Concise synthesis of a probe molecule enabling analysis and imaging of vizantin

Article abstract of DOI:10.1248/cpb.c13-00006

Trehalose 6,6'-dicorynomycolate (TDCM) was first characterized in 1963 as a cell surface glycolipid of Corynebacterium spp. by Ioneda and co-workers. TDCM shows potent anti-tumor activity due to its immunoadjuvant properties. Furthermore, the toxicity of

Full text of DOI:10.1248/cpb.c13-00006

4
52
Regular Article
Chem. Pharm. Bull. 61(4) 452–459 (2013)
Vol. 61, No. 4
Concise Synthesis of a Probe Molecule Enabling Analysis and Imaging of
Vizantin
#
Hirofumi Yamamoto,* Masataka Oda, Mayo Nakano, Kenta Yabiku, Masahiro Shibutani,
Toshiyuki Nakanishi, Midori Suenaga, Masahisa Inoue, Hiroshi Imagawa, Masahiro Nagahama,
Yoichi Matsunaga, Seiichiro Himeno, Kojun Setsu, Jun Sakurai, and Mugio Nishizawa
Faculty of Pharmaceutical Sciences, Tokushima Bunri University; Yamashiro-cho, Tokushima 770–8514, Japan.
Received December 30, 2012; accepted January 28, 2013
Trehalose 6,6′-dicorynomycolate (TDCM) was first characterized in 1963 as a cell surface glycolipid of
Corynebacterium spp. by Ioneda and co-workers. TDCM shows potent anti-tumor activity due to its immuno-
adjuvant properties. Furthermore, the toxicity of TDCM in mice is much weaker than the related trehalose
diester of mycolic acid; trehalose 6,6′-dimycolate (TDM, formerly known as cord factor). We have investi-
gated the chemical modification of this class of compound to generate novel agents that display increased
immunoadjuvant activity with minimal associated toxicity. During the course of this work we recently devel-
oped 6,6′-bis-O-(3-nonyldodecanoyl)-α,α′-trehalose (designated as vizantin). Our results show that vizantin
exhibited a potent prophylactic effect on experimental lung metastasis of B16-F0 melanoma cells without a
loss of body weight and death in mice. Furthermore, vizantin effectively stimulated human macrophages in
an in vitro model, making it a promising candidate for a safe adjuvant in clinical applications. In order to
elucidate the pharmacokinetics of vizantin, a probe molecule with similar activity was developed on the basis
of a structure–activity relationship (SAR) study with vizantin. The distribution of the probe molecule after
intravenous administration into a mouse was assessed by macro confocal microscopy, where it was found to
accumulate in the lungs and liver.
Key words vizantin; concise synthesis; probe molecule; pharmacokinetics
The innate immune system plays a key role in biophylaxis human macrophages (a type of phagocyte critical to the innate
by detecting the presence of microbial invaders and activating immune system), it is a promising candidate for clinical appli-
1)
a protective reaction. Compounds that form integral parts of cations. In order to investigate the pharmacokinetics of vizan-
the pathogens are rapidly perceived as danger signals by the tin, we decided to synthesize a fluorescent-labeled derivative
host innate immune system. Recognition of these molecular of vizantin 4 as a probe molecule that retains a similar activity
2)
patterns is mediated by sets of highly conserved receptors,
whose activation results in acute defense responses. These
responses include the production of a diverse set of cytokines, Results and Discussion
to the non-fluorescent form 3.
direct local attack against the invading pathogen, and the
To identify a suitable position for the installation of a
activation of phagocytes and/or leukocytes. In 1982, Azuma fluorescent functional group into vizantin by focusing on
and co-worker reported that trehalose 6,6ꢀ-dimycolate (TDM), the sugar unit, specifically, sugar isomers 11, 18 and 25 that
which is a major constituent of the outer surface membrane of
3,4)
Mycobacterium tuberculosis, boosted the potency of these
immune responses and enhanced host resistance to patho-
5,6)
gens. The activated phagocytes and leukocytes act nonspe-
cifically not only against microorganisms but also abnormal
cells. Thus, TDM shows potent anti-tumor activity. This effect
was elucidated with in vivo models of several murine tumor
7
–9)
cells. However, TDM is unsuitable for practical use because
it over-activates the immune response in mice leading to seri-
10,11)
ous symptoms of septic shock.
We are interested in identifying compounds that display
increased immunological activity with no associated toxic-
1
2,13)
ity, and recently developed vizantin (3)
munostimulating compound through
relationship (SAR) study with trehalose 6,6ꢀ-dicorynomycolate
as a safe im-
a
structure–activity
14,15)
(
TDCM),
which is a shorter carbon chain analog of TDM.
Vizantin can be synthesized easily on a large scale and ex-
hibits a more potent prophylactic effect on experimental lung
metastasis of B16-F0 melanoma cells than TDCM without
12)
causing toxicity.
Because vizantin effectively stimulates
The authors declare no conflict of interest.
#
Equal contribution to first author.
Fig. 1. TDM, TDCM, Vizantin, and the Fluorescent-Labeled Vizantin 4
*
To whom correspondence should be addressed. e-mail: hirofumi@ph.bunri-u.ac.jp
© 2013 The Pharmaceutical Society of Japan

April 2013
453
Reagents and conditions: (a) 2-ethynylbenzoic acid, EDCI, DMAP, MS 4A, CH Cl , rt, 8h; (b) Hg(OTf) , (CH Cl) , 60°C, 15min; (c) MeONa, MeOH, rt, 30min; (d)
2
2
2
2
2
3
-nonyldodecanoic acid, EDCI, DMAP, MS 4A, CH Cl , reflux, 4h; (e) Pd(OH) /C, CHCl –MeOH (2:1), rt, 4h.
2 2 2 3
Chart 1. Synthesis of Mannosyl Type Analog 11
Reagents and conditions: (a) Hg(OTf) , toluene, 60°C, 20min; (b) MeONa, MeOH, rt, 30min; (c) 3-nonyldodecanoic acid, EDCI, DMAP, MS 4A, CH Cl , reflux, 4h; (d)
2
2
2
Pd(OH) /C, CHCl –MeOH (2:1), rt, 6h; (e) Hg(OTf) , (CH Cl) , 60°C, 15min.
2
3
2
2
2
Chart 2. Syntheses of Allosyl and Galactosyl Type Analogs 18 and 25
consist of α-d-mannose, α-d-allose and α-d-galactose were anomeric hydroxyl group of 6 was condensed with 2-ethy-
17)
prepared. Initially, the synthetic route to 11 is illustrated in nylbenzoic acid to provide 7 as a glycosyl donor mol-
Chart 1. 2,3,4-Tri-O-benzyl-6-acetyl-α-d-mannnose (6) was ecule, and the coupling reaction of 6 with 7 was carried out
1
8)
19)
prepared from commercially available α-methyl-d-mannoside using 5mol% of mercuric triflate in 1,2-dichloroethane.
(
16)
5) in four-steps according to the reported procedure. The This reaction proceeded smoothly at 60°C and the desired

4
54
Vol. 61, No. 4
Fig. 2. MIP-1β and TNF-α Releasing Activities
5
The THP-1 (5.0×10 cells) was incubated with 100µm vizantin (3), 11, 18 or 25 at 37°C for 3h. MIP-1β and TNF-α in culture supernatants were determined using ELISA
kits. In assay B, E. coli LPS (0.5µg) was employed as a positive control that caused the release of 782± 15pg/mL of TNF-α from THP-1. Values represent mean± S.D.
(n=5). *p<0.01, compared with the vehicle group (DMSO).
α,α-dimer 8 was obtained selectively in 92% yield. The acyl
groups of 8 were hydrolyzed with excess sodium methylate
(
MeONa), and the resulting hydroxyl groups were condensed
2
0)
with 3-nonyldodecanoic acid by the Keck procedure with
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
EDCI) and 4-dimethylaminopyridine (DMAP) to give 10.
1
(
Reaction was conducted with DAOC (17.4µmol), vizantin (3) (86.9µmol), EDCI
86.9µmol), and DMAP (17.4µmol).
Finally, hydrogenolysis over 20mol% of palladium hydroxide
resulted in the deprotection of all benzyl ethers to provide the
desired mannosyl type 11.
(
Chart 3. Concise Synthesis of Compound 4
The synthesis of allosyl type 18 was also achieved by fol-
lowing a similar procedure to that used for 11 (Chart 2A).
When anomeric mixtures 12 (α/β=3:2) and 13 (α/β=2:1)
were used as glycosyl donor and acceptor for the coupling
reaction, a 2:1 mixture of α,α-dimer 14 and α,β-dimer 15
21)
was generated in 66% yield. However, the desired 14 was
easily separated by column chromatography and hydrolyzed
using MeONa to give 16. Condensation was then performed
with 3-nonyldodecanoic acid using EDCI and DMAP. After
cleavage of the benzyl groups in 17 the allosyl type 18 was af-
forded. The glycosylation of 19 (α/β=3:2) with 20 (α/β=1:0)
also gave the α,α-dimer 21 along with the α,β-dimer 22 as a
minor product (Chart 2B). However, 21 could be easily iso-
lated by column chromatography using the same procedure as
described for 14, leading to the formation of 25 in good yield.
In the course of investigating the adjuvant properties of
vizantin, we found that this compound activated human mono-
cyte macrophage cells (THP-1), which induce macrophage
Fig. 3. MIP-1β Releasing Activities of Vizantin (3) and 4
5
The THP-1 (5.0×10 cells) was incubated with 100µm 3 or 4 at 37°C for 3h.
MIP-1β in culture supernatants was determined using ELISA kit. Values represent
mean± S.D. (n=5). *p<0.01, compared with the vehicle group (DMSO).
2
3)
inflammatory protein-1β (MIP-1β) but not tumor necrosis methylamino)-2-oxo-2H-chroman-4-yl] acetic acid (hereafter
12)
factor-α (TNF-α). TNF-α has substantial proinflammatory referred to as DAOC).
activity, which may lead to serious complications such as a The reactivity order for the secondary hydroxyl groups on
2
2)
24)
cytokine storm. Thus, it is a significant finding that vizantin each pyranoside was reported by Williams and Richardson.
induces almost no TNF-α. The sugar isomers 11, 18 and 25 Their study suggested that the 2-hydroxyl group displayed
were investigated with respect to the induction of TNF-α as the highest reactivity of all the secondary hydroxyl groups
well as MIP-1β from THP-1 cells. As can be seen in Fig. 2A, on α-d-glucoside. Thus, we hypothesized that compound 4
only 11 showed comparable potency to vizantin for MIP-1β might be directly derived from vizantin by the esterification of
inducing activity, although the efficacy of 11 was slightly DAOC. Initially, the esterification of DAOC with five equiva-
lower than that of vizantin. Even in the assay of TNF-α induc- lents of vizantin was carried out using EDCI and DMAP in
tion, 11 acted like vizantin to show very little activity (Fig. dichloromethane. However, a complex mixture including a
2
B). Allosyl type 18 as well as galactosyl type analog 25 were small amount of 4 was generated because of low selectivity.
practically inactive in these assays. The use of diethyl ether, nitromethane, and aromatic solvents
The observation that the mannosyl type analog 11 showed such as toluene and benzene also afforded a complex mixture.
similar activity to vizantin suggested that the 2-hydroxyl group Intriguingly, by contrast N,N-dimethylformamide showed
on the trehalose unit of vizantin might be a suitable position remarkable reactivity giving rise to the desired 4 as a major
for the installation of a fluorescent functional group. Therefore, product (Chart 3). Although the exact effect of the solvent was
2
5)
we decided to aim for the synthesis of 4 linked with 2-[7-(di- unclear, 4 could be derived from vizantin in 34% yield.

April 2013
455
Despite substitution with a sterically bulky functional performed using 63–210 mesh silica gel 60 (Kanto Chemical
group, 4 displayed MIP-1β inducing activity as effective as Co., Inc.). Specific rotations were determined on a JASCO
vizantin without TNF-α induction (Fig. 3).
Inspired by this finding, the localization of 4 after admin- 589nm) at specific temperatures and are reported as fol-
istration to a mouse was examined (Fig. 4). When 500µg lows: [α]D , concentration (c=g/100mL), and solvent. FT-IR
of 4 was administered intravenously into a C57BL/6 mouse, spectra were measured on a JASCO FT/IR-410 infrared
P-1030 digital polarimeter using the sodium D line (λ=
temp.
−1
most of the injected 4 accumulated in lung and liver tissue spectrophotometer and reported as wavenumber (cm ) of sig-
1
13
within 24h. Other tissues (brain, spleen and kidney) were also nificant peaks. H- and C-NMR spectra were recorded on a
evaluated using macro confocal microscopy, but no fluorescent Varian Mercury plus-300-4N spectrometer, a Varian 400-MR
signal corresponding to 4 could be detected. In addition, the spectrometer, a Varian 500-MR spectrometer and a Varian
1
administration of DAOC did not show fluorescent signal in Unity-600 spectrometer. The H chemical shifts are reported
lung and liver. The tissue distribution of 4, especially its ac- in parts per million (ppm) from an internal standard of tetra-
1
2)
cumulation in the lungs, are consistent with the observation
methylsilane (TMS) (0.00ppm), residual CHCl (7.26ppm),
3
13
that vizantin showed strong inhibition against the lung metas- or C H N (7.19ppm, right peak), and the C chemical shifts
5
5
tasis of B16-F0 melanoma cells. Hence, it is conceivable that 4 were reported using an internal standard of CDCl (77.03ppm,
3
acts as an accurate probe molecule of vizantin even in the in central peak) or C D N (123.50ppm, central peak in right-side
5
5
1
vivo model. We believe these findings are significant in terms triplet). The H-NMR spectra are reported as follows: chemi-
of improving our understanding of the pharmacokinetics of cal shift, multiplicity (s=singlet, d=doublet, t=triplet, q=
vizantin.
quartet, qui=quintet and br=broad), relative integral, coupling
In conclusion, a novel vizantin derivative 4 with a fluores- constants J (Hz). High-resolution mass spectra (HR-MS) were
cent functional group was developed through a SAR study recorded on a JEOL the Mstation JMS-700.
with sugar isomers of vizantin. The distribution of 4 after in-
6-O-Acetyl-2,3,4-tri-O-benzyl-α-mannosyl
2-Ethynyl-
travenous administration into a mouse was assessed by macro benzoate (7) The mixture of 6 (100mg, 203µmol), 2-ethy-
confocal microscopy, where it was found to accumulate in the nylbenzoic acid (36mg, 244µmol), molecular sieve 4A pow-
lungs and liver. We believe 4 is a promising tool for inves- der (MS 4A) (200mg), DMAP (5mg, 40.6µmol) and EDCI
tigating the target site(s) of vizantin. These experiments are (58mg, 305µmol) in CH Cl2 (2mL) was stirred for 8h at
2
currently underway.
room temperature. The resulting mixture was filtered using
celite 535RVS, and the filtrate was washed with brine. The
separated organic layer was dried over anhydrous MgSO4
Experimental
General Reactions were performed under argon unless and concentrated under reduced pressure. The residue was
otherwise stated. Commercially available chemicals were used purified by flash column chromatography (silica gel, hexane–
1
9
as purchased. Special grade and dehydrated solvents were EtOAc, 10:1) to give 7 (101mg, 80%) as a brown syrup. [α]
D
purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). The +22.4 (c=1.0, CHCl ); FT-IR (neat) 3280, 3088, 3063, 3031,
3
−1
1
progress of the reaction was monitored by thin layer chro- 2871, 2107, 1955, 1879, 1787, 1739cm ; H-NMR (500MHz
matography (TLC) using precoated silica gel 60 F254 plates in CDCl ) δ: 2.05 (3H, s), 3.26 (1H, s), 3.90 (1H, t, J=2.0Hz),
3
(
Merck, Darmstadt, Germany). Spots were visualized under 4.05 (1H, q, J=9.0Hz), 4.09 (1H, m), 4.12 (1H, dd, J=9.0,
UV illumination (254nm) after immersion in 2% p-anisalde- 3.0Hz), 4.34 (2H, m), 4.56 (1H, d, J=11.5Hz), 4.60 (1H, d,
hyde and 5% H SO in EtOH, followed by heating on a hot J=11.5Hz), 4.62 (1H, d, J=10.5Hz), 4.78 (1H, d, J=12.0Hz),
2
4
plate. Solvents were removed under reduced pressure using a 4.84 (1H, d, J=12.0Hz), 4.98 (1H, d, J=10.5Hz), 6.50 (1H,
standard rotary evaporator. Flash column chromatography was d, J=2.0Hz), 7.26–7.44 (16H, m), 7.52 (1H, dt, J=7.5, 1.0Hz),
Fig. 4. Analyses of the Localization of Fluorescent-Labeled Vizantin 4
Right pictures are lung and liver tissue treated with 4, respectively. Middle pictures are lung and liver tissue treated with DAOC instead of 4, and left is the vehicle con-
trol (saline including 1.1% Tween 80, 5.6% mannitol and 20% mineral oil). These localizations were evaluated using a macro confocal microscope 24 h after administration
of the compound.

4
56
Vol. 61, No. 4
7
.62 (1H, dd, J=7.5, 1.0Hz), 7.89 (1H, dd, J=7.5, 1.0Hz); J=9.6Hz), 4.19 (2H, dd, J=12.0, 1.8Hz), 4.32 (2H, dd, J=12.0,
1
3
C-NMR (100MHz in CDCl ) δ: 20.92, 63.22, 71.85, 72.48, 4.8Hz), 4.55 (2H, d, J=10.2Hz), 4.55 (4H, s), 4.63 (2H, d,
3
7
1
1
2.85, 73.22, 73.85, 75.36, 78.79, 82.50, 83.04, 92.64, 122.67, J=12.6Hz), 4.69 (2H, d, J=12.6Hz), 4.93 (2H, d, J=10.2 Hz),
13
27.80, 127.82, 127.86, 127.89, 127.98, 128.11, 128.43, 128.48, 5.10 (2H, d, J=2.1Hz), 7.22–7.37 (30H, m); C-NMR (75MHz
28.69, 131.02, 131.40, 132.42, 135.29, 137.73, 137.93, 138.07, in CDCl ) δ: 14.14, 22.69, 26.54, 29.34, 29.65, 29.95, 31.91,
3
+
164.03, 170.92; HR-MS (FAB ) m/z Calcd for C H O Na 33.78, 34.87, 39.13, 62.93, 70.62, 72.11, 72.50, 73.74, 74.34,
38
36
8
+
(
M +Na) 643.2308, Found 643.2332.
75.55, 79.30, 93.18, 127.79, 127.91, 127.97, 128.22, 128.45,
+
6
-O-Acetyl-2,3,4-tri-O-benzyl-α-mannosyl 6′-O-Acetyl- 128.50, 137.65, 137.97, 173.48; HR-MS (FAB ) m/z Calcd for
+
2′,3′,4′-tri-O-benzyl-α′-mannoside (8)
A
mixture of
6
C H O Na (M +Na) 1522.0010, Found 1521.9985.
96 138 13
(48mg, 96.7µmol) and 7 (50mg, 80.6µmol) in (CH Cl)2
6-(3-Nonyldodecanoyl)-α-mannosyl 6′-(3-Nonyldodeca-
2
(
50 µL) was added to mercuric triflate (2.0mg, 4.03µmol) at noyl)-α′-mannoside (11) Pd(OH) /C (60mg, 20w/w%) was
2
room temperature. The mixture was stirred for 15min at 60°C added to a solution of 10 (300mg, 200µmol) in CHCl –MeOH
3
and then quenched with triethylamine. After filtration through (2:1, 2mL). The mixture was stirred vigorously for 4h at
silica gel using AcOEt, the filtrate was concentrated under room temperature under H (1atm) atmosphere, filtered and
2
reduced pressure. The residue was purified by flash column the concentrated residue purified by flash chromatography
chromatography (silica gel, hexane–EtOAc, 4:1) to give 8 (silica gel, CH Cl –MeOH, 10:1) to give 11 (135mg, 71%)
2
2
2
6
19
(
72mg, 92%) as a colorless syrup. [α]D +39.8 (c=0.6, CHCl3); as a colorless syrup. [α]D +30.8 (c=1.0, CHCl ); FT-IR (neat)
3
−1 1
FT-IR (neat) 3087, 3063, 3030, 2939, 2871, 1955, 1878, 1806, 3479, 3347, 2953, 2925, 2854, 1714cm ; H-NMR (400MHz
−1 1
1
(
(
740 cm ; H-NMR (600MHz in CDCl ) δ: 2.03 (6Η, s), 3.55 in C D N) δ: 0.85 (12H, t, J=6.4Hz), 1.25 (64H, m), 2.02
3
5
5
2H, ddd, J=9.5, 5.0, 2.2Hz), 3.58 (2H, dd, J=3.2, 1.7Hz), 3.72 (2H, m), 2.29 (4H, dd, J=6.8, 3.2Hz), 4.44 (2H, m), 4.52
2H, dd, J=9.5, 3.2Hz), 3.91 (2H, dd, J=9.5, 9.5Hz), 4.20 (2H, (2H, dd, J=9.2, 3.2Hz), 4.60 (4H, m), 4.78 (2H, dd, J=12.0,
dd, J=12.0, 2.2Hz), 4.28 (2H, dd, J=12.0, 5.0Hz), 4.56 (2H, 6.4Hz, H-6), 4.96 (2H, dd, J=12.0, 2.0Hz, H-2), 5.94 (2H,
13
s), 4.57 (2H, d, J=10.5Hz), 4.65 (2H, d, J=12.2Hz), 4.69 (2H, d, J=1.2Hz, H-1); C-NMR (100MHz in C D N) δ: 14.29,
5
5
d, J=12.2Hz), 4.93 (2H, d, J=10.5Hz), 5.10 (2H, d, J=1.7Hz), 22.94, 26.84, 26.88, 29.63, 29.94, 30.25, 32.14, 34.10, 35.16,
13
7
6
1
(
.24–7.36 (30H, m); C-NMR (50MHz in CDCl ) δ: 20.9, 39.26, 64.65, 68.61, 71.94, 72.79, 72.96, 96.94, 173.63; HR-MS
3
+
+
3.3, 70.6, 72.1, 72.5, 73.9, 74.2, 75.4, 79.3, 93.2, 127.8–128.5, (FAB ) m/z Calcd for C H O Na (M +Na) 981.7214, Found
54 102 13
+
37.6, 137.9, 170.7; HR-MS (FAB ) m/z Calcd for C H O Na 981.7210.
58 62 13
+
M +Na) 989.4084, Found 989.4085.
,3,4-Tri-O-benzyl-α-mannosyl 2′,3′,4′-Tri-O-benzyl-α′- (13) This compound was synthesized from 12 (200mg,
mannoside (9) 28% MeONa solution in MeOH (4.6mL, 406µmol) according to a procedure similar to that described
6-O-Acetyl-2,3,4-tri-O-benzylallosyl 2-Ethynylbenzoate
16)
2
1
9
2
4.8mmol) was added to a solution of 8 (1.60g, 1.65mmol) for 7. 13: 79% yield, brown syrup, [α]D +56.1 (c=1.0, CHCl3);
in MeOH (20mL) at room temperature. The mixture was FT-IR (neat) 3273, 3087, 3064, 3031, 3005, 2939, 2871, 2106,
stirred for 30min and then amberlite IR-120B was added. The 1958, 1875, 1806, 1740, 1732cm ; H-NMR (400MHz in
−1
1
filtered and concentrated residue was purified by flash column CDCl ) δ: 1.97 (3H, s), 2.03 (3H, s), 3.10 (1H, s), 3.40 (1H, s),
3
chromatography (silica gel, hexane–EtOAc, 2:1) to give 9 3.69 (1H, brq, J=4.8Hz), 3.92 (1H, m), 4.03 (1H, dd, J=5.6,
2
2
(
1.32g, 92%) as a colorless syrup. [α]D +59.7 (c=1.1, CHCl3); 4.0Hz), 4.10 (2H, m), 4.20 (2H, m), 4.28 (1H, dd, J=12.0,
FT-IR (neat) 3482, 3087, 3062, 3030, 2925, 2874, 1953, 1878, 4.4Hz), 4.40 (1H, dd, J=7.4, 4.4Hz), 4.57 (10H, m), 4.78 (1H,
−1
1
1
813, 1724cm ; H-NMR (400MHz in CDCl ) δ: 3.36 (2H, d, J=11.6Hz), 4.82 (1H, d, J=12.0Hz), 6.49 (1H, s), 6.58 (1H,
3
m), 3.58 (2H, dd, J=3.2, 1.7Hz), 3.72 (6H, m), 3.98 (2H, t, d, J=4.0Hz), 7.13–7.47 (32H, m), 7.59 (2H, t, J=8.0Hz), 7.78
13
J=9.4Hz), 4.58 (4H, s), 4.60 (2H, d, J=12.1Hz), 4.65 (2H, d, (1H, d, J=8.0Hz), 8.06 (1H, d, J=8.0Hz); C-NMR (100MHz
J=10.4Hz), 4.74 (2H, d, J=12.1Hz), 4.94 (2H, d, J=11.0Hz), in CDCl ) δ: 20.86, 62.67, 63.08, 72.13, 72.25, 72.72, 73.02,
3
13
5
.00 (2H, d, J=1.7Hz), 7.24–7.35 (30H, m); C-NMR (50MHz 73.53, 74.92, 76.29, 76.44, 78.73, 79.05, 81.96, 81.99, 82.59,
in CDCl ) δ: 61.9, 72.3, 72.9, 73.0, 74.0, 74.3, 75.4, 79.3, 93.5, 82.74, 85.73, 94.89, 99.72, 122.68, 123.01, 127.50, 127.59,
3
+
1
27.7–128.4, 137.8, 138.0, 138.1; HR-MS (FAB ) m/z Calcd for 127.64, 127.74, 127.90, 127.91, 127.94,127.99, 128.10, 128.20,
+
C H O Na (M +Na) 905.3871, Found 905.3865.
128.29, 128.38, 128.42, 128.45, 128.47, 128.51, 128.58, 130.62,
5
4
58 11
6
-(3-Nonyldodecanoyl)-2,3,4-tri-O-benzyl-α-mannosyl 131.38, 131.42, 131.86, 132.12, 134.80, 135.09, 137.13, 137.34,
6
′-(3-Nonyldodecanoyl)-2′,3′,4′-tri-O-benzyl-α′-mannoside 137.48, 137.64, 138.13, 138.25, 164.51, 164.67, 170.59, 170.73;
+
+
(
10) The mixture of 3-nonyldodecanoic acid (92mg, HR-MS (FAB ) m/z Calcd for C H O Na (M +Na) 643.2308,
38
36
8
2
83µmol), 9 (100mg, 280µmol), MS 4A (300mg), DMAP Found 643.2290.
6-O-Acetyl-2,3,4-tri-O-benzyl-α-allosyl6′-O- A c e t y l - 2 ′ ,3 ′ ,4 ′ -
3mL) was stirred for 6h under reflux. The resulting mixture tri-O-benzyl-α -alloside (14), 6-O-Acetyl-2,3,4-tri-O-
(14mg, 280µmol) and EDCI (87mg, 453µmol) in CH Cl2
2
(
′
was filtered using celite 535RVS, and the filtrate was washed benzyl-α-allosyl 6′-O-Acetyl-2′,3′,4′-tri-O-benzyl-α′-alloside
with brine. The separated organic layer was dried over an- (15) These compounds were synthesized from 12 (19mg,
hydrous MgSO4 and concentrated under reduced pressure. 38.7 µmol) and 13 (20mg, 32.2µmol) according to a procedure
The residue was purified by flash chromatography (silica gel, similar to that described for 8. 14: 44% yield, colorless syrup,
2
1
hexane/EtOAc, 20:1) to give 10 (167mg, 99%) as a color- [α] +111.6 (c=0.4, CHCl ); FT-IR (neat) 3087, 3062, 3030,
D
3
19
−1
1
less syrup. [α] +27.8 (c=1.0, CHCl ); FT-IR (neat) 3087, 2931, 2871, 1957, 1876, 1809, 1741cm ; H-NMR (300MHz
063, 3031, 2925, 2853, 1949, 1871, 1806, 1737cm ; H-NMR in CDCl ) δ: 1.97 (6H, s), 3.73 (2H, m), 3.90 (2H, dd, J=6.0,
300MHz in CDCl ) δ: 0.87 (12H, t, J=6.3Hz), 1.21 (64H, m), 3.9Hz), 4.08 (4H, m), 4.14 (2H, dd, J=11.7, 4.8Hz), 4.34
.85 (2H, m), 2.24 (4H, d, J=6.9Hz), 3.53 (2H, m), 3.57 (2H, (2H, dd, J=4.5, 3.6Hz), 4.48 (2H, d, J=11.7Hz), 4.55 (2H, d,
D
3
−1 1
3
3
(
3
1
dd, J=2.7, 2.1Hz), 3.71 (2H, dd, J=9.6, 2.7Hz), 3.93 (2H, t, J=11.7Hz,), 4.57 (2H, d, J=11.7Hz), 4.63 (2H, d, J=11.7Hz),

April 2013
457
2
1
4
.66 (2H, d, J=11.7Hz), 4.85 (2H, d, J=11.7Hz), 5.51 (2H, d, that described for 11. 18: 60% yield, colorless syrup, [α]
D
13
J=3.9Hz), 7.18–7.36 (30H, m); C-NMR (75MHz in CDCl3) +69.0 (c=0.3, CHCl ); FT-IR (neat) 3398, 2953, 2924, 2854,
δ: 20.85, 63.35, 72.25, 72.29, 73.37, 75.58, 76.94, 77.35, 82.44, 1739 cm ; H-NMR (400MHz in C D N) δ: 0.83 (12H, t,
3
−
1
1
5
5
9
1
7.78, 127.44, 127.49, 127.73, 127.85, 127.93, 128.14, 128.19, J=6.4Hz), 1.27 (64H, m), 2.02 (2H, m), 2.34 (4H, d, J=6.8Hz),
+
28.34, 138.08, 138.19, 138.23, 170.70; HR-MS (FAB ) m/z 4.37 (2H, dd, J=11.2, 4.4Hz), 4.59 (2H, dd, J=6.4, 4.4Hz),
+
Calcd for C H O Na (M +Na) 989.4102, Found 989.4116. 4.63 (2H, dd, J=11.2, 7.2Hz), 4.70 (2H, dd, J=11.2, 3.6Hz),
58
62 13
19
1
5: 22% yield, colorless syrup, [α]D +32.9 (c=0.3, CHCl3); 4.81 (2H, dd, J=5.2, 2.0Hz), 4.87 (2H, dd, J=6.8, 2.0Hz), 5.87
13
FT-IR (neat) 3087, 3063, 3031, 2928, 2870, 1955, 1875, 1810, (2H, d, J=4.4Hz); C-NMR (100MHz in C D N) δ: 14.29,
5
5
−1 1
1739 cm ; H-NMR (500MHz in CDCl ) δ: 1.99 (3H, s), 2.00 22.93, 26.86, 29.60, 29.89, 29.92, 30.22, 32.11, 34.14, 35.25,
3
(
3H, s), 3.60 (1H, q, J=4.0Hz), 3.70 (1H, m), 3.77 (1H, dd, 39.31, 66.60, 70.24, 70.35, 73.22, 87.65, 98.55, 173.54; HR-MS
+
+
J=6.0, 4.0Hz), 3.98 (1H, dd, J=6.5, 2.5Hz), 4.02 (2H, m), (FAB ) m/z Calcd for C H O Na (M +Na) 981.7233, Found
4
dd, J=12.0, 4.5Hz), 4.27 (1H, dd, J=4.0, 2.5Hz), 4.29 (1H,
dd, J=6.5, 5.0Hz), 4.38 (1H, dd, J=12.0, 3.0Hz), 4.46 (1H, d, benzoate (20) This compound was synthesized from 19
J=11.5Hz), 4.46 (1H, d, J=11.5Hz), 4.52 (1H, d, J=11.5Hz), (200mg, 406µmol) using toluene as a reaction solvent, ac-
4
5
4
102 13
.11 (1H, q, J=4.5Hz), 4.13 (1H, dd, J=11.5, 4.5Hz), 4.21 (1H, 981.7248.
6-O-Acetyl-2,3,4-tri-O-benzyl-α-galactosyl
2-Ethynyl-
16)
.52 (5H, m), 4.58 (1H, d, J=11.5Hz), 4.60 (1H, d, J=11.5Hz), cording to a procedure similar to that described for 7. 20:
14
4
.63 (1H, d, J=11.5Hz), 4.64 (1H, d, J=11.5Hz), 5.14 (1H, 84% yield, brown syrup, [α]D +56.0 (c=0.8, CHCl ); FT-IR
3
d, J=4.5Hz), 5.29 (1H, d, J=2.5Hz), 7.20–7.34 (30H, m); (neat) 3280, 3087, 3063, 3031, 2878, 2107, 1954, 1879, 1806,
13
−1 1
C-NMR (125MHz in CDCl ) δ: 20.83, 20.97, 62.5, 62.99, 1741 cm ; H-NMR (500MHz in CDCl ) δ: 1.96 (3H, s), 3.21
3
3
7
7
1
1
2.02, 72.06, 72.26, 72.38, 72.60, 73.49, 74.68, 76.96, 77.64, (1H, s), 3.95 (1H, d, J=2.0Hz), 4.05 (1H, dd, J=10.5, 3.0Hz),
8.27, 78.93, 80.75, 81.17, 83.72, 127.55, 127.62, 127.70, 127.74, 4.13 (2H, m), 4.21 (1H, brt, J=6.0Hz), 4.29 (2H, dd, J=10.0,
27.83, 127.92, 127.95, 128.03, 128.10, 128.27, 128.29, 128.34, 4.0Hz), 4.66 (1H, d, J=11.5Hz), 4.72 (1H, d, J=11.5Hz),
28.40, 128.46, 137.72, 137.80, 137.89, 138.10, 138.36, 170.66, 4.79 (2H, t, J=11.5Hz), 4.88 (1H, d, J=11.5Hz), 5.02 (1H, d,
+
+
170.86; HR-MS (FAB ) m/z Calcd for C H O Na (M +Na) J=11.5Hz), 6.72 (1H, d, J=4.0Hz), 7.26–7.42 (16H, m), 7.50
58
62 13
989.4102, Found 989.4131.
(1H, dt, J=7.5, 1.0Hz), 7.63 (1H, dd, J=7.5, 1.0Hz), 7.93 (1H,
13
2
,3,4-Tri-O-benzyl-α-allosyl-2′,3′,4′-tri-O-benzyl-α′- dd, J=7.5, 1.0Hz); C-NMR (125MHz in CDCl ) δ: 20.84,
3
alloside (16) This compound was synthesized from 14 63.26, 71.69, 73.22, 73.29, 74.39, 74.71, 75.56, 78.27, 82.47,
37mg, 38.7µmol) according to a procedure similar to that 82.54, 91.92, 122.62, 127.58, 127.69, 127.90, 127.98, 128.32,
(
2
2
described for 9. 16: 99% yield, colorless syrup, [α]D +135.5 128.42, 128.44, 128.60, 130.96, 132.05, 132.16, 135.25, 137.99,
+
(
2
c=0.3, CHCl ); FT-IR (neat) 3479, 3087, 3062, 3030, 2929, 138.01, 138.52, 164.49, 170.68; HR-MS (FAB ) m/z Calcd for
3
−
1
1
+
875, 1954, 1877, 1815cm ; H-NMR (300MHz in CDCl ) δ: C H O Na (M +Na) 643.2308, Found 643.2311.
.12 (2H, brt, J=5.4Hz), 3.61 (6H, m), 3.92 (2H, dd, J=5.7,
3
38 36
8
2
6-O-Acetyl-2,3,4-tri-O-benzyl-α-galactosyl 6′-O-Acetyl-
3
4
.6Hz), 4.09 (2H, t, J=5.7Hz), 4.35 (2H, dd, J=5.7, 3.3Hz), 2′,3′,4′-tri-O-benzyl-α′-galactoside (21), 6-O-Acetyl-2,3,4-
.45 (2H, d, J=11.7Hz), 4.53 (2H, d, J=11.7Hz), 4.60 (2H, d, tri-O-benzyl-α-galactosyl 6′-O- A c e t y l - 2 ′ ,3 ′ ,4 ′ - t r i - O-benzyl-
J=11.7Hz), 4.64 (2H, d, J=11.7Hz), 4.65 (2H, d, J=11.7Hz), α′-galactoside (22) These compounds were synthesized from
.88 (2H, d, J=11.7Hz), 5.23 (2H, d, J=3.6Hz), 7.20–7.37 19 (45mg, 90.9µmol) and 20 (47mg, 75.7µmol) according to
4
1
3
(
30H, m); C-NMR (75MHz in CDCl ) δ: 61.72, 72.30, 72.43, a procedure similar to that described for 8. 21: 65% yield,
3
2
4
7
1
3.35, 75.48, 79.07, 82.77, 97.76, 127.57, 127.63, 127.84, 128.12, colorless syrup, [α]D +83.5 (c=1.2, CHCl ); FT-IR (neat)
3
+ −1
28.17, 128.26, 128.47, 137.94, 138.13; HR-MS (FAB ) m/z 3087, 3062, 3030, 2912, 2866, 1954, 1879, 1811, 1745cm ;
+
1
Calcd for C H O Na (M +Na) 905.3884, Found 905.3891.
H-NMR (600MHz in CDCl ) δ: 1.85 (6H, s), 3.87 (2H, dd,
5
4
58 11
3
6
-(3-Nonyldodecanoyl)-2,3,4-tri-O-benzyl-α-allosyl J=2.7, 1.2Hz), 3.93 (2H, dd, J=11.0, 6.4Hz), 4.03 (2H, dd,
′-(3-Nonyldodecanoyl)-2′,3′,4′-tri-O-benzyl-α′-alloside (17) J=10.0, 2.7Hz), 4.11 (2H, dd, J=11.0, 6.6Hz), 4.13 (2H, dd,
6
This compound was synthesized from 16 (37mg, 38.3µmol) J=10.0, 3.7Hz), 4.21 (2H, brdd, J=6.6, 6.4Hz), 4.58 (2H, d,
according to a procedure similar to that described for 10. J=11.5Hz), 4.71 (2H, d, J=12.0Hz), 4.74 (2H, d, J=12.0Hz),
2
0
1
7: 97% yield, colorless syrup, [α]D +84.0 (c=1.0, CHCl3); 4.79 (2H, d, J=11.7Hz), 4.84 (2H, d, J=11.7Hz), 4.94 (2H,
FT-IR (neat) 3087, 3063, 3031, 2925, 2854, 1949, 1875, 1806, d, J=11.5Hz), 5.28 (2H, d, J=3.7Hz), 7.24–7.41 (30H, m);
739 cm ; H-NMR (300MHz in CDCl ) δ: 0.87 (12H, t,
−
1
1
13
1
C-NMR (50MHz in CDCl ) δ: 20.8, 63.1, 68.7, 73.0, 74.3,
3
3
J=6.3Hz), 1.24 (64H, m), 1.82 (2H, m), 2.17 (2H, d, J=6.9Hz), 74.6, 76.0, 78.7, 93.0, 127.3–128.3, 138.2, 138.4, 138.6, 170.2;
+
+
3
4
4
.74 (2H, m), 3.90 (2H, dd, J=6.0, 3.9Hz), 4.07 (4H, m), FAB-MS m/z (%) 989 (M +Na); HR-MS (FAB ) m/z Calcd
+
.20 (2H, dd, J=11.7, 4.2Hz), 4.34 (2H, dd, J=4.5, 3.6Hz), for C H O Na (M +Na) 989.4084, Found 989.4067. 22:
.47 (2H, d, J=11.7Hz), 4.54 (2H, d, J=11.7Hz), 4.57 (2H, d, 7% yield, colorless syrup, [α]D +228.2 (c=1.3, CHCl3);
58
62 13
2
3
J=11.7Hz), 4.65 (4H, d, J=11.7Hz), 4.85 (2H, d, J=11.7Hz), FT-IR (neat) 3087, 3062, 3030, 2912, 2871, 1954, 1875, 1811,
13
−1 1
5
.52 (2H, d, J=3.9Hz), 7.17–7.35 (30H, m); C-NMR (75MHz 1742 cm ; H-NMR (600MHz in CDCl ) δ: 1.91 (3H, s), 1.93
3
in CDCl ) δ: 14.14, 22.70, 26.54, 29.36, 29.64, 29.67, 29.94, (3H, s), 3.53 (1H, dd, J=10.0, 2.7Hz), 3.55 (1H, brt, J=6.4Hz),
3
3
8
1
1.92, 33.78, 34.95, 39.12, 63.36, 72.21, 72.30, 73.46, 75.72, 3.78 (1H, brd, J=2.7Hz), 3.87 (1H, dd, J=10.0, 7.8Hz), 3.93
2.49, 97.78, 127.37, 127.43, 127.65, 127.77, 127.88, 128.16, (1H, dd, J=2.4, 1.2Hz), 4.04 (1H, dd, J=10.5, 7.8Hz), 4.06
+
28.31, 138.17, 138.27, 138.31, 173.31; HR-MS (FAB ) m/z (1H, dd, J=7.6, 2.7Hz), 4.08 (1H, dd, J=5.4, 3.2Hz), 4.09–4.11
+
Calcd for C H O Na (M +Na) 1522.0020, Found 1522.0004. (3H, m), 4.30 (1H, brt, J=7.1Hz), 4.53 (1H, d, J=7.8Hz),
9
6
138 13
6
-(3-Nonyldodecanoyl)-α-allosyl 6′-(3-Nonyldodecanoyl)- 4.59 (1H, d, J=11.2Hz), 4.64 (1H, d, J=11.5Hz), 4.68 (1H, d,
α′-alloside (18) This compound was synthesized from J=12.0Hz), 4.72 (1H, d, J=11.7Hz), 4.75 (1H, d, J=12.0Hz),
7 (200mg, 133µmol) according to a procedure similar to 4.77 (1H, d, J=11.5Hz), 4.79 (2H, d, J=11.7Hz), 4.85 (1H, d,
1

4
58
Vol. 61, No. 4
J=11.7Hz), 4.94 (1H, d, J=11.5Hz), 4.95 (1H, d, J=11.2Hz), 86.9mmol) in DMF (0.35mL) was stirred for 24h at room
.05 (1H, d, J=11.5Hz), 5.19 (1H, d, J=3.2Hz), 7.17–7.40 (30H, temperature. After addition of distilled water the reaction
5
13
m); C-NMR (50MHz in CDCl ) δ: 20.7, 62.3, 63.3, 68.8, mixture was extracted with CH Cl . The extract was dried
3
2
2
72.6, 73.0, 73.3, 73.5, 74.3, 74.4, 74.6, 74.7, 75.8, 78.7, 78.9, over anhydrous MgSO4 and concentrated under reduced
8
2.2, 100.1, 103.8, 127.4–128.6, 138.0, 138.2, 138.3, 138.6, pressure. The residue was purified by flash chromatography
+
138.8, 170.3, 170.6; HR-MS (FAB ) m/z Calcd for C H O Na (silica gel, CH Cl –MeOH, 15:1) and then HPLC (YMC Pack
5
8
62 13
2
2
+
(
M +Na) 989.4084, Found 989.4023.
D-SIL-5 250×20mm column, CHCl –MeOH, 20:1, 1.0mL/
3
2
,3,4-Tri-O-benzyl-α-galactosyl-2′,3′,4′-tri-O-benzyl-α′- min, detection at 245nm, t 22.2min) to give mono-labeled 4
R
2
3
galactoside (23) This compound was synthesized from (7.0mg, 34%) as a yellow syrup. [α]D +50.0 (c=0.3, CHCl3);
1 (500mg, 505µmol) according to a procedure similar to λex 430nm, λem 463nm; FT-IR (neat) 3398, 2953, 2925, 2854,
2
2
2
−1
1
that described for 9. 23: 92% yield, colorless syrup, [α]D
1731, 1695, 1614 cm ; H-NMR (500MHz in C D N) δ: 0.85
5 5
+
116.5 (c=1.6, CHCl ); FT-IR (neat) 3481, 3087, 3063, 3031, (6H, t, J=6.5Hz), 0.86 (6H, t, J=6.5Hz), 1.25 (64H, m), 2.03
3
−1
1
2
935, 2885, 1953, 1877, 1812, 1722cm ; H-NMR (400MHz (1H, m), 2.09 (1H, m), 2.37 (2H, dd, J=7.0, 4.5Hz), 2.49
in CDCl ) δ: 3.44 (2H, br dd, J=9.9, 2.8Hz), 3.54 (2H, dd, (2H, dd, J=7.0, 3.0Hz), 2.76 (6H, s), 4.02 (1H, d, J=16.0Hz),
3
J=11.0, 5.5Hz), 3.93 (2H, brs), 3.97 (2H, dd, J=9.9, 2.8Hz), 4.08 (1H, d, J=16.0Hz), 4.09 (1H, t, J=9.5Hz), 4.22 (1H, t,
4
(
.03 (2H, brt, J=5.5Hz), 4.10 (2H, dd, J=9.9, 3.3Hz), 4.61 J=9.5Hz), 4.31 (1H, dd, J=9.5, 3.5Hz), 4.71 (1H, m), 4.78
2H, d, J=11.6Hz), 4.69 (2H, d, J=11.6Hz), 4.75 (2H, d, (1H, t, J=9.5Hz), 4.83 (2H, dd, J=12.0, 5.0Hz), 4.87 (1H,
J=12.1Hz), 4.77 (2H, d, J=12.1Hz), 4.83 (2H, d, J=11.6Hz), dd, J=11.5, 7.0Hz), 4.88 (1H, t, J=9.5Hz), 5.01 (1H, dd,
.95 (2H, d, J=11.6Hz), 5.27 (2H, d, J=3.3Hz), 7.23–7.46 J=11.5, 1.5Hz), 5.11 (2H, m), 5.63 (1H, dd, J=10.0, 3.5Hz),
4
13
(
30H, m); C-NMR (50MHz in CDCl ) δ: 62.2, 70.6, 73.0, 5.84 (1H, d, J=3.5Hz), 5.98 (1H, d, J=3.5Hz), 6.48 (1H, d,
3
7
3.1, 74.7, 75.2, 76.3, 78.8, 98.3, 127.4–128.4, 138.2, 138.5, J=2.5Hz), 6.51 (1H, s), 6.85 (1H, dd, J=9.0, 2.5Hz), 7.64 (1H,
+
+
13
1
38.6; HR-MS (FAB ) m/z Calcd for C H O Na (M +Na) d, J=9.0Hz); C-NMR (125MHz in C D N) δ: 14.30, 22.96,
54 58 11 5 5
9
05.3859, Found 905.3841.
-(3-Nonyldodecanoyl)-2,3,4-tri-O-benzyl-α-galactosyl 30.32, 32.14, 32.15, 34.11, 34.17, 35.24, 35.33, 37.89, 39.28,
′-(3-Nonyldodecanoyl)-2′,3′,4′-tri-O-benzyl-α′-galactoside 39.37, 39.63, 63.87, 64.86, 71.57, 71.98, 72.17, 72.27, 72.98,
26.88, 26.87, 26.91, 29.64, 29.66, 29.91, 29.96, 29.99, 30.24,
6
6
(24) This compound was synthesized from 23 (224mg, 75.07, 92.24, 95.62, 98.28, 108.89, 109.35, 111.31, 126.11,
2
54µmol) according to a procedure similar to that described 148.84, 153.14, 156.42, 161.35, 169.66, 173.45, 173.73; HR-MS
19
+
+
for 10. 24: 98% yield, colorless syrup, [α]D +61.9 (c=1.0, (FAB ) m/z Calcd for C H NO Na (M +Na) 1210.7965,
CHCl ); FT-IR (neat) 3087, 3063, 3031, 2925, 2854, 1949, 1871, Found 1210.7973.
67
113
16
3
−1
1
1
806, 1740cm ; H-NMR (300MHz in CDCl ) δ: 0.87 (12H,
In Vivo Assay to Determine Tissue Distribution Emul-
t, J=6.3Hz), 1.22 (64H, m), 1.76 (2H, m), 2.08 (4H, dd, J=6.9, sions of 11 and DAOC (500µg) were intravenously adminis-
.1Hz), 3.88 (2H, brs), 3.97 (2H, dd, J=10.8, 6.0Hz), 4.04 tered to C57BL/6 mice, respectively. After 24h, localization
2H, dd, J=10.2, 2.7Hz), 4.12 (2H, dd, J=10.2, 3.3Hz), 4.18 of the probe was evaluated using a macro confocal microscope
2H, dd, J=10.8, 6.9Hz), 4.25 (2H, brt, J=6.3Hz), 4.55 (2H, (Nicon AZ-C1, Tokyo, Japan). Six-week old male wild-type
3
5
(
(
d, J=11.1Hz), 4.72 (4H, s), 4.77 (2H, d, J=12.0Hz), 4.83 (2H, mice of the C57BL/6 strains (Nihon SLC, Shizuoka, Japan)
d, J=12.0Hz), 4.93 (2H, d, J=11.1Hz), 5.27 (2H, d, J=3.3Hz), were used for the assay. This protocol was approved by the In-
13
7
.22–7.40 (30H, m); C-NMR (75MHz in CDCl ) δ: 14.14, stitute Animal Care and Use Committee at Tokushima Bunri
3
22.70, 26.54, 29.35, 29.63, 29.66, 29.93, 31.91, 33.74, 34.83, University. The mice were housed in plastic cages under con-
3
9.00, 62.93, 68.72, 72.96, 74.81, 75.79, 78.65, 92.80, 127.34, trolled environmental conditions (temperature 22°C, humidity
127.52, 127.69, 128.21, 128.30, 128.36, 138.26, 138.38, 138.66, 55%). Food and water were freely available.
+
+
1
72.93; HR-MS (FAB ) m/z Calcd for C H O Na (M +Na)
Statistics Results are expressed as the mean± S.D., with n
9
6
138 13
1522.0051, Found 1522.0067.
representing the sample size. Statistical comparisons were per-
6
-(3-Nonyldodecanoyl)-α-galactosyl 6′-(3-Nonyldodecan formed using an unpaired t-test or one-way analysis of vari-
oyl)-α′-galactoside (25) This compound was synthesized ance (ANOVA) with Bonferroni correction. p values of less
from 24 (300mg, 200µmol) according to a procedure simi- than 0.05 were considered statistically signification.
lar to that described for 11. 25: 65% yield, colorless syrup,
1
8
[
2
α] +97.3 (c=0.5, CHCl ); FT-IR (neat) 3444, 2953, 2926,
Acknowledgment This study was financially supported
D
3
−1
1
854, 1715cm ; H-NMR (400MHz in C D N) δ: 0.84 by a Grant-in-Aid (Senryaku-project, No. S0891078) from the
5
5
(12H, t, J=5.6Hz), 1.22 (64H, m), 2.03 (2H, m), 2.36 (4H, Ministry of Education, Culture, Sports, Science and Technol-
dd, J=7.2, 1.6Hz), 4.45 (2H, brd, J=2.4Hz), 4.67 (2H, dd, ogy (MEXT) of Japan.
J=10.0, 3.2Hz), 4.77 (2H, dd, J=10.0, 3.6Hz), 4.86 (2H, dd,
J=11.2, 5.2Hz), 4.92 (2H, dd, J=11.2, 7.2Hz), 5.14 (2H, brt, References and Notes
1
3
J=6.8Hz) 5.94 (2H, d, J=3.6Hz); C-NMR (100MHz in
C D N) δ: 14.29, 22.94, 26.87, 29.62, 29.91, 29.94, 30.22, 32.13,
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4.11, 35.21, 39.37, 64.84, 69.94, 70.08, 70.94, 71.10, 95.67,
73.48; HR-MS (FAB ) m/z Calcd for C H O Na (M +Na)
81.7234, Found 981.7250.
,6′-Bis-O-(3-nonyldodecanoyl)-2-{2-[7-(dimethy-
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+
+
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25) 82% of vizantin (71µmol) was recovered in this esterification.