Synthesis of lipid A type carboxymethyl derivatives with ether chains instead of ester chains and their LPS-antagonistic activities

Article abstract of DOI:10.1016/S0008-6215(02)00357-9

Synthesis of lipid A type carboxymethyl derivatives having ether chains at both the C-3 and C-3′ positions and their LPS-antagonistic activities toward human U937 cells are described.

Full text of DOI:10.1016/S0008-6215(02)00357-9

Carbohydrate Research 338 (2003) 47–54
www.elsevier.com/locate/carres
Synthesis of lipid A type carboxymethyl derivatives with ether
chains instead of ester chains and their LPS-antagonistic activities
Yukiko Watanabe,a Kumiko Miura,a Masao Shiozaki,a,* Saori Kanai,b
Shin-ichi Kurakata,b Masahiro Nishijimac
aExploratory Chemistry Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
bBiological Research Laboratories, Sankyo Co. Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan
cDepartment of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
Received 18 July 2002; accepted 18 September 2002
Abstract
Synthesis of lipid A type carboxymethyl derivatives having ether chains at both the C-3 and C-3% positions and their
LPS-antagonistic activities toward human U937 cells are described. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Lipid A; Endotoxin; LPS-agonist; LPS-antagonist; TNFa production inhibitor
1. Introduction
endotoxic shock (bacterial sepsis) caused as a conse-
quence of acute inflammatory response.
Endotoxin (lipopolysaccharide; LPS)1 is a toxic sub-
stance from Gram-negative bacteria and one of the
components of their outer surface membrane. A variety
of responses, both beneficial and harmful, can be elic-
ited by LPS. It is also a highly potent stimulator of the
immune system.2 Therefore, LPS and its related com-
pounds have mainly been investigated as anti-cancer
drugs3 that function as LPS-agonists by activating
macrophages. Most of the biological activities of LPS
reside in a relatively small portion of the molecule, that
is, the terminal disaccharide phospholipid subunit
known as lipid A,4 which is a hydrophobic anchor
substance holding an essentially linear polysaccharide
chain to the cell wall. Many lipid A type disaccharide
analogues were synthesized to investigate their biologi-
cal activities.5 Monosaccharide analogues of both the
non-reducing distal subunit and the reducing sugar part
of Lipid A are usually still biologically active.6 How-
ever, many of these compounds usually show fatal
In recent years, lipid A-related compounds have been
studied as LPS-antagonists,7 which may have potential
as immunosuppressants,8 and in autoimmune diseases8
and septicemia9 by deactivating LPS-induced aggressive
macrophages. For example, Qureshi’s group10 isolated
a non-toxic lipid A-related compound from Rhodobac-
ter sphaeroides as an LPS antagonist, and an Eisai
group recently developed
a
related compound,
E5564,9,11 as a highly potent anti-septicemia drug.
On the other hand, during our investigation of the
biological activities of compounds related to GLA-60,6a
which is a non-reducing distal subunit analogue in the
Lipid A molecule, we also found that most of them had
LPS-agonistic activity, but a few of them behaved as
LPS antagonists. The a anomeric carboxymethyl GLA-
60 analogue A12 exhibited fairly strong LPS-antagonis-
tic activity (IC50:5 nM), and also lipid A-type
disaccharide B13 constructed from anomeric pyrancar-
boxylic acid and O-ether side chains showed a strong
LPS-antagonistic activity (IC50=0.6 nM) toward hu-
man U937 cells. Therefore, we are interested in the
activity of lipid A type a-carboxymethyl derivatives,
which have partially each component of compounds A
and B. And also we anticipated that the O-ether chains
instead of ester chains would stabilize the compound
* Corresponding author. Present address: Chemistry De-
partment, Chemtech Labo., Inc., Hiromachi 1-2-58, Shina-
gawa-ku, Tokyo 140-8710, Japan. Tel.: +81-3-34923131; fax:
+81-3-54368570
E-mail address: shioza@shina.sankyo.co.jp (M. Shiozaki).
0008-6215/03/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00357-9

48
Y. Watanabe et al. / Carbohydrate Research 338 (2003) 47–54
Fig. 1. Structures of compounds A, B, 18, 19 and 20.
Scheme 1. Reagents and conditions: (a) (R)-3-benzyloxy-1-
(methylsulfonyloxy)tetradecane, NaH, DMF, rt, 6 h, 74%; (b)
(1) 1 M aq NaOH, EtOH, 60 °C, 5 h; (2) (R)-3-(benzy-
loxy)tetradecanoic acid, DCC, DMAP, CH2Cl2, rt, 18 h, two
steps 89%; (c) (1) OsO4, NMO, THF–t-BuOH–H2O, rt, 3 h;
(2) Pb(OAc)4, benzene, rt, 1 h; (3) NaClO2, NaH2PO4, 2-
methyl-2-butene, t-BuOH–H2O, rt, 18 h; (4) Ph2CN2, EtOAc,
rt, 18 h, four steps 76%; (d) 80% aq AcOH, 60 °C, 4 h, 86%.
and increase the activity. Herein we report the synthesis
of compounds 18, 19 and 20 and their activities (Fig. 1).
2. Results and discussion
Firstly, the carboxymethyl glucosamine derivative 5 on
the right half moiety was synthesized from trifluoroac-
etamide 1 as shown in Scheme 1. Compound 114 was
alkylated
with
(R)-3-benzyloxy-1-(methylsulfony-
loxy)tetradecane15 and NaH in N,N-dimethylfor-
mamide (DMF) to yield ether 2, and the protecting
trifluoroacetyl group was cleaved by aqueous NaOH at
60 °C, and then the liberated amine was treated with
(R)-3-benzyloxytetradecanoic acid using dicyclohexyl-
carbodiimide (DCC) as a dehydrating agent and 4-
dimethylaminopyridine (DMAP) as a catalyst to give
amide 3. The double bond of allyl group was oxidized
with OsO4 and 4-methylmorpholine N-oxide (NMO),
and the liberated vicinal diol was cleaved with
Pb(OAc)4 to afford the aldehyde, which was further
oxidized to carboxylic acid by NaClO2 according to the
reported procedure.5 Finally the carboxylic acid was
esterified with Ph2CN2 to give benzhydryl ester 4 in
four steps in 76% yield. The isopropylidene group was
deprotected with aqueous 80% AcOH at 60 °C to give
diol 5.
Secondly, the left half compounds 6, 7 and 8 synthe-
sized from a common starting material 1 according to
the reported method13 were treated with trichloroace-
tonitrile using Cs2CO3 as a catalyst to yield correspond-
ing imidates, which were reacted with the diol 5
obtained above to give corresponding disaccharides 9,
10 and 11 as shown in Scheme 2 according to the
reported method.4a,4c Treatment of each compound (9,
10 or 11) with Zn–acetic acid, and successive acetyla-
tion with acetic anhydride–pyridine gave the corre-
sponding acetamide (12, 13 or 14, respectively), which
was hydrogenolyzed with 20% Pd(OH)2–C to give the
acid (15, 16 or 17, respectively). Finally, each acid (15,
16 or 17) was treated with hydrogen using Pt as a
catalyst to give phosphoric acid (18, 19 or 20,
respectively).
Scheme 2. Reagents and conditions: (a) (1) Cl3CCN, cat.
Cs2CO3, CH2Cl2, rt, 1 h; (2) 5, TMSOTf, MS 4 A, CH2Cl2,
−40 °C, 1 h; (b) (1) Zn, AcOH, rt, 2 h; (2) Ac2O, pyridine,
THF–H2O, rt, 1 h; (c) H2, 20% Pd(OH)2–C, EtOH, rt, 18 h;
(d) H2, PtO2, THF, rt, 20 h.
,
2.1. Biological activity
The inhibitory activity of compounds 18, 19 and 20 on
LPS-induced TNFa production was investigated in

Y. Watanabe et al. / Carbohydrate Research 338 (2003) 47–54
49
vitro using human monoblastic U937 cells. Compounds
18, 19 and 20, which have four chains in their
molecules, inhibited TNFa production as LPS-antago-
nists toward human monoblastic U937 cells, and the
IC50 values of these three compounds were 6.5, 6.1 and
12.4 nM, respectively. Judging from the results reported
in Ref. 13 (that is, the IC50 values of the corresponding
pyran–carboxylic acid analogues of compounds 18, 19
and 20 were 11, 6.4 and 10 nM, respectively), the values
were almost the same. Therefore, the difference of the
anomeric substituents between the pyrancarboxylic acid
type acid in Ref. 13 and the carboxymethyl type acid in
this paper did not greatly affect the inhibitory activity
toward human monoblastic U937 cells. The C-6
methoxy group11 of compound 19 did not enhance the
activity compared with C-6 hydroxyl group of com-
pound 18. The C-6 fluoride16 of compound 20 weak-
ened the activity in comparison with compounds 18 and
19. However, the LPS-antagonistic activity of these
compounds was much less than that of compound B
(IC50=0.6 nM). The structural feature of compound B
possessed an ester side chain in 3-(tetra-
decanoyloxy)tetradecyl group instead of the corre-
sponding ether side chain. The existence of an ester
bond in the side chain in the 3-position may suggest a
significant role for this activity.
9.93 mmol) in DMF (40 mL) was gradually added NaH
(60% oil dispersion, 482 mg, 12.0 mmol) at 0 °C with
stirring. After 15 min, (R)-3-benzyloxy-1-methyl-
sulfonyloxytetradecane (3.31 g, 8.30 mmol) was added
to this solution, which was stirred at room temperature
(rt) for 6 h. The reaction mixture was quenched with
water, extracted with EtOAc, washed with water and
brine, dried over MgSO4, and filtered. The filtrate was
concentrated in vacuo to give a mixture that was chro-
matographed on a silica gel column. Elution with 4:1
C6H14–EtOAc gave 2 (4.05 g, 74%). IR (cm−1):
wmax(CHCl3) 3430, 2928, 2856, 1734. 1H NMR (400
MHz, CDCl3): l 0.88 (3 H, t, J 6.6 Hz), 1.26 (18 H,
brs), 1.40 (3 H, s), 1.48 (3 H, s), 1.51–1.75 (4 H, m),
3.43–3.49 (2 H, m), 3.59 (1 H, m), 3.66–3.69 (2 H, m),
3.75 (1 H, t, J 10.3 Hz), 3.84–3.91 (2 H, m), 3.98 (1 H,
dd, J 5.9, 12.5 Hz), 4.14–4.20 (2 H, m), 4.42, 4.51 (2 H,
AB-q, J 11.7 Hz), 4.86 (1 H, d, J 3.7 Hz), 5.25–5.31 (2
H, m), 5.87 (1 H, m), 6.42 (1 H, d, J 9.5 Hz, NH),
7.26–7.34 (5 H, m). FABMS (positive-ion): m/z 658
(M+H)+. HRFABMS (positive-ion); Calcd for
C35H55F3NO7: 658.3931. Found: 658.3904. Anal. Calcd
for C35H54F3NO7 (657.8): C, 63.91; H, 8.28; F, 8.66; N,
2.13. Found: C, 64.09; H, 8.30; F, 8.46; N, 2.11.
3.2. Allyl 2-[(R)-3-(benzyloxy)tetradecanamido]-3-O-
[(R)-3-(benzyloxy)tetradecyl]-2-deoxy-4,6-O-isopropyli-
dene-a-D-glucopyranoside (3)
3. Experimental
A solution of 2 (2.73 g, 4.15 mmol) in EtOH (10 mL)
and 1 M aq NaOH (10 mL) was stirred at 60 °C for 5
h. The solution was concentrated in vacuo, diluted with
EtOAc, washed with water and brine, dried over
Na2SO4, filtered, and concentrated to give an amine,
which was dissolved in CH2Cl2 (30 mL). (R)-3-(Benzy-
loxy)tetradecanoic acid (1.50 g, 4.48 mmol), DCC (963
mg, 4.67 mmol), and DMAP (574 mg, 4.70 mmol) were
added to this solution, which was stirred for 18 h at rt,
filtered, and concentrated in vacuo to give a mixture.
The mixture was chromatographed on a silica gel
column. Elution with 4:1 C6H14–EtOAc gave 3 (3.25 g,
89%) as a gum. IR (cm−1): wmax(CHCl3) 3441, 3363,
Melting points are uncorrected. Optical rotations were
obtained by the use of a JASCO P-1030 polarimeter. IR
absorption spectra were recorded on a JASCO IR A-2
spectrophotometer. 1H NMR spectra were recorded
with a JEOL-GSX 400 spectrometer using Me4Si as an
internal standard, and mass spectra were obtained with
a JMS-700 mass spectrometer. Separation of the com-
pounds by column chromatography was carried out
with silica gel 60 (E. Merck, 0.040–0.063 mm) at
slightly elevated pressure (1.1–1.8 atm) for easy elution,
and the quantity of the used silica gel was 50–100 times
the weight of the purified compounds. Thin-layer chro-
matography was performed on E. Merck silica gel
60-F254 (cat. no. 5715) plates. Tetrahydrofuran was
distilled in the presence of radical anions generated by
Na–benzophenone ketyl. Dichloromethane was dried
by being passed through an ICN Alumina B-Super I,
1
2928, 2856, 1666. H NMR (400 MHz, CDCl3): l 0.88
(6 H, t, J 6.6–7.3 Hz), 1.24–1.81 (48 H, m, containing
two 3 H, s, at 1.46 and 1.38 ppm), 2.32 (1 H, dd, J 7.3,
14.6 Hz), 2.43 (1 H, dd, J 3.7, 14.6 Hz), 3.40 (1 H, t, J
10.3 Hz), 3.45–3.56 (2 H, m), 3.61–3.85 (7 H, m), 4.04
(1 H, dd, J 5.9, 12.5 Hz), 4.20 (1 H, td, J 9.5, 3.7 Hz),
4.45, 4.47 (2 H, AB-q, J 11.0 Hz), 4.51, 4.54 (2 H,
AB-q, J 11.0 Hz), 4.78 (1 H, d, J 3.7 Hz), 5.12–5.23 (2
H, m), 5.77 (1 H, m), 6.40 (1 H, d, J 9.5 Hz, NH),
7.26–7.35 (10 H, m). FABMS (positive-ion): m/z 900
(M+Na)+, 878 (M+H)+. HRFABMS (positive-ion);
Calcd for C54H88NO8: 878.6510. Found: 878.6506.
Anal. Calcd for C54H87NO8 (878.3): C, 73.86; H, 9.98;
N, 1.59. Found: C, 73.92; H, 9.84; N, 1.67.
,
and DMF and pyridine were dried by storage over 4 A
molecular sieves.
3.1. Allyl 3-O-[(R)-3-(benzyloxy)tetradecyl]-2-deoxy-
4,6-O-isopropylidene-2-trifluoroacetamido-a-D-glucopy-
ranoside (2)
To a solution of allyl 2-deoxy-4,6-O-isopropylidene-2-
trifluoroacetamido-a- -glucopyranoside (1) (3.53 g,
D

50
Y. Watanabe et al. / Carbohydrate Research 338 (2003) 47–54
3.3. (Diphenylmethoxycarbonyl)methyl 2-[(R)-3-(benzyl-
oxy)tetradecanamido]-3-O-[(R)-3-(benzyloxy)tetra-
decyl]-2-deoxy-4,6-O-isopropylidene-a-D-gluco-
vacuo, and chromatographed on a silica gel column.
Elution with 2:3 C6H14–EtOAc gave 5 (1.95 mg, 86%)
as a white powder. IR (cm−1): wmax(KBr) 3319, 3065,
3033, 2925, 2854, 1756, 1642. 1H NMR (400 MHz,
CDCl3): l 0.88 (6 H, t, J 6.6 Hz), 1.26–1.78 (42 H, m),
2.40–2.43 (2 H, m), 3.40 (1 H, t, J 9.5 Hz), 3.46–3.56
(3 H, m), 3.62–3.75 (4 H, m), 3.84 (1 H, m), 4.06 (2 H,
s), 4.19 (1 H, td, J 10.3, 3.7 Hz), 4.40–4.48 (4 H, m),
4.74 (1 H, d, J 3.7 Hz), 6.83 (1 H, d, J 9.5 Hz, NH),
6.92 (1 H, s), 7.20–7.37 (20 H, m). FABMS (positive-
ion): m/z 1060 (M+K)+ (on addition of KI), 1044
(M+Na)+, 1022 (M+H)+. HRFABMS (positive-
ion); Calcd for C63H91KNO10: 1060.6280. Found:
1060.6273. Anal. Calcd for C63H91NO10 (1022.4): C,
74.01; H, 8.97; N, 1.37. Found: C, 73.82; H, 8.90; N,
1.48.
pyranoside (4)
To a solution of 3 (2.80 g, 3.19 mmol) in THF (10
mL)–t-BuOH (10 mL)–water (1 mL) were added 4-
methylmorpholine N-oxide (1.12 g, 9.56 mmol) and
OsO4 in t-BuOH (2.5%, 6.5 mL, 0.518 mmol). After
vigorous stirring for 3 h at rt, the mixture was
quenched with satd aq Na2S2O3, and extracted with
EtOAc. The organic layer was washed with satd aq
Na2S2O3 and brine, dried over Na2SO4, and concen-
trated in vacuo to give the crude diol, which was used
without further purification for the following oxidation.
The crude diol thus obtained was dissolved in C6H6
(20 mL). To this solution was added Pb(OAc)4 (1.88 g,
3.82 mmol). After stirring for 1 h at rt, the mixture was
filtered through a silica gel column using EtOAc as an
eluent. After removal of the solvent in vacuo, the crude
aldehyde was dissolved in t-BuOH (16 mL) and water
(4 mL). To this solution was added NaH2PO4 (561 mg,
3.60 mmol), 2-methyl-2-butene (1.06 g, 15.1 mmol), and
NaClO2 (1.05 g, 9.17 mmol) at rt. After 18 h, the
reaction mixture was acidified with 1 M aq HCl and
extracted with EtOAc. The extract was washed with
water and brine, dried over Na2SO4, filtered, and con-
centrated in vacuo to give a residue, which was dis-
solved in EtOAc (20 mL). To this solution was added
Ph2CN2 (1.15 g, 5.92 mmol) at rt. After stirring for 18
h, the reaction mixture was quenched with AcOH and
concentrated in vacuo to give a mixture, which was
chromatographed on a silica gel column. Elution with
9:1“7:3 C6H14–EtOAc gave 4 (2.57 g, 76%) as a solid.
IR (cm−1): wmax(CHCl3) 3692, 3360, 2927, 2855, 1755,
1666. 1H NMR (400 MHz, CDCl3): l 0.88 (6 H, t, J 6.6
Hz), 1.25–1.79 (48 H, m, containing two 3 H, s, at 1.44
and 1.36 ppm), 2.36 (1 H, dd, J 6.6, 14.6 Hz), 2.43 (1
H, dd, J 4.4, 14.6 Hz), 3.38–3.57 (3 H, m), 3.63–3.84 (6
H, m), 4.01 (2 H, s), 4.22 (1 H, td, J 10.3, 3.7 Hz), 4.44
(2 H, s), 4.46 (2 H, s), 4.73 (1 H, d, J 3.7 Hz), 6.74 (1
H, d, J 9.5 Hz, NH), 6.91 (1 H, s), 7.19–7.36 (20 H, m).
FABMS (positive-ion): m/z 1084 (M+Na)+, 1062
(M+H)+. HRFABMS (positive-ion); Calcd for
C66H96NO10: 1062.7034. Found: 1062.7009. Anal. Calcd
for C66H95NO10 (1062.5): C, 74.61; H, 9.01; N, 1.32.
Found: C, 74.26; H, 8.72; N, 1.35.
3.5. (Diphenylmethoxycarbonyl)methyl 6-O-[6-O-benzyl-
oxycarbonyl-2-deoxy-4-O-diphenylphosphono-3-O-[(R)-
3-(dodecyloxy)tetradecyl]-2-[(2,2,2-trichloroethoxycar-
bonyl)amino]-b-
tetradecanamido]-3-O-[(R)-3-(benzyloxy)tetradecyl]-2-
deoxy-a- -glucopyranoside (9)
D-glucopyranosyl]-2-[(R)-3-(benzyloxy)-
D
To a solution of 6 (352 mg, 0.320 mmol) in CH2Cl2 (5
mL) were added Cl3CCN (0.32 mL, 3.20 mmol) and
Cs2CO3 (53 mg, 0.163 mmol). After stirring for 1 h at
rt, the reaction mixture was quenched with satd aq
NaHCO3 (40 mL), and extracted with EtOAc. The
extract was washed with brine and dried over Na2SO4.
Removal of the solvent in vacuo gave the crude imidate
(399 mg), which was immediately used for subsequent
glycosylation without further purification. In a nitrogen
atmosphere, a solution of the imidate (339 mg) thus
obtained, diol 5 (299 mg, 0.292 mmol), and molecular
,
sieves 4 A (420 mg) in CH2Cl2 (5 mL) was stirred at rt.
After stirring for 1 h, a catalytic amount of TMSOTf (6
mL, 0.033 mmol) was added to the mixture at −40 °C.
After stirring for 1 h at −40 °C, the mixture was
quenched with satd aq NaHCO3, diluted with EtOAc,
washed with water and brine, dried over MgSO4,
filtered, and concentrated in vacuo to give a mixture,
which was chromatographed on a silica gel column.
Elution with 3:2 C6H14–EtOAc gave 9 (471 mg, 77%)
as a gum. IR (cm−1): wmax(CHCl3) 3442, 2928, 2855,
1
1748, 1667. H NMR (400 MHz, CDCl3): l 0.88 (12 H,
t, J 6.6 Hz), 1.25–1.77 (84 H, m), 2.36–2.38 (2 H, m),
3.08 (1 H, brs, OH), 3.20–3.27 (4 H, m), 3.33 (1 H, t,
J 9.5 Hz), 3.41–3.45 (2 H, m), 3.57–3.76 (7 H, m), 3.82
(1 H, m), 3.89–3.96 (2 H, m), 4.04 (2 H, s), 4.17 (1 H,
td, J 10.3, 3.7 Hz), 4.23 (1 H, dd, J 5.1, 12.5 Hz),
4.36–4.56 (6 H, m, containing 1 H, d, J 3.7 Hz, l 4.43),
4.67–4.74 (3 H, m), 4.86 (1 H, m), 5.04, 5.09 (2 H,
AB-q, J 12.5 Hz), 5.52 (1 H, m), 6.75 (1 H, d, J 9.5 Hz,
NH), 6.89 (1 H, s), 7.11–7.34 (35 H, m). FABMS
(positive-ion): m/z 2141 (M+K)+ (on addition of KI),
3.4. (Diphenylmethoxycarbonyl)methyl 2-[(R)-3-(benzyl-
oxy)tetradecanamido]-3-O-[(R)-3-(benzyloxy)tetra-
decyl]-2-deoxy-a-D-glucopyranoside (5)
A solution of 4 (2.35 g, 2.21 mmol) in 80% aq AcOH
(20 mL) was stirred at 60 °C for 4 h. The solution was
diluted with EtOAc, washed with aq NaHCO3 and
brine, dried over MgSO4, filtered, concentrated in

Y. Watanabe et al. / Carbohydrate Research 338 (2003) 47–54
51
2125 (M+Na)+. HRFABMS (positive-ion); Calcd for
3.8. (Diphenylmethoxycarbonyl)methyl 6-O-[2-acetam-
ido-6-O-benzyloxycarbonyl-2-deoxy-4-O-diphenylphos-
C118H170Cl3KN2O22P: 2142.0686. Found: 2142.0625.
Anal. Calcd for C118H170Cl3N2O22P (2105.9): C, 67.30;
H, 8.14; Cl, 5.05; N, 1.33; P, 1.47. Found: C, 67.10; H,
7.95; Cl, 5.02; N, 1.31; P, 1.53.
phono-3-O-[(R)-3-(dodecyloxy)tetradecyl]-b-
pyranosyl]-2-[(R)-3-(benzyloxy)tetradecanamido]-3-O-
D-gluco-
[(R)-3-(benzyloxy)tetradecyl]-2-deoxy-a-D-glucopyrano-
side (12)
3.6. (Diphenylmethoxycarbonyl)methyl 2-[(R)-3-(benzyl-
oxy)tetradecanamido]-3-O-[(R)-3-(benzyloxy)tetra-
decyl]-2-deoxy-6-O-[2-deoxy-4-O-diphenylphosphono-
3-O-[(R)-3-(dodecyloxy)tetradecyl]-6-O-methyl-2-(2,2,2-
To a solution of 9 (271 mg, 0.128 mmol) in AcOH (3
mL) was added Zn dust (168 mg, 2.57 mmol). After
vigorous stirring for 2 h at rt, the solution was filtered
to remove Zn and concentrated in vacuo to give a crude
product. The product was diluted with EtOAc, washed
with satd aq NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated in vacuo to give a crude
product, which was dissolved in THF (2 mL). Water (1
mL), pyridine (52 mL) and Ac2O (60 mL) were added to
this solution. After stirring for 1 h at rt, the mixture
was diluted with EtOAc, washed with water and brine,
dried over MgSO4, and filtered. The filtrate was concen-
trated in vacuo to give a residue, which was chro-
matographed on a silica gel column. Elution with 1:1
C6H14–EtOAc gave 12 (212 mg, two steps, 84%) as a
gum. IR (cm−1): wmax(CHCl3) 3450, 2928, 2855, 1751,
trichloroethoxycarbonylamino)-b-
D-glucopyranosyl]-a-
D-glucopyranoside (10)
Compound 7 (410 mg, 0.418 mmol) was treated as
described in the formation of 9 from 6 to give 10 (537
mg, 71%) as a gum. IR (cm−1): wmax(CHCl3) 3441,
1
2928, 2855, 1747, 1667. H NMR (400 MHz, CDCl3): l
0.88 (12 H, t, J 6.6 Hz), 1.25–1.78 (84 H, m), 2.36–2.37
(2 H, m), 3.12–3.29 (8 H, m, containing 3 H, s, l 3.20),
3.34 (1 H, t, J 9.5 Hz), 3.46–3.97 (14 H, m), 4.04 (2 H,
s), 4.17 (1 H, td, J 10.3, 3.7 Hz), 4.38–4.48 (4 H, m),
4.55 (1 H, q, J 9.5 Hz), 4.71–4.74 (3 H, m), 4.84 (1 H,
m), 5.45 (1 H, m), 6.74 (1 H, d, J 9.5 Hz, NH), 6.90 (1
H, s), 7.15–7.36 (30 H, m). FABMS (positive-ion): m/z
2005 (M+Na)+. HRFABMS (positive-ion); Calcd for
C111H166Cl3N2NaO20P: 2006.0735. Found: 2006.0745.
Anal. Calcd for C111H166Cl3N2O20P (1985.84): C, 67.14;
H, 8.43; Cl, 5.36; N, 1.41; P, 1.56. Found: C, 67.19; H,
8.11; Cl, 5.59; N, 1.68; P, 1.68.
1
1668. H NMR (400 MHz, CDCl3): l 0.88 (12 H, t, J
6.6 Hz), 1.20–1.75 (84 H, m), 1.95 (3 H, s), 2.36 (2 H,
d, J 6.6 Hz), 3.08 (1 H, m), 3.24–3.36 (4 H, m),
3.46–3.48 (3 H, m, containing OH), 3.62–3.73 (7 H,
m), 3.83 (1 H, m), 3.94 (1 H, m), 4.02–4.06 (3 H, m,
containing 2 H, s, at 4.02 ppm), 4.15–4.23 (2 H, m),
4.36–4.51 (6 H, m), 4.73 (1 H, d, J 3.7 Hz), 5.04, 5.09
(2 H, AB-q, J 12.1 Hz), 5.22 (1 H, d, J 8.1 Hz), 6.16 (1
H, d, J 6.6 Hz, NH), 6.72 (1 H, d, J 9.5 Hz, NH), 6.89
(1 H, s), 7.11–7.34 (35 H, m). FABMS (positive-ion):
m/z 1993 (M+Na)+. HRFABMS (positive-ion); Calcd
for C117H171N2NaO21P: 1994.2010. Found: 1994.2020.
Anal. Calcd for C117H171N2O21P (1972.6): C, 71.24; H,
8.74; N, 1.42; P, 1.57. Found: C, 71.70; H, 8.69; N,
1.39; P, 1.56.
3.7. (Diphenylmethoxycarbonyl)methyl 2-[(R)-3-(benzyl-
oxy)tetradecanamido]-3-O-[(R)-3-(benzyloxy)tetra-
decyl]-2-deoxy-6-O-[2,6-dideoxy-4-O-diphenylphos-
phono-3-O-[(R)-3-(dodecyloxy)tetradecyl]-6-fluoro-2-
(2,2,2-trichloroethoxycarbonylamino)-b-
D-glucopyrano-
syl]-a- -glucopyranoside (11)
D
Compound 8 (420 mg, 0.433 mmol) was treated as
described in the formation of 9 from 6 to give 11 (570
mg, 73%) as a gum. IR (cm−1): wmax(CHCl3) 3443,
3358, 2928, 2855, 1745, 1667. 1H NMR (400 MHz,
CDCl3): l 0.88 (12 H, t, J 6.6 Hz), 1.25–1.77 (84 H, m),
2.33–2.38 (2 H, m), 3.21–3.27 (5 H, m), 3.34 (1 H, t, J
9.5 Hz), 3.43 (1 H, t, J 9.5 Hz), 3.48–3.75 (8 H, m),
3.82 (1 H, m), 3.90–4.05 (2 H, m), 4.05 (2 H, s), 4.17 (1
H, td, J 10.3, 3.7 Hz), 4.37–4.59 (7 H, m), 4.71–4.73 (3
H, m), 4.90 (1 H, m), 5.54 (1 H, m), 6.77 (1 H, d, J 9.5
Hz, NH), 6.90 (1 H, s), 7.17–7.35 (30 H, m). FABMS
(positive-ion): m/z 1993 (M+Na)+. HRFABMS (posi-
tive-ion); Calcd for C110H163Cl3FN2NaO19P: 1994.0535.
3.9. (Diphenylmethoxycarbonyl)methyl 6-O-[2-acet-
amido-2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-
(dodecyloxy)tetradecyl]-6-O-methyl-b-
2-[(R)-3-(benzyloxy)tetradecanamido]-3-O-[(R)-3-
(benzyloxy)tetradecyl]-2-deoxy-a- -glucopyranoside (13)
D-glucopyranosyl]-
D
Compound 10 (340 mg, 0.171 mmol) was treated as
described in the formation of 12 from 9 to give 13 (280
mg, 88%) as a gum. IR (cm−1): wmax(CHCl3) 3692,
3451, 3363, 2928, 2855, 1755, 1668. 1H NMR (400
MHz, CDCl3): l 0.88 (12 H, t, J 6.6 Hz), 1.20–1.76 (84
H, m), 1.96 (3 H, s), 2.36 (2 H, d, J 5.9 Hz), 3.14–3.20
(4 H, m, containing 3 H, s, at 3.20 ppm), 3.22–3.37 (5
H, m), 3.46–3.77 (11 H, m, containing OH), 3.83 (1 H,
m), 3.96–4.05 (4 H, m, containing 2 H, s, at 4.02 ppm),
4.18 (1 H, dt, J 10.3, 3.7 Hz), 4.38–4.45 (4 H, m), 4.51
(1 H, q, J 9.5 Hz), 4.73 (1 H, d, J 3.7 Hz), 5.16 (1 H,
Found: 1994.0587. Anal. Calcd for
C110H163-
Cl3FN2O19P (1973.8): C, 66.94; H, 8.32; Cl, 5.39; N,
1.42; P, 1.57. Found: C, 67.03; H, 8.24; Cl, 5.36; N,
1.43; P, 1.65.

52
Y. Watanabe et al. / Carbohydrate Research 338 (2003) 47–54
d, J 8.1 Hz), 6.09 (1 H, d, J 6.6 Hz, NH), 6.73 (1 H, d,
J 9.5 Hz, NH), 6.89 (1 H, s), 7.16–7.36 (30 H, m).
FABMS (positive-ion): m/z 1873 (M+Na)+. HR-
FABMS (positive-ion); Calcd for C110H167N2NaO19P:
1874.1798. Found: 1874.1810. Anal. Calcd for
C110H167N2O19P (1852.5): C, 71.32; H, 9.09; N, 1.51; P,
1.67. Found: C, 71.68; H, 9.18; N, 1.54; P, 1.72.
3.12. Carboxymethyl 6-O-[2-acetamido-2-deoxy-4-O-
diphenylphosphono-3-O-[(R)-3-(dodecyloxy)tetradecyl]-
6-O-methyl-b-
hydroxytetradecanamido]-3-O-[(R)-3-hydroxytetra-
decyl]-a- -glucopyranoside (16)
D-glucopyranosyl]-2-deoxy-2-[(R)-3-
D
Compound 13 (231 mg, 0.125 mmol) was treated as
described in the formation of 15 from 12 to give 16 (143
mg, 76%) as an amorphous. IR (cm−1): wmax(KBr)
3308, 3073, 2924, 2854, 1730, 1657. 1H NMR (400
MHz, CD3OD): l 0.90 (12 H, t, J 6.6 Hz), 1.21–1.70
(84 H, m), 2.01 (3 H, s), 2.33–2.43 (2 H, m), 3.21 (3 H,
s), 3.22–3.34 (5 H, m), 3.38 (1 H, t, J 9.5 Hz), 3.48 (2
H, m), 3.56 (1 H, m), 3.63–3.93 (8 H, m), 3.96–4.05 (2
H, m), 4.05, 4.17 (2 H, AB-q, J 16.8 Hz), 4.21 (1 H, m),
4.55 (1 H, q, J 8.8 Hz), 4.63 (1 H, d, J 8.1 Hz), 4.73 (1
H, d, J 2.9 Hz), 7.20–7.41 (10 H, m). FABMS (posi-
tive-ion): m/z 1527 (M+Na)+. HRFABMS (positive-
ion); Calcd for C83H145N2NaO19P: 1528.0077. Found:
1528.0076. Anal. Calcd for C83H145N2O19P (1506.0): C,
66.19; H, 9.71; N, 1.86; P, 2.06. Found: C, 65.86; H,
9.86; N, 1.58; P, 2.00.
3.10. (Diphenylmethoxycarbonyl)methyl 6-O-[2-acetam-
ido-2,6-dideoxy-4-O-diphenylphosphono-3-O-[(R)-3-
(dodecyloxy)tetradecyl]-6-fluoro-b-
[(R)-3-(benzyloxy)tetradecanamido]-3-O-[(R)-3-(benzyl-
oxy)tetradecyl]-2-deoxy-a- -glucopyranoside (14)
D-glucopyranosyl]-2-
D
Compound 11 (339 mg, 0.172 mmol) was treated as
described in the formation of 12 from 9 to give 14 (279
mg, 88%) as a gum. IR (cm−1): wmax(CHCl3) 3692,
3451, 3360, 2928, 2855, 1754, 1669. 1H NMR (400
MHz, CDCl3): l 0.88 (12 H, t, J 6.6 Hz), 1.20–1.77 (84
H, m), 1.95 (3 H, s), 2.36–2.37 (2 H, m), 3.08 (1 H, m),
3.26–3.37 (5 H, m), 3.48–3.50 (3 H, m, containing
OH), 3.61–3.72 (6 H, m), 3.83 (1 H, m), 3.97 (1 H, m),
4.02 (2 H, d, J 4.4 Hz), 4.10 (1 H, dt, J 9.5, 10.3 Hz),
4.18 (1 H, dt, J 10.3, 3.7 Hz), 4.38–4.58 (7 H, m), 4.73
(1 H, d, J 3.7 Hz), 6.18 (1 H, d, J 5.9 Hz, NH), 6.75 (1
H, d, J 9.5 Hz, NH), 6.89 (1 H, s), 7.17–7.34 (30 H, m).
FABMS (positive-ion): m/z 1862 (M+Na)+. HR-
FABMS (positive-ion); Calcd for C109H164FN2NaO18P:
1862.1599. Found: 1862.1622. Anal. Calcd for
3.13. Carboxymethyl 6-O-[2-acetamido-2,6-dideoxy-4-O-
diphenylphosphono-3-O-[(R)-3-(dodecyloxy)tetradecyl]-
6-fluoro-b- -glucopyranosyl]-2-deoxy-2-[(R)-3-hydroxy-
D
tetradecanamido]-3-O-[(R)-3-hydroxytetradecyl]-a-D-
glucopyranoside (17)
C
109H164FN2O18P (1840.4): C, 71.13; H, 8.98; F, 1.03;
Compound 14 (236 mg, 0.128 mmol) was treated as
described in the formation of 15 from 12 to give 17 (125
mg, 66%) as an amorphous. IR (cm−1): wmax(KBr) 3305
N, 1.52; P, 1.68. Found: C, 71.41; H, 9.01; F, 1.08; N,
1.50; P, 1.77.
1
(broad), 3074, 2924, 2854, 1731, 1646. H NMR (400
3.11. Carboxymethyl 6-O-[2-acetamido-2-deoxy-4-O-
diphenylphosphono-3-O-[(R)-3-(dodecyloxy)tetradecyl]-
MHz, CD3OD): l 0.89 (12 H, t, J 6.6 Hz), 1.29–1.70
(84 H, m), 2.00 (3 H, s), 2.33–2.42 (2 H, m), 3.27–3.39
(4 H, m), 3.54 (1 H, t, J 11.0–8.8 Hz), 3.65–3.82 (8 H,
m), 3.90 (1 H, m), 4.00–4.04 (2 H, m), 4.09–4.27 (4 H,
m, containing 2 H, AB-q, J 16.8 Hz, l 4.10, 4.25),
4.36–4.56 (3 H, m), 4.64 (1 H, d, J 8.1 Hz), 4.78 (1 H,
d, J 2.9 Hz), 7.18–7.39 (10 H, m). FABMS (positive-
ion): m/z 1515 (M+Na)+. HRFABMS (positive-ion);
Calcd for C82H142FN2NaO18P: 1515.9877. Found:
1515.9871. Anal. Calcd for C82H142FN2O18P (1494.0):
C, 65.92; H, 9.58; F; 1.27; N, 1.88; P, 2.07. Found: C,
66.09; H, 9.60; F, 1.27; N, 1.59; P, 1.96.
b- -glucopyranosyl]-2-deoxy-2-[(R)-3-hydroxytetra-
D
decanamido]-3-O-[(R)-3-hydroxytetradecyl]-a-D-gluco-
pyranoside (15)
A solution of 12 (159 mg, 0.081 mmol) in EtOH (5 mL)
containing 20% Pd(OH)2-on-carbon (88.5 mg) was
stirred vigorously under hydrogen for 18 h at rt. The
reaction mixture was filtered and concentrated in vacuo
to give a crude product, which was purified by prepara-
tive silica gel thin-layer chromatography developed by
8:1 CHCl3–MeOH to give 15 (65.4 mg, 55%) as an
amorphous. IR (cm−1): wmax(CH3OH) 3430 (broad),
3.14. Carboxymethyl 6-O-[2-acetamido-2-deoxy-3-O-
1
3326 (broad), 2927, 2855, 1729, 1653. H NMR (400
[(R)-3-(dodecyloxy)tetradecyl]-4-O-phosphono-b-
D-
MHz, CDCl3): l 0.90 (12 H, t, J 6.6 Hz), 1.29–1.73 (84
H, m), 2.00 (3 H, s), 2.36–2.41 (2 H, m), 3.26–3.42 (5
H, m), 3.52–3.80 (11 H, m), 3.91 (1 H, m), 4.00–4.03 (2
H, m), 4.12 (1 H, m), 4.10, 4.23 (2 H, AB-q, J 16.8 Hz),
4.54 (1 H, m), 4.59 (1 H, d, J 7.3 Hz), 4.78 (1 H, d, J
3.7 Hz), 7.19–7.40 (10 H, m). FABMS (positive-ion):
m/z 1513 (M+Na)+. HRFABMS (positive-ion); Calcd
for C82H143N2NaO19P: 1513.9920. Found: 1513.9908.
glucopyranosyl]-2-deoxy-2-[(R)-3-hydroxytetradecan-
amido]-3-O-[(R)-3-hydroxytetradecyl]-a-D-glucopyrano-
side (18)
A solution of 15 (55 mg, 0.037 mmol) in THF (5 mL)
containing PtO2 (28 mg) as a catalyst was stirred vigor-
ously under hydrogen for 20 h at rt. The reaction
mixture was filtered and concentrated in vacuo to give

Y. Watanabe et al. / Carbohydrate Research 338 (2003) 47–54
53
a residue. The residue was dissolved in CHCl3 (5 mL),
MeOH (10 mL) and 0.1 M aq HCl (4 mL). To this
solution was added another volume of CHCl3 (5 mL)
and 0.1 M aq HCl (5 mL) to separate the solution into
two phases. The lower CHCl3 phase was collected and
concentrated to give 18 (49 mg, 98%) as a white pow-
der: mp 194.0–196.5 °C; [h]2D3 −13.0° (c 0.3, CHCl3).
IR (cm−1): wmax(KBr) 3289 (broad), 3087, 2923, 2853,
MHz, 5:1 CD3OD–CDCl3): l 0.89 (12 H, t, J 6.8 Hz),
1.20–1.59 (82 H, m), 1.71–1.83 (2 H, m), 2.01 (3 H, s),
2.34 (1 H, dd, J 8.7, 14.6 Hz), 2.41 (1 H, dd, J 3.8, 14.6
Hz), 3.38–3.47 (4 H, m), 3.55 (1 H, t, J 9.3–10.2 Hz),
3.61–3.69 (5 H, m), 3.75–3.92 (4 H, m), 3.95–4.14 (5
H, m), 4.08, 4.25, (2 H, AB-q, J 16.8 Hz), 4.56–4.71 (2
H, m), 4.63 (1 H, d, J 7.6 Hz), 4.78 (1 H, d, J 3.3 Hz).
FABMS (positive-ion): m/z 1363 (M+Na)+, 1341
(M+H)+. HRFABMS (positive-ion); Calcd for
C70H134FN2NaO18P: 1363.9251. Found: 1363.9291.
Anal. Calcd for C70H134FN2O18P (1341.8): C, 62.66; H,
10.07; F, 1.42; N, 2.09; P, 2.31. Found: C, 62.58; H,
10.05; F, 1.34; N, 1.95; P, 2.14.
1
1733, 1654. H NMR (400 MHz, 1:1 CD3OD–CDCl3):
l 0.89 (12 H, t, J 6.8 Hz), 1.16–1.55 (82 H, m),
1.74–1.88 (2 H, m), 2.01 (3 H, s), 2.30–2.43 (2 H, m),
3.39–3.47 (5 H, m), 3.54 (1 H, t, J 10.7–8.8 Hz),
3.61–3.71 (6 H, m), 3.86–3.87 (5 H, m), 3.98 (1 H, m),
4.05–4.15 (3 H, m), 4.08, 4.25 (2 H, AB-q, J 16.6 Hz),
4.61 (1 H, d, J 4.9 Hz), 4.77 (1 H, d, J 2.9 Hz). FABMS
(positive-ion): m/z 1361 (M+Na)+, 1339 (M+H)+.
HRFABMS (positive-ion); Calcd for C70H135N2-
NaO19P: 1361.9294. Found: 1361.9294. Anal. Calcd for
C70H135N2O19P (1339.8): C, 62.75; H, 10.16; N, 2.09; P,
2.31. Found: C, 62.64; H, 10.01 N, 2.02; P, 2.21.
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(broad), 3091, 2924, 2854, 1734, 1655. H NMR (400
MHz, 5:1 CD3OD–CDCl3): l 0.89 (12 H, t, J 6.8 Hz),
1.28–1.59 (82 H, m), 1.75–1.83 (2 H, m), 2.01 (3 H, s),
2.34 (1 H, dd, J 8.6, 14.6 Hz), 2.41 (1 H, dd, J 3.7, 14.6
Hz), 3.35–3.46 (7 H, m, containing 3 H, s, at 3.41
ppm), 3.52–3.75 (9 H, m), 3.81–3.91 (4 H, m), 3.93–
4.11 (4 H, m), 4.07, 4.24 (2 H, AB-q, J 16.7 Hz), 4.58
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NaO19P: 1375.9451. Found: 1375.9497. Anal. Calcd for
C71H137N2O19P (1353.8): C, 62.99; H, 10.20; N, 2.07; P,
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phono-b- -glucopyranosyl]-2-deoxy-2-[(R)-3-hydroxy-
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glucopyranoside (20)
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+19.1° (c 0.3, CHCl3). IR (cm−1): wmax(KBr) 3280
1
(broad), 3093, 2924, 2854, 1733, 1654. H NMR (400

54
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