m, 7H, H-5, CH2 of formylmethyl, C3H of acyl x 2, H-6b, H-4), 2.70-2.65 (dd, Jjem
= 14.8 Hz, Jvic = 6.0 Hz, 1H, C2H2 of acyl), 2.46-2.28 (m, 3H, C2H2of acyl x 3), 1.47-1.18 (m, 40H, C4-
13H2 of acyl x 2), 0.90-0.86 (t, 6H, C14H3 of acyl x 2).
Benzyloxycarbonylmethyl 4,6-O-benzylidene-3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-benzyl-
oxytetradecanoylamino)-2-deoxy-α-D-glucopyranoside (11). To a suspension of 10 (942 mg, 1.00
mmol), NaH2PO4 (120 mg, 1.0 mmol), and 2-methyl-2-butene (530 mL, 5.00 mol) in t-BuOH/H2O = 4/1
(10 mL) was added NaClO2 (80% purity, 113 mg, 1.00 mmol) at room temperature. After being stirred
over night, the mixture was neutralized with aqueous 1 M HCl and extracted with AcOEt three times.
The organic layer was washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and con-
centrated. The residue was freezed-dried from dioxane to give the crude carboxylic acid. To the suspen-
sion of the carboxylic acid in CH2Cl2 (10 mL) was added the Et2O solution of phenyl diazomethane (0.46
M) at room temperature until the suspension turned a pale red. The excess reagent was quenched with
AcOH and AcOEt was added. The organic solution was washed with water and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica-gel column chromatography (40 g, tolu-
ene/AcOEt = 10/1) to give 11 as a white solid (886 mg, 84%). MALDI-TOF (positive) m/z 1070.7
1
[(M+Na)+]. H NMR (400 MHz, CDCl3) δ 7.40-7.21 (m, 20H, OCH2Ph x 3, PhCH), 6.71-6.69 (d, JN,2 =
9.5 Hz, 1H, NH), 5.44 (s, 1H, PhCH), 5.43-5.38 ( dd, J3,2 = 9.9 Hz, J3,4 = 10.1 Hz, 1H, H-3), 5.12 (s, 2H,
COOCH2Ph), 4.80-4.79 (d, J1,2 = 3.7 Hz, 1H, H-1), 4.59-4.47 (m, 3H, OCH2Ph, OCH2Ph), 4.45-4.39 (ddd,
J2,1 = 3.7 Hz, J2,3 = 9.9 Hz, J2,N = 9.5 Hz, 1H, H-2), 4.38-4.35 (d, Jjem = 11.6 Hz, 1H, OCH2Ph), 4.22-4.18
(dd, J6a,5 = 4.9 Hz, J6a,6b = 10.4 Hz, 1H, H-6a), 4.01-3.92 (m, 3H, CH2 of CM, H-6b), 3.85-3.78 (m, 2H,
H-5, H-4), 2.70-2.64 (m, Jjem = 15.1 Hz, Jvic = 6.6 Hz, 1H, C2H2 of acyl), 2.43-2.37 (m, Jjem = 15.1 Hz, Jvic
= 5.8 Hz, 3H, C2H2 of acyl x 3), 1.48-1.43 (m, 40H, C4-13H2 of acyl x 2), 0.90-0.86 (m, 6H, C14H3 of acyl
x 2). Anal. Calcd for C87H119N2O17P: C, 73.32; H, 8.56; N, 1.34%. Found: C, 72.94; H, 5.89; N, 1.36.
[α]D25 = +34.8 (c 1.00, CHCl3).

HETEROCYCLES, Vol. 69, 2006
405
Benzyloxycarbonylmethyl
3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-benzyloxytetradecanoyl-
amino)-2-deoxy-α-D-glucopyranoside (12). To the suspension of 11 (56.0 mg, 53.4 mmol) in
CH2Cl2/H2O = 50/1 (0.25 mL) was added the CH2Cl2 solution of trifluoroacetic acid (10%, 250 mL) at
room temperature under N2 atmosphere. After being stirred for 40 min, the reaction was quenched with
aqueous saturated NaHCO3 (1 mL) and extracted with AcOEt. The organic layer was washed with water
and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica-gel column
chromatography (5 g, CHCl3/acetone = 10/1) to give 12 as a white solid (46.2 mg, 90%). MALDI-TOF
1
(positive) m/z 982.7 [(M+Na)+]. H NMR (400 MHz, CDCl3) δ 7.44-7.20 (m, 15H, OCH2Ph x 3),
6.68-6.66 (d, JN,2 = 9.5 Hz, 1H, NH), 5.16-5.11 (dd, J3,2 = 10.7 Hz, J3,4 = 9.0 Hz, 1H, H-3), 5.11 (s, 2H,
COOCH2Ph), 4.79-4.78 (d, J1,2 = 3.7 Hz, 1H, H-1), 4.55-4.50 (m, 4H, OCH2Ph x 2), 4.32-4.26 (ddd, J2,1 =
3.7 Hz, J2,3 = 10.7 Hz, J2,N = 9.5 Hz, 1H, H-2), 4.03-3.98 (d, Jjem = 16.6 Hz, 1H, CH2 of CM) , 3.97-3.93 (d,
Jjem = 16.6 Hz, 1H, CH2 of CM), 3.90-3.84 (m, 1H, C3H of acyl), 3.83-3.79 (m, 1H, C3H of acyl),
3.75-3.68 (m, 3H, H-5, H-6a, H-6b), 3.65-3.60 (dd, J4,3 = 9.3 Hz, J4,5 = 9.3 Hz, 1H, H-4), 2.64-2.59 (dd,
Jjem = 14.7 Hz, Jvic = 8.1 Hz, 1H, C2H2 of acyl), 2.48-2.44 (dd, Jjem = 14.7 Hz, Jvic = 4.6 Hz, 1H, C2H2 of
acyl), 2.38-2.37 (d, Jvic = 5.9 Hz, 2H, C2H2 of acyl), 1.65-1.42 (m, 2H, C4H2 of acyl x 2), 1.30-0.90 (m,
36H, C5-13H2 of acyl x 2), 0.88-0.86 (t, Jvic = 6.1 Hz, 6H, C14H3 of acyl x 2). Anal. Calcd for
C87H119N2O17P: C, 71.29; H, 8.92; N, 1.46%. Found: C, 70.92 ; H, 8.94; N, 1.51. [α]D25 = +34.2 (c 1.00,
CHCl3).
Allyl
6-O-benzyl-3-O-((R)-3-benzyloxytetradecanoyl)-2-deoxy-2-(2,2,2-trichloroethoxycarboyl-
amino)-α-D-glucopyranoside (13). To a solution of 8 (2.00 g, 2.56 mmol) in dry CH2Cl2 (24 mL) were
added triethylsilane (4.8 mL, 30.0 mmol) and boron trifruoride diethyl etherate (614 mL, 5.0 mmol) at 0
˚C under N2 atmosphere. After being stirred for 5 h at room temperature, the reaction was quenched with
saturated aqueous NaHCO3 (20 mL) and extracted with AcOEt. 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 column chromatography (180 g, toluene/AcOEt = 15/1) to give 13 as colorless oil
(1.70 g, 85%). A part of the product was acetylated for NMR analysis. MALDI-TOF (positive) m/z 822.3
[(M+Na)+]. 1H NMR (400 MHz, CDCl3) of 4-O-acetylate: δ 7.35-7.22 (m, 10H, OCH2Ph x 2), 5.95-5.85
(m, 1H, OCH2CH=CH2), 5.34-5.29 (dd, J3,2 = 11.0 Hz, J3,4 = 9.6 Hz, 1H, H-3), 5.33-5.28 (dd, Jjem = 1.4 Hz,
Jvic = 17.1 Hz, 1H, OCH2CH=CH2), 5.27-5.25 (d, JN,2 = 10.1 Hz, 1H, NH), 5.25-5.22 (dd, Jjem = 1.2 Hz, Jvic
= 10.7 Hz, 1H, OCH2CH=CH2), 5.17-5.12 (dd, J4,3 = 9.7 Hz, J4,5 = 9.9 Hz, 1H, H-4), 4.96-4.95 (d, J1,2 =
3.7 Hz, 1H, H-1), 4.70-4.67 (d, Jjem = 12.1 Hz, 1H, CH2 of Troc), 4.60-4.58 (d, Jjem = 12.1 Hz, 1H, CH2 of
Troc), 4.57-4.56 (m, 4H, OCH2Ph x 2), 4.24-4.19 (dd, Jjem = 11.4 Hz, Jvic = 5.3 Hz, 1H, OCH2CH=CH2),
4.08-4.00 (m, Jjem = 11.4 Hz, Jvic = 6.1 Hz, 2H, OCH2CH=CH2, H-2), 3.98-3.93 (ddd, J5,4 = 10.1 Hz, J5,6a =
3.7 Hz, J5,6b = 4.1 Hz, 1H, H-5), 3.83-3.77 (m, 1H, C3H of acyl), 3.56-3.49 (m, Jjem = 2.1 Hz, J6a,5 = 3.7 Hz,

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HETEROCYCLES, Vol. 69, 2006
J6b,5 = 4.1 Hz, 2H, H-6a, H-6b), 2.59-2.53 (dd, Jjem = 15.7 Hz, Jvic = 7.0 Hz, 1H, C2H2 of acyl), 2.45-2.40
(dd, Jjem = 15.7 Hz, Jvic = 5.2 Hz, 1H, C2H2 of acyl), 1.75 (s, 3H, CH3 of Ac), 1.71-1.38 (m, 2H, C4H2 of
acyl), 1.32-1.25 (m, 18H, C5-13H2 of acyl), 0.90-0.86 (t, Jvic = 6.7 Hz, 3H, C14H3 of acyl). Anal. Calcd for
C87H119N2O17P: C, 59.96; H, 7.04; N, 1.75%. Found: C, 59.97; H, 7.07; N, 1.78. [α]D25 = +36.7 (c 1.00,
CHCl3).
Allyl
6-O-benzyl-4-O-benzyloxycarbonylmethyl-3-O-((R)-3-benzyloxytetradecanoyl)-2-deoxy-2-
(2,2,2-trichloroethoxycarboylamino)-α-D-glucopyranoside (14). To the suspension of 13 (335 mg,
0.418 mmol), silver oxide (484 mg, 2.09 mmol) and MS4A in anhydrous toluene (4.2 mL) was added
benzyl iodoacetate (571 mg, 2.09 mmol) at room temperature under N2 atmosphere. After being stirred
for 10 d, AcOEt was added to the reaction mixture and the insoluble materials were removed by filtration.
The filtrate was washed with water and brine, dried over MgSO4, and concentrated in vacuo. The residue
was purified by silica-gel column chromatography (80 g, toluene/AcOEt = 20/1) and by recycling HPLC
to give 14 as a colorless oil (282 mg, 79%). MALDI-TOF (positive) m/z 970.4 [(M+Na)+]. 1H NMR (400
MHz, CDCl3) δ 7.34-7.20 (m, 15H, OCH2Ph x 3), 5.91-5.81 (m, 1H, OCH2CH=CH2), 5.39-5.34 (dd, J3,2
= 10.4 Hz, J3,4 = 9.5 Hz, 1H, H-3), 5.31-5.25 (m, JN,2 = 10.1 Hz Jjem = 1.5 Hz, Jvic = 17.2 Hz, 2H, NH,
OCH2CH=CH2), 5.24-5.18 (dd, Jjem = 1.3 Hz, Jvic = 10.3 Hz, 1H, OCH2CH=CH2), 5.06-5.03 (d, Jjem = 12.3
Hz, 1H, COOCH2Ph), 5.02-4.99 (d, Jjem = 12.3 Hz, 1H, COOCH2Ph), 4.90-4.89 (d, J1,2 = 3.5 Hz, 1H, H-1),
4.69-4.44 (m, 6H, OCH2Ph x 2, CH2 of Troc), 4.22-4.04 (m, Jjem of CM = 16.1 Hz, Jjem = 12.6 Hz, Jvic = 5.3
Hz, 3H, CH2 of CM, OCH2CH=CH2), 4.01-3.97 (dd, Jjem = 12.6 Hz, Jvic = 6.2 Hz, 1H, OCH2CH=CH2),
3.96-3.82 (m, 4H, H-2, H-5, H-6a, C3H of acyl), 3.74-3.67 (m, J4,3 = 9.5 Hz, 2H, H-4, H-6b), 2.64-2.58
(dd, Jjem = 15.8 Hz, Jvic = 7.1 Hz, 1H, C2H2 of acyl), 2.46-2.41 (dd, Jjem = 15.8 Hz, Jvic = 5.1 Hz, 1H, C2H2of
acyl), 1.56-1.45 (m, 2H, C4H2 of acyl), 1.30-1.25 (m, 18H, C5-13H2 of acyl), 0.90-0.86 (t, Jvic = 6.8 Hz, 3H,
C14H3 of acyl). Anal. Calcd for C87H119N2O17P: C, 61.99; H, 6.79; N, 1.48%. Found: C, 61.91; H, 6.75; N,
1.47.
6-O-Benzyl-4-O-benzyloxycarbonylmethyl-3-O-((R)-3-benzyloxytetradecanoyl)-2-deoxy-2-(2,2,2-
trichloroethoxycarboylamino)-α-D-glucopyranose (15). To a degassed solution of 14 (606 mg, 0.638
mmol) in anhydrous THF (4 mL) was added [bis(methyldiphenylphosphine)](1,5-cyclooctadiene)-
iridium(I) hexafluorophosphate (13.4 mg, 15.8 mmol). After activation of the iridium catalyst with hy-
drogen three times (each 30 sec), the mixture was stirred under nitrogen atmosphere at room temperature
for 30 min. To the mixture were added successively H2O (5 mL) and iodine (335 mg, 1.32 mmol), and
the mixture was stirred for additional 30 min. After quenching with aqueous 10% Na2S2O3 (8 mL), the
mixture was extracted with AcOEt. Combined extracts were washed with aqueous 10% Na2S2O3 and
brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by silica-gel column
chromatography (40 g, toluene/AcOEt = 7/1) to give 15 as a white solid (575 mg, 99%). MALDI-TOF

HETEROCYCLES, Vol. 69, 2006
407
1
(positive) m/z 930.4 [(M+Na)+]. H NMR (400 MHz, CDCl3) for α-anomer: δ 7.36-7.19 (m, 15H,
OCH2Ph x 3), 5.45-5.39 (m, JN,2 = 9.5 Hz, J3,2 = 11.0 Hz, J3,4 = 9.5 Hz, 2H, NH, H-3), 5.26-5.26 (d, J1,2 =
3.2 Hz, 1H, H-1), 5.07-5.04 (d, Jjem = 12.2 Hz, 1H, COOCH2Ph), 5.03-4.99 (d, Jjem = 12.2 Hz, 1H,
COOCH2Ph), 4.66-4.45 (m, 6H, CH2 of Troc, OCH2Ph x 2), 4.21-4.17 (m, Jjem of CM = 16.4 Hz, 1H, CH2 of
CM), 4.14-4.11 (ddd, J5,4 = 10.0 Hz, J5,6a = 4.2 Hz, J5,6b = 1.7 Hz, 1H, H-5), 4.10-4.06 (m, Jjem of CM = 16.4
Hz, 1H, CH2 of CM), 3.92-3.85 (m, 2H, H-2, C3H of acyl), 3.84-3.80 (dd, J6a,5 = 4.2 Hz, J6a,6b = 11.0 Hz,
1H, H-6a), 3.77-3.74 (dd, J6b,5 = 1.7 Hz, J6b,6a = 11.0 Hz, 1H, H-6b), 3.66-3.61 (m, J4,3 = 9.5 Hz, J4,5 = 10.0
Hz, 2H, H-4,), 3.16 (s, 1H, OH-1), 2.64-2.58 (dd, Jjem = 15.9 Hz, Jvic = 7.6 Hz, 1H, C2H2 of acyl),
2.46-2.41 (dd, Jjem = 15.9 Hz, Jvic = 5.1 Hz, 1H, C2H2 of acyl), 1.53-1.45 (m, 2H, C4H2 of acyl), 1.32-1.25
(m, 18H, C5-13H2 of acyl), 0.90-0.86 (t, Jvic = 7.1 Hz, 3H, C14H3 of acyl). Anal. Calcd for C87H119N2O17P: C,
60.76; H, 6.65; N, 1.54%. Found: C, 60.31; H, 6.58; N, 1.55.
Benzyloxycarbonylmethyl
6-O-[6-O-benzyl-4-O-benzyloxycarbonylmethyl-3-O-((R)-3-benzyloxy-
tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-deoxy-β-D-glucopyranosyl]-3-O-((R)-3-
benzyloxytetradecanoyl)-2-deoxy-2-(2,2,2-trichloroethoxycarboylamino)-α-D-glucopyranoside (17).
To a suspension of 15 (341 mg, 0.375 mmol) and MS4A in dry CH2Cl2 (3.7 mL) were added
trichloroacetonitrile (376 mL, 3.75 mmol) and cesium carbonate (61.3 mg, 0.188 mmol) at room tem-
perature under N2 atmosphere. After being stirred for 1 h, the reaction was quenched with aqueous satu-
rated NaHCO3 (10 mL) and extracted with AcOEt. The organic layer was washed with aqueous saturated
NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo to give the crude imidate (16) (396 mg,
quant.). To the suspension of 16 and the glycosyl acceptor (12) (293 mg, 0.306 mmol) and MS4A in dry
CH2Cl2 (4 mL) was added trimethylsilyl triflate (11.9 mL, 61.2 mmol) at -20 ˚C under N2 atmosphere.
After being stirred for 10 min, the mixture was quenched by addition of aqueous saturated NaHCO3 (10
mL) and extracted with AcOEt. The organic layer was washed with aqueous saturated NaHCO3 and brine,
dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica-gel column chroma-
tography (30 g, toluene/AcOEt = 7/1) to give 17 as a white solid (427 mg, 71%). MALDI-TOF (positive)
m/z 1873.4 [(M+Na)+]. 1H NMR (400 MHz, CDCl3) δ 7.37-7.20 (m, 15H, OCH2Ph x 6), 6.61-6.59 (d, JN,2
= 9.3 Hz, 1H, NH), 5.22-5.18 (m, 2H, N’H, H-3’), 5.15-5.07 (m, J3,2 = 10.5 Hz, J3,4 = 9.3 Hz, Jjem = 11.9
Hz, 3H, H-3, COOCH2Ph), 5.03-5.00 (d, Jjem = 12.5 Hz, 1H, COOCH2Ph’), 4.76-4.75 (d, J1,2 = 3.5 Hz, 1H,
H-1), 4.68-4.43 (m, J1’,2’ = 8.5 Hz, 11H, CH2 of Troc, H-1’, OCH2Ph x 4), 4.30-4.24 (ddd, J2,1 = 3.7 Hz, J2,
3 = 10.8 Hz, J2, N = 9.3 Hz, 1H, H-2), 4.18-4.14 (m, Jjem = 16.2 Hz, 1H, CH2 of 4-O-carboxymethyl(CM’)),
4.09-4.05 (m, Jjem = 16.2 Hz, 1H, CH2 of CM’), 4.02-3.98 (m, Jjem = 16.8 Hz, 2H, H-6a, CH2 of CM),
3.96-3.92 (m, Jjem of CM = 16.8 Hz, 1H, CH2 of CM), 3.83-3.80 (m, 6H, H-5, H-6a, H-6a’, C3H of acyl x 3),
3.73-3.69 (dd, J6b,5 = 5.0 Hz, J6b,6a = 10.8 Hz, 1H, H-6b), 3.64-3.52 (m, 4H, H-4, H-4’, H-2’, H-5’),
2.65-2.56 (m, Jjem = 15.3 Hz, Jjem = 15.4 Hz , Jvic = 7.8 Hz, Jvic = 7.0 Hz, 2H, C2H2 of acyl x 2), 2.50-2.42

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HETEROCYCLES, Vol. 69, 2006
(m, Jjem = 15.4 Hz, Jjem = 15.3 Hz, Jvi c= 5.0 Hz, Jvic = 4.9 Hz, 2H, C2H2 of acyl x 2), 2.35-2.31 (m, 2H,
C2H2 of acyl), 1.59-1.48 (m, 6H, C4H2 of acyl x 3), 1.30-1.25 (m, 54H, C5-13H2 of acyl x 3), 0.90-0.86 ( t,
Jvic = 7.1 Hz, 9H, C14H3 of acyl x 3). Anal. Calcd for C87H119N2O17P: C, 66.81; H, 7.78; N, 1.51%. Found:
C, 66.63; H, 7.76; N, 1.58. [α]D25 = +20.9 (c 1.00, CHCl3).
Benzyloxycarbonylmethyl
6-O-[6-O-benzyl-4-O-benzyloxycarbonylmethyl-3-O-((R)-3-benzyloxy-
tetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-deoxy-β-D-glucopyranosyl]-3-O-((R)-3-
benzyloxytetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-deoxy-α-D-glucopyranoside
(18). To the solution of 17 (281 mg, 0.152 mmol) in AcOH (3 mL) was added zinc-copper couple [Zn
(200 mg) was activated with aqueous 10% CuSO4 solution]. The mixture was stirred for 30 min and
AcOEt was added. After the in soluble materials were removed by filtration, the filtrate was washed with
saturated aqueous NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo. To the solution of
the residue, (R)-3-benzyloxytetradecanoic acid (62.8 mg, 0.182 mmol), and HOAt (31.0 mg, 28.0 mmol)
in dry CHCl3 (2 mL) was added WSCI･HCl (43.7 mg, 0.228 mmol) at room temperature under N2 at-
mosphere. After being stirred for 2 d, the mixture was purified by silica-gel column chromatography (40
g, CHCl3/ acetone = 20/1) and recycling HPLC to give 18 as a white solid (137 mg, 45%). MALDI-TOF
1
(positive) m/z 2013.98 [(M+Na)+]. H NMR (400 MHz, CDCl3) δ 7.37-7.20 (m, 15H, OCH2Ph x 7),
6.62-6.60 (d, JN,2 = 9.3 Hz, 1H, NH), 6.29-6.27 (d, JN’,2’ = 9.3 Hz, 1H, N’H), 5.17-5.12 (dd, J3,2 = 10.5 Hz,
J3,4 = 9.3 Hz, 1H, H-3), 5.08 ( s, 1H, COOCH2Ph), 5.06-4.97 (m, 3H, COOCH2Ph’, H-3’), 4.74-4.73 (d,
J1,2 = 3.7 Hz, 1H, H-1), 4.54-4.39 (m, 10H, OCH2Ph x 5), 4.29-4.23 (m, J2,1 = 3.7 Hz, J2,3 = 10.7 Hz, J1’,2’
=
8.3 Hz, 2H, H-2 , H-1’), 4.15-4.11 (m, Jjem = 16.4 Hz, 1H, CH2 of CM’), 4.04-4.00 (m, Jjem = 16.4 Hz, 1H,
CH2 of CM’), 3.97-3.62 (m, 12H, CH2 of CM, H-2’, C3H of acyl x 3, H-6a, H-5, H-6a’, H-6b’, H-6b,
H-4’), 3.60-3.55 (dd, J4, 3 = 9.0 Hz, J4, 5 = 9.0 Hz, 1H, H-4), 3.48-3.44 (m, 1H, H-5’), 2.64-2.54 (m, Jjem =
15.6 Hz, Jjem = 15.6 Hz , Jvic = 7.3 Hz, Jvic = 7.6 Hz, 2H, C2H2 of acyl x 2), 2.46-2.40 (m, 2H, C2H2 of acyl
x 2), 2.39-2.17 (m, 4H, C2H2 of acyl x 2), 1.59-1.24 (m, 80H, C5-13H2 of acyl x 4), 0.90-0.86 ( t, Jvic= 6.8
Hz, 12H, C14H3 of acyl x 4). Anal. Calcd for C87H119N2O17P: C, 72.93; H, 8.80; N, 1.41%. Found: C,
72.32; H, 8.78; N, 1.43. [α]D25 = +16.4 (c 1.00, CHCl3).
Carboxymethyl 6-O-[4-O-carboxymethyl-3-O-((R)-3-hydroxytetradecanoyl)-2-((R)-3-hydroxytetra-
decanoylamino)-2-deoxy-β-D-glucopyranosyl]-3-O-((R)-3-hydroxytetradecanoyl)-2-((R)-3-hydroxy-
tetradecanoylamino)-2-deoxy-α-D-glucopyranoside (Bis-CM-406, 6). To a solution of 18 (61.5 mg,
30.9 mmol) in distilled THF (0.7 mL) was added Pd-black (90.3 mg). The mixture was stirred under 7
kg/cm2 of hydrogen at room temperature overnight. After removal of the Pd catalyst by filtration, the so-
lution was concentrated in vacuo. To the residue was added distilled water and t-BuOH and the suspen-
sion was lyophilized to give Bis-CM-406 (6) as a white powder (41.5 mg, quant.). ESI-MS (negative)
m/z 1360.04[(M-H)-], 679.45 [(M-2H)2-]. 1H NMR (500 MHz, CDCl3/CD3OD = 1/1) δ 5.16-5.10 (m, J3’,2’

HETEROCYCLES, Vol. 69, 2006
409
= 10.5 Hz, J3’,4’ = 9.1 Hz J3,2 = 10.5 Hz, J3,4 = 9.3 Hz, 2H, H-3’, H-3), 4.70-4.69 (d, J1,2 = 3.5 Hz, 1H, H-1),
4.12-4.07 (m, Jjem of CM = 16.3 Hz, J2,1 = 3.7 Hz, J2,3 = 10.6 Hz, 2H, CH2 of CM’, H-2), 4.05-4.01 (m, Jjem of
CM = 15.8 Hz, Jjem of CM’ = 16.3 Hz, 2H, CH2 of CM’, CH2 of CM), 4.00-3.98 (m, J6a,6b = 10.8 Hz, 1H, H-6a),
3.94-3.78 (m, Jjem of CM’ = 16.3 Hz, J2’,1’ = 8.5 Hz, J2’,3’ = 10.6 Hz, 7H, C3H of acyl x 4, CH2 of CM, H-6a’,
H-2’), 3.76-3.71 (m, 2H, H-6b’, H-5), 3.70-3.67 (dd, J6b,5 = 4.5 Hz , J6b,6a = 10.8 Hz, 1H, H-6b), 3.60-3.56
(dd, J4’,3’ = 9.3 Hz, J4’,5’ = 9.3 Hz, 1H, H-4’), 3.55-3.51 (dd, J4,3 = 9.5 Hz, J4,5 = 9.6 Hz, 1H, H-4), 3.35-3.32
(m, J5’,4’ = 9.4 Hz, 1H, H-5’), 2.45-2.10 (m, 8H, C2H2 of acyl x 4), 1.37-1.11 (m, 80H, C4-13H2 of acyl x 4),
0.81-0.78 (t, 12H, C14H3 of acyl x 4).
Allyl
6-O-benzyl-4-O-benzyloxycarbonylmethyl-3-O-((R)-3-hydroxytetradecanoyl)-2-deoxy-2-
(2,2,2-trichloroethoxycarboylamino)-α-D-glucopyranoside (19). To a suspension of 14 (1.38 g, 1.45
mmol) in CH2Cl2/H2O = 20/1 (14.5 mL) was added DDQ (330 mg, 1.45 mmol) at room temperature. Af-
ter being stirred for 1 d, the reaction was quenched with aqueous 10% Na2S2O3 (10 mL) and extracted
with CH2Cl2. Combined extracts were washed with aqueous saturated NaHSO4, water, and brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified by silica-gel column chromatography
(40 g, toluene/AcOEt = 20/1) to give 19 as colorless oil (601 mg, 48%). MALDI-TOF (positive) m/z
1
880.32 [(M+Na)+]. H NMR (400 MHz, CDCl3) δ 7.38-7.23 (m, 10H, OCH2Ph x 2), 5.92-5.82 (m, 1H,
OCH2CH=CH2), 5.40-5.35 (dd, J3,2 = 10.3 Hz, J3,4 = 9.9 Hz, 1H, H-3), 5.34-5.31 (d, JN,2 = 10.1 Hz, 2H,
NH), 5.30-5.26 (dd, Jgem = 1.5 Hz, Jtrans = 17.3 Hz, OCH2CH=CH2), 5.23-5.20 (dd, Jgem = 1.5 Hz, Jcis = 10.3
Hz, 1H, OCH2CH=CH2), 5.16-5.12 (d, Jjem = 12.2 Hz, 1H, COOCH2Ph), 5.12-5.09 (d, Jjem = 12.2 Hz, 1H,
COOCH2Ph), 4.90-4.90 (d, J1,2 = 3.4 Hz, 1H, H-1), 4.73-4.70 (d, Jjem = 12.0 Hz, 1H, OCH2Ph), 4.68-4.65
(d, Jjem = 12.0 Hz, 1H, OCH2Ph), 4.63-4.60 (d, Jjem = 12.0 Hz, 1H, CH2 of Troc), 4.50-4.46 (d, Jjem = 12.0
Hz, 1H, CH2 of Troc), 4.23- 4.15 (m, Jjem of CM = 16.1 Hz, Jjem = 12.7 Hz, Jvic = 5.4 Hz, 2H, CH2 of CM,
OCH2CH=CH2), 4.13-4.09 (d, Jjem = 16.1 Hz, 1H, CH2 of CM), 4.04-3.94 (m, 3H, OCH2CH=CH2, H-2,
C3H of acyl), 3.89-3.81 (m, J5, 4 = 10.5 Hz, J5, 6a = 3.2 Hz, J5, 6b = 1.5 Hz, J6a, 5 = 3.2 Hz, 2H, H-5, H-6a),
3.74-3.70 (m, 2H, H-4, H-6b), 2.81-2.80 (d, JOH, C3H = 3.9 Hz, 1H, C3OH of acyl), 2.48-2.44 (dd, Jjem =
16.1 Hz, Jvic = 2.7 Hz, 1H, C2H2 of acyl), 2.40-2.34 (dd, Jjem = 16.3 Hz, Jvic = 9.4 Hz, 1H, C2H2 of acyl),
1.49-1.26 (m, 20H, C4-13H2 of acyl), 0.90-0.86 (t, Jvic= 7.1 Hz, 3H, C14H3 of acyl). Anal. Calcd for
C87H119N2O17P: C, 58.71; H, 6.80; N, 1.63%. Found: C, 58.56; H, 6.77; N, 1.62. [α]D25 = +53.9 (c 1.00,
CHCl3).
Allyl
6-O-benzyl-4-O-benzyloxycarbonylmethyl-3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2-
deoxy-2-(2,2,2-trichloroethoxycarboylamino)-α-D-glucopyranoside (20). To the solution of 19 (520
mg, 0.605 mmol) and tetradecanoic acid (276 mg, 1.21 mmol) in dry CH2Cl2 (6 mL) were added DCC
(312 mg, 1.51 mmol) and DMAP (14.8 mg, 0.121 mmol) at room temperature under N2 atmosphere. Af-
ter being stirred for 4 h, the mixture was filtered through silica gel pad and concentrated in vacuo. The

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HETEROCYCLES, Vol. 69, 2006
residue was purified by silica-gel column chromatography (10 g, toluene/AcOEt = 20/1) and recycling
1
HPLC to give 20 as a white solid (636 mg, 98%). MALDI-TOF (positive) m/z 1090.70 [(M+Na)+]. H
NMR (400 MHz, CDCl3) δ 7.34-7.23 (m, 10H, OCH2Ph x 2), 5.89-5.79 (m, 1H, OCH2CH=CH2),
5.35-5.29 (m, JN,2 = 9.7 Hz, 2H, NH, H-3), 5.28-5.23 (dd, Jgem = 1.4 Hz, Jtrans = 17.3 Hz, OCH2CH=CH2),
5.20-5.14 (dd, Jgem = 1.4 Hz, Jcis = 10.2 Hz, 2H, OCH2CH=CH2, C3H of acyl), 5.12 (s, 2H, COOCH2Ph),
4.89-4.89 (d, J1,2 = 3.6 Hz, 1H, H-1), 4.73-4.70 (d, Jjem = 12.0 Hz, 1H, OCH2Ph), 4.66-4.63 (d, Jjem = 12.0
Hz, 1H, OCH2Ph), 4.61-4.58 (d, Jjem = 12.0 Hz, 1H, CH2 of Troc), 4.48-4.45 (d, Jjem = 12.0 Hz, 1H, CH2
of Troc), 4.26-4.22 (d, Jjem = 16.0 Hz, 1H, CH2 of CM), 4.17-4.09 (m, Jjem = 12.8 Hz, Jvic = 5.3 Hz, Jjem of
= 16.0 Hz, 2H, OCH2CH=CH2, CH2of CM), 3.99-3.94 (dd, Jjem = 12.7 Hz, Jvic = 6.3 Hz, 1H,
CM
OCH2CH=CH2), 3.93-3.82 (m, 3H, H-2, H-6a, H-6b), 3.72-3.66 (m, 2H, H-4, H-5), 2.62-2.56 (dd, Jjem =
16.2 Hz, Jvic = 7.2 Hz, 1H, C2H2 of acyl), 2.53-2.57 (dd, Jjem = 16.2 Hz, Jvic = 5.4 Hz, 1H, C2H2 of acyl),
2.24-2.21 (t, Jvic = 6.6 Hz, 2H, C2’H2 of acyl), 1.55-1.25 (m, 42H, C4-13H2 of acyl, C3’-13’H2 of acyl),
0.89-0.86 (t, Jvic = 6.6 Hz, 6H, C14H3 of acyl, C14’H3 of acyl). Anal. Calcd for C87H119N2O17P: C, 62.88; H,
7.92; N, 1.31%. Found: C, 62.82; H, 7.92; N, 1.27. [α]D25 = +55.0 (c 1.00, CHCl3).
6-O-Benzyl-4-O-benzyloxycarbonylmethyl-3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-2-deoxy-2-
(2,2,2-trichloroethoxycarboylamino)-α-D-glucopyranose (21). To a degassed solution of 20 (518 mg,
0.484 mmol) in anhydrous THF (3 mL) was added [bis(methyldiphenylphosphine)](1,5-cyclooctadiene)-
iridium(I) hexafluorophosphate (13.4 mg, 15.8 mmol). After activation of the iridium catalyst with hy-
drogen three times (each 30 sec), the mixture was stirred under nitrogen atmosphere at room temperature
for 30 min. To the mixture were added successively H2O (4 mL) and iodine (305 mg, 1.20 mmol), and
stirred for additional 25 min. After the reaction was quenched with aqueous 10% Na2S2O3 (10 mL), the
mixture was extracted with AcOEt. Combined extracts were washed with aqueous 10% Na2S2O3, aque-
ous saturated NaHCO3, and brine, dried over Na2SO4, and concentrated in vacuo. The residue was puri-
fied by silica-gel column chromatography (40 g, toluene/AcOEt = 10/1) to give 21 as a white solid (417
1
mg, 84%). MALDI-TOF (positive) m/z 1050.05 [(M+Na)+]. H NMR (400 MHz, CDCl3) for α-anomer:
δ 7.38-7.22 (m, 10H, OCH2Ph x 2), 5.53-5.50 (d, JN,2 = 9.8 Hz, 1H, NH), 5.39-5.34 (dd, J3,2 = 10.7 Hz, J3,4
= 9.5 Hz, 1H, H-3), 5.28-5.26 (dd, J1,2 = 3.7 Hz, J1,OH = 3.4 Hz, 1H, H-1), 5.17-5.12 (m, 3H, C3H of acyl,
COOCH2Ph), 4.69 (s, 2H, OCH2Ph), 4.59-4.56 (d, Jjem = 11.9 Hz, 1H, CH2 of Troc), 4.49-4.46 (d, Jjem =
11.9 Hz, 1H, CH2 of Troc), 4.26-4.08 (m, Jjem = 16.2 Hz, J5,4 = 9.9 Hz, J5,6a = 4.3 Hz, J5,6b = 1.8 Hz, 3H,
CH2 of CM, H-5), 3.90-3.85 (m, 1H, H-2), 3.82-3.78 (dd, J6a,5 = 4.3 Hz, J6a,6b = 11.0 Hz, 1H, H-6a),
3.75-3.73 (m, 1H, H-6b), 3.66-3.61 (dd, J4,3 = 9.5 Hz, J4,5 = 9.8 Hz, 1H, H-4), 3.29-3.29 (d, JOH,1= 3.4 Hz,
1H, OH-1), 2.62-2.56 (dd, Jjem = 16.2 Hz, Jvic = 7.3 Hz, 1H, C2H2 of acyl), 2.55-2.47 (dd, Jjem = 16.2 Hz,
Jvic = 5.2 Hz, 1H, C2H2 of acyl), 2.25-2.21 (t, Jvic = 7.9 Hz, 2H, C2’H2 of acyl), 1.56-1.55 (m, 4H, C4H2 of
acyl, C3’H2 of acyl), 1.25-1.21 (m, 38H, C5-13H2 of acyl, C4’-13’H2 of acyl), 0.90-0.86 (t, 6H, C14H3 of acyl,

HETEROCYCLES, Vol. 69, 2006
411
C14’H3 of acyl). Anal. Calcd for C87H119N2O17P: C, 61.83; H, 7.83; N, 1.36%. Found: C, 61.82; H, 7.80; N,
1.36.
Benzyloxycarbonylmethyl
6-O-[6-O-benzyl-4-O-benzyloxycarbonylmethyl-3-O-[(R)-3-(tetrade-
canoyloxy)tetradecanoyl]-2-deoxy-2-(2,2,2-trichloroethoxycarboylamino)-β-D-glucopyranosyl]-3-
O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-deoxy-α-D-gluco-
pyranoside (23). To a suspension of 21 (37.9 mg, 36.8 µmol) in anhydrous CH2Cl2 (0.4 mL) and MS4A
were added trichloroacetonitrile (36.9 µL, 0.368 mmol) and cesium carbonate (6.0 mg, 18.4 µmol) at
room temperature under argon atmosphere. After being stirred for 1 h, the mixture was filtered with sili-
ca gel and concentrated in vacuo to give the imidate (22) (44 mg, quant.), which was subjected to the
following glycosylation without further purification. To the suspension of imidate (22), the glycosyl ac-
ceptor (12) (23.7 mg, 24.7 µmol) and MS4A in anhydrous CH2Cl2 (0.3 mL) was added trimethylsilyl tri-
flate (1.0 µL, 4.9 µmol) at -15 ˚C under argon atmosphere. After stirring for 15 min, the mixture was
quenched with aqueous saturated NaHCO3 (1.5 mL) and extracted with AcOEt. Combined extracts were
washed with aqueous saturated NaHCO3 and brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica-gel column chromatography (30 g, CHCl3/acetone = 20/1) to give 23 as a
1
white solid (45.2 mg, 93%). MALDI-TOF (positive) m/z 1991.89 [(M+Na)+]. H NMR (400 MHz,
CDCl3) δ 7.38- 7.19 (m, 25H, OCH2Ph x 5), 6.65- 6.63 (d, JN,2 = 9.3 Hz, 1H, NH), 5.45- 5.43 (d, JN’,2’
=
8.5 Hz, 1H, N’H), 5.20- 5.06 (m, 7H, C3H, H-3, H-3’, COOCH2Ph x 2), 4.76- 4.74 (m, J1,2 = 3.7 Hz, 2H,
H-1, OCH2Ph), 4.68- 4.44 (m, J1’,2’ = 9.2 Hz Jjem of Troc = 12.1 Hz, 8H, H-1’, OCH2Ph, OCH2Ph x 2, CH2 of
Troc), 4.29- 4.33 (ddd, J2,1 = 3.7 Hz, J2,3 = 10.7 Hz, J2,N = 9.5 Hz, 1H, H-2), 4.23-4.19 (d, Jjem = 16.2 Hz,
1H, CH2 of CM), 4.14-4.10 (d, Jjem = 16.2 Hz, 1H, CH2 of CM), 4.02-3.98 (m, Jjem of CM = 16.9 Hz, J6a,5 =
2.7 Hz, 2H, CH2 of CM, H-6a), 3.97-3.93 (d, Jjem = 16.9 Hz, 1H, CH2 of CM), 3.88- 3.78 (m, 5H, C3H of
acyl × 2, H-5, H-6a’, H-6b’), 3.74-3.70 (dd, J6a,5 = 4.9 Hz, J6a,6b = 11.3 Hz, 1H, H-6b), 3.65-3.58 (m, 3H,
H-4’, H-2’, H-4), 3.54-3.50 (ddd, J5,’4’ = 9.5 Hz, J5’,6a’ = 2.7 Hz, J5,6b’ = 3.2 Hz, 1H, H-5’), 3.03 (s, 1H,
OH-4), 2.65- 2.59 (dd, Jjem = 15.1 Hz, Jvic = 7.9 Hz, 1H, C2H2 of acyl), 2.60-2.54 (dd, Jjem = 15.9 Hz, Jvic =
7.2 Hz, 1H, C2H2 of acyl), 2.54-2.48 (dd, Jjem = 16.0 Hz, Jvic = 5.0 Hz, 1H, C2H2 of acyl), 2.47-2.42 (dd,
Jjem = 15.1 Hz, Jvic = 4.7 Hz, 1H, C2H2 of acyl), 2.35-2.33 (m, 2H, C2H2 of acyl), 2.31-2.22 (m, 2H, C2’H2
of acyl), 1.61-1.42 (m, 8H, C4H2 of acyl × 3, C3’H2 of acyl), 1.30-1.25 (m, 76H, C5-13H2 of acyl × 3,
C4’-13’H2 of acyl), 0.92-0.86 (t, Jvic = 7.0 Hz, 12H, C14H3 of acyl × 3, C14’H3 of acyl). Anal. Calcd for
C87H119N2O17P: C, 67.00; H, 8.33; N, 1.42%. Found: C, 66.83; H, 8.35; N, 1.42. [α]D25 = +18.2 (c 1.00,
CHCl3).
Benzyloxycarbonylmethyl 6-O-[6-O-benzyl-4-O-benzyloxycarbonylmethyl-2-deoxy-2-[(R)-3-
(dodecanoloxy)tetradecanoylamino]-3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-β-D-gluco-
pyranosyl]-3-O-((R)-3-benzyloxytetradecanoyl)-2-((R)-3-benzyloxytetradecanoylamino)-2-deoxy-

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HETEROCYCLES, Vol. 69, 2006
α-D-glucopyranoside (24). To a solution of 23 (43.8 mg, 22.2 µmol) in AcOH/MeOH/THF = 15/10/3
(280 µL) was added zinc powder (50 mg). The suspension was sonicated for 30 sec and stirred at room
temperature for 20 min. After additional sonication for 30 min, the mixture was stirred for 2h and then
filtered through cerite pad. The filtrate was concentrated and co-evaporated with toluene three times. The
residue was purified by silica-gel column chromatography (5 g, CHCl3/acetone = 10/1). To the solution
of the residue, (R)-3-(dodecanoyloxy)tetradecanoic acid (18.9 mg, 22.2 µmol) and HOAt (7.6 mg, 5.5
µmol) in dry CHCl3 (0.22 mL) was added WSCD•HCl (10.6 mg, 55.5 µmol) at room temperature under
N2 atmosphere. The mixture was stirred for 2 h and then applied to silica-gel column chromatography
(80 g, toluene/AcOEt = 50/1) to give 24 as a white solid (30.2 mg, 62%). MALDI-TOF (positive) m/z
2225.69 [(M+Na)+]. 1H NMR (400 MHz, CDCl3) δ 7.38-7.19 (m, 25H, OCH2Ph × 5), 6.64-6.61 (d, JN,2 =
9.2 Hz, 1H, NH), 6.11-6.09 (d, JN’,2’ = 8.2 Hz, 1H, N’H), 5.20-5.05 (m, J3,2 = 10.7 Hz, J3,4 = 8.9 Hz, 8H,
C3H x 2, H-3, H-3’, COOCH2Ph × 2), 4.78-4.77 (d, J1,2 = 3.7 Hz, 1H, H-1), 4.62-4.60 (d, J1’,2’ = 7.9 Hz,
1H, H-1’), 4.57-4.41 (m, 6H, OCH2Ph × 3), 4.31- 4.25 (ddd, J2,1 = 3.7 Hz, J2,3 = 10.7 Hz, J2,N = 9.2 Hz, 1H,
H-2), 4.21-4.17 (d, Jjem = 16.2 Hz, 1H, CH2 of CM), 4.12-4.08 (d, Jjem = 16.2 Hz, 1H, CH2 of CM),
3.98-3.91 (m, 3H, CH2 of CM, H-6a), 3.88-3.69 (m, 7H, C3H of acyl × 2, H-2’, H-5, H-6a’, H-6b, H-4),
3.63-3.53 (m, 3H, H-4’, H-6b’, H-5’), 2.66-2.60 (dd, Jjem= 15.3 Hz, Jvic= 7.3 Hz, 1H, C2H2 of acyl),
2.55-2.54 (m, 2H, C2H2 of acyl), 2.45-2.21 (m, 9H, C2H2 of acyl × 2, C2H2 of acyl, C2’H2 of acyl × 2),
1.56-1.44 (m, 12H, C4H2 of acyl × 4, C3’H2 of acyl × 2), 1.25-1.21 (m, 108H, C5- 13H2 of acyl × 4, C4’-13’H2
of acyl, C4’-11’H2 of acyl), 0.89-0.86 (t, 18H, C14H3 of acyl × 4, C14’H3 of acyl, C12’H3 of acyl). Calcd for
C87H119N2O17P: C, 72.44; H, 9.62; N, 1.27%. Found: C, 71.94; H, 9.60; N, 1.27. [α]D25 = +17.0 (c 1.00,
CHCl3).
Carboxymethyl 6-O-[4-O-carbooxymethyl-2-deoxy-2-[(R)-3-(dodecanoyloxy)tetradecanoylamino]-
3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-β-D-glucopyranosyl]-3-O-((R)-3-hydroxytetra-
decanoyl)-2-((R)-3-hydroxytetradecanoylamino)-2-deoxy-α-D-glucopyranoside (Bis-CM-506, 5). To
a solution of 24 (55.2 mg, 25.0 µmol) in distilled THF (1 mL) was added Pd-black (53.4 mg). The mix-
ture was stirred under 7 kg cm-2 of hydrogen at room temperature overnight. After the removal of the Pd
catalyst by filtration, the solvent was evaporated in vacuo. The residue was purified by silica-gel column
chromatography [silica-gel (4 g) inactivated H2O (150 µL) overnight, 1% hexafluoroisopropanol,
CHCl3/MeOH = 7/1, then 1% hexafluoroisopropanol, MeOH] and concentrated. The residue was dis-
solved in CHCl3/H2O = 5/4 (9mL). The organic layer was washed with aqueous 0.1 N HCl and water,
and concentrated in vacuo. Lyophilization with water gave Bis-CM-506 (5) as a white powder (24.3
1
mg, 55%). ESI-TOF Mass (negative) m/z 1753.1 [(M-H)-], 876.1 [(M-2H)2-]. H NMR (500 MHz,
CDCl3/CD3OD = 1/1) δ 5.16-5.05 (m, 4H, H-3’, H-3, C3H × 2 of acyl), 4.70 (m, 1H, H-1), 4.52 (m, 1H,
H-1’), 4.20-4.00 (m, 4H, CH2 of CM’, CH2 of CM, H-2), 3.93-3.91 (m, 4H, H-5, H-6a, H-6a’, C3H of

HETEROCYCLES, Vol. 69, 2006
413
acyl), 3.81 (m, 3H, H-6b’ CH2 of CM, C3H of acyl), 3.71 (m, 3H, H-2’, H-4’, H-6b), 3.30-3.26 (m, 2H,
H-5’, H-4), 2.58-2.18 (m, 10H, C2H2 of acyl × 4, C2’H2 of acyl × 2), 1.52 (m, 4H, C4H2 of acyl × 2),
1.37-1.20 (m, 116H, C4-13H2 of acyl × 2, C5- 13H2 of acyl × 2, C3’- 13’H2 of acyl, C3’- 11’H2 of acyl), 0.82-0.79
(t, 18H, C14H3 of acyl × 4, C14’H3 of acyl, C12’H3 of acyl).
Cytokine induction in human peripheral whole-blood cell cultures. Aqueous 5% DMSO solution (50
mg/mL) of each sample was prepared by dilution of DMSO solution (1 mg/mL) with saline (Otsuka
Pharmaceuticals Co., Ltd.). Each sample was further diluted stepwise with saline on a 96-well plastic
plate (#2870-096, Iwaki Glass Co., Ltd). The mixture consisting of test sample (25 mL), RPMI 1640
medium (75 mL; Flow Laboratories, Irvine, Scotland), and heparinized human peripheral whole-blood
(25 mL) collected from an adult volunteer was incubated in triplicate at 37°C in 5% CO2 for 24 h. The
plate was centrifuged at 300 X g for 2 min. The levels of TNF-α and IL-6 in culture supernatant were
measure by means of enzyme-linked immunosorbent assay (ELISA).
Inhibition assay in cytokine induction. The mixture consisting of test sample (25 µL) prepared in the
same manner as above, LPS (25 µL, E. coli 0111:B4; Sigma Chemicals Co.), RPMI 1640 medium (75
µL; Flow Laboratories, Irvine, Scotland), and heparinized human peripheral whole-blood (25 µL) col-
lected from an adult volunteer was incubated in triplicate at 37°C in 5% CO2 for 24 h. The plate was
centrifuged at 300 X g for 2 min. The levels of TNF-α and IL-6 in culture supernatant were measured by
means of ELISA.
ACKNOWLEDGEMENTS
We are grateful to Prof. T. Tamura and Ms. K. Aoyama at Hyogo medical college for their assistance in
the cytokine induction assay. We also thank Mr. S. Adachi for NMR measurements, and Ms. K. Hayashi
and Ms. T. Ikeuchi for elemental analysis at Osaka University. The present work was financially sup-
ported in part by Grants-in Aid for Scientific Research (No.13NP0401, No.17035050, No.17310128)
from the Japan Society for the Promotion of Science, CREST of JST, Suntory Institute for Bioorganic
Research (SUNBOR) Grant, and Houansha Foundation Grant.
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