Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses and biological activities of amphoteric glycolipids containing a phosphocholine residue from the earthworm Pheretima hilgendorfi

Article abstract of DOI:10.1016/j.carres.2008.05.001

Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(→6)-β-d-Galp-(1→6)-β-d-Galp-(1→1)Cer (1) and PC(→6)-β-d-Galp-(1→6)-β-d-Galp-(1→6)-β-d-Galp-(1→1)Cer (2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis, in a TNFα-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers of IL-8 production.

Full text of DOI:10.1016/j.carres.2008.05.001

Carbohydrate Research 343 (2008) 2221–2228
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses
and biological activities of amphoteric glycolipids containing a
phosphocholine residue from the earthworm Pheretima hilgendorfi
a
a
a
Noriyasu Hada a, , Yukihiko Shida , Hiroshi Shimamura , Yoshiko Sonoda ,
*
Tadashi Kasahara a, Mutsumi Sugita b, Tadahiro Takeda a,
*
a Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
b Department of Chemistry, Faculty of Liberal Arts and Education, Shiga University, 2-5-1, Hiratsu, Otsu-shi, Shiga-ken 520-0862, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(?6)-b-D-
Galp-(1?6)-b-D-Galp-(1?1)Cer (1) and PC(?6)-b-D-Galp-(1?6)-b-D-Galp-(1?6)-b-D-Galp-(1?1)Cer
(2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance
production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis,
in a TNFa-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers
of IL-8 production.
Received 25 February 2008
Received in revised form 30 April 2008
Accepted 1 May 2008
Available online 9 May 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Amphoteric glycosphingolipid
Pheretima hilgendorfi
IL-8 production
Phosphocholine
1. Introduction
and co-workers examined the induced production of IL-12 and
TNFa by macrophages using our synthetic PC-containing oligosac-
Sialic acid-containing oligosaccharides are important constitu-
ents of gangliosides. Many carbohydrate scientists have taken an
interest in the structure and functional role of mammalian glycos-
phingolipids, and they have been synthesized by various groups.1
As part of our investigation of the relationship between the struc-
ture and biological function of glycolipids from invertebrate animal
species that do not have gangliosides, we have synthesized glyco-
lipids found in various protostomia phyla.2 These compounds may
function as alternative compounds to gangliosides. Sugita et al. re-
ported3 the neogala series of glycosphingolipids, whose structures
charide compounds.5
In this study, we attempted total syntheses of glyco-
sphingolipids 1 and 2 (a disaccharide- and a trisaccharide-contain-
ing PC, Fig. 1), in order to elucidate their biological functions as
immunomodulatory substances in detail. In the course of these
studies, 4 and 5 which were synthesized as a precursor of 2 and
1, respectively, and 3,2f as well as a commercial ceramide 6, were
used to establish the structure–activity relationships. The key reac-
tion was coupling of the hydroxyl group at the C-6 position of the
galactose moiety with phosphocholine. In our previous study,2f we
used 2-chloro-2-oxo-1,3,2-dioxaphospholane and trimethylamine;
however, this time we used a method employing a phosphorodi-
amidite compound.6 We also attempted to carry out the phospho-
choline coupling reaction using phosphoryl chloride and choline
tosylate in order to develop a more economical method for this
reaction.7
contain a b-D-Galp-(1?6)-b-D-Galp- core and a mannose, glucose
and a phosphocholine residue that are found in the earthworm
Pheretima (P.) hilgendorfi, and completed a systematic diagram of
the family of compounds. In our previous paper,2f we reported
the synthesis of two phosphocholine (PC) glycolipid analogues
containing octyl residues in place of ceramide, PC(?6)-b-
D-Galp-
1?Oct and PC(?6)-b- -Galp-(1?6)-b- -Galp-1?Oct, in order to
D
D
IL-8 is a cytokine that exerts chemotactic effects on neutrophils,
T-cells, and basophils.8 These cells play important roles in the host
defense against microbial infections. IL-8 belongs to the C–X–C
chemokine family and is produced by a variety of cells in response
to stimulation with pro-inflammatory cytokines, such as IL-1 and
TNFa.9 In this study, we attempted to examine whether the newly
synthesized glycosphingolipids and analogues have IL-8-inducing
capacity using TNFa-stimulated granulocytic HL-60 cells.
investigate the biological function of zwitterionic oligosaccharides.
A number of researchers have reported the structural elucidation
and immunomodulatory properties of zwitterionic glyco-
sphingolipids from parasitic nematodes.4 Subsequently, Harnett
* Corresponding authors. Tel.: +81 3 5400 2666; fax: +81 3 5400 2556 (N.H.).
E-mail address: hada-nr@kyoritsu-ph.ac.jp (N. Hada).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.05.001

2222
N. Hada et al. / Carbohydrate Research 343 (2008) 2221–2228
O-
O
O-
O
N+(CH3)3
P
N+(CH3)3
P
O-
O
O
OH
O
OH
O
N+(CH3)3
O
P
O
O
O
OH
O
O
O
HO
HO
O
OH
OH
O
HO
HO
HO
HO
O
O
O
HO
O
O
OH
HN C15H31
O
HO
HO
O
OH
HN C15H31
O
C13H27
OH
OH
O
3
O
C13H27
OH
HO
OH
2
OH
1
OH
O-
O
N+(CH3)3
P
O
OH
O
OH
O
OH
HO
O
O
HO
O
OH
O
HO
O
HN C15H31
C13H27
O
OH
HN C15H31
O
OH
HO
HO
HO
O
OH
O
C13H27
OH
O
OH
4
O
5
OH
HO
OH
TMS
6
OH
Figure 1. Target compounds.
Selective removal of the 2-(trimethylsilyl)ethyl group with trifluo-
roacetic acid in CH2Cl2, followed by reaction with trichloroaceto-
nitrile in the presence of DBU,12 gave the corresponding
a-trichloroacetimidate 10. Coupling of (2S,3R,4E)-3-O-benzoyl-2-
hexadecanamido-4-octadecene-1,3-diol (11)2c with the glycosyl
donor 10 was carried out in the presence of trimethylsilyl trifluo-
romethanesulfonate (TMSOTf) and 4 Å MS to afford the desired
glycoconjugate 12 (62%). Next, selective removal of the chloroace-
tyl group in 12 with thiourea gave 13. Treatment with 2-cyano-
ethyl N,N,N0,N0-tetraisopropylphosphorodiamidite, followed by
choline tosylate, gave a product that was oxidized in situ by
2. Results and discussion
2.1. Syntheses of glycosphingolipids 1 and 2 and analogues 4
and 5
Syntheses of glycosphingolipid 1 and its precursor 5 were con-
ducted as shown in Scheme 1. Glycosylation of 72h with 810 in the
presence of N-iodosuccinimide (NIS), trifluoromethanesulfonic
acid (TfOH),11 and 4 Å molecular sieves (4 Å MS) in CH2Cl2 at
ꢀ60 °C to afforded the desired disaccharide 9 in 74% yield, as
1
confirmed by H NMR spectroscopy (H-10, d 4.87 ppm, J 7.9 Hz).
O
OBz
OH
C15H31
(CH2)12CH3
HN
O
OBz
O
OClAc
O
HO
BzO
TMS
OBz
OBz
OBz
11
7
O
BzO
a
OBz
O
c
+
R1
BzO
BzO
OBz
OClAc
O
R2
R1 = OSE R2 = H
SPh
9
BzO
b
OBz
R
1 = H
R2 = OC(NH)CCl3
10
8
OR2
OR1
O
SE = 2-(trimethylsilyl)ethyl
PC = phosphocholine
O
OR2
O
R2O
C15H31
HN
OR2
R2O
O
(CH2)12CH3
O
OR2
OR2
R
1 = ClAc R2 = Bz
12
13
14
1
d
e
R
R
1 = H
1 = PC R2 = Bz
1 = PC R2 = H
R1 = R2 = H
R2 = Bz
f
f
R
5
Scheme 1. Reagents: (a) NIS/TfOH, CH2Cl2, 4 Å MS, 74%; (b) (i) CF3CO2H, CH2Cl2; (ii) CCl3CN, DBU, CH2Cl2, 97%; (c) TMSOTf, CH2Cl2, 4 Å MS, 62%; (d) thiourea, 5:3 EtOH–Pyr,
65%; (e) (i) 2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodiamidite, 1H-tetrazole, CH2Cl2, 3 Å MS; (ii) 1H-tetrazole, choline tosylate; (iii) m-CPBA, MeOH; (iv) aq NH3, 66%;
(f) NaOMe, MeOH, 1: 62%, 5: 88%.

N. Hada et al. / Carbohydrate Research 343 (2008) 2221–2228
2223
m-CPBA.6a After removal of the cyanoethyl group in aqueous
2.2. Biological activities
ammonia and methanol, followed by chromatographic purifica-
tion, 6-O-phosphocholine disaccharide 14 was obtained in 66%
yield. Finally, removal of the acyl groups in 14 under Zemplén con-
ditions, followed by column chromatography (Sephadex LH-20),
furnished the target glycolipid 1. Complete deprotection of a small
amount of 12 was carried out to give PC-free glycolipid 5. The
structure and purity of 1 and 5 were demonstrated by 1H NMR
spectroscopy and HRFABMS.
Granulocytic HL-60 cells were prepared by differentiation with
all-trans-retinoic acid (ATRA) of HL-60 cells. These cells did not
produce significant amounts of IL-8, and none of the four glycolip-
ids containing phosphocholine (1–4) alone induced production of
IL-8 in the culture supernatant (data not shown). However, when
the cells were stimulated with TNFa for two days, significant IL-8
production (ꢁ29 ng/mL) was observed. Glycolipids 1, 2, and 3 that
contained phosphocholine significantly enhanced IL-8 production
in conjunction with TNFa. This enhancement was found to be
dose-dependent. To examine the relationship between structure
and activity, we conducted further tests of IL-8 production using
glycolipid 5 and ceramide 6, in which compound 1 showed stron-
ger activity than 5, indicating that the presence of phosphocholine
residue increases the activity. Similarly, 1 showed stronger activity
than 3, suggesting that the ceramide is better than an octyl residue
to increase activity. On the other hand, comparison of activities of 4
and 6 with 1 led to the conclusion that the ceramide and glycosyl
part are necessary for IL-8 production. These results indicated that,
in general, glycosyl residues and ceramide, as well as the phospho-
choline residue, are the most suitable part of the structure to en-
hance IL-8 producing activity. In inflammatory or infected
lesions, inflammatory cytokines including TNFa and IL-1 are re-
leased from activated macrophages, and then these inflammatory
cytokines stimulate macrophages and granulocytes to produce IL-
8. Since IL-8 is a potent neutrophil and T-lymphocyte chemotactic
factor, 1 and 2 may thus lead to protection from mycobacterial13
and fungal14 infections by recruiting neutrophils and enhancing
bactericidal activity (Fig. 2).
Glycosphingolipid 2 and analogue 4 were synthesized as fol-
lows: selective removal of the chloroacetyl group in 9 with thio-
urea gave disaccharide acceptor 15, which was subjected to
glycosylation by 8 in the presence of NIS/TfOH to afford the desired
trisaccharide 16 in 60% yield, as confirmed by 1H NMR spectro-
scopy (H-10, d 4.82, J 7.9 Hz). Compound 16 was also converted to
the trichloroacetimidate derivative 19, which was then coupled
using 11 in the presence of TMSOTf to give the glycoconjugate 20
(60%). Compounds 16 and 20 were then easily converted into 2
and 4, respectively, in a few steps as follows: (1) removal of chlo-
roacetate with thiourea (17 and 21), (2) phosphorylation of the free
hydroxy group (18 and 22) employing a phosphorodiamidite
method, and (3) Zemplén O-deacylation (Scheme 2). The structures
of 2 and 4 were elucidated by 1H NMR spectroscopy and HRFABMS.
Finally, we attempted coupling with a phosphocholine group by
an alternative method as follows: (1) glycosylceramide derivative
13 was esterified with phosphoryl chloride and (2) the resulting
dichloroester was immediately converted to the phosphocholine
derivative 14 using choline tosylate (79% yield, Scheme 3). The
reagent used in this method (phosphoryl chloride) is not only inex-
pensive compared with the other reagent used (2-cyanoethyl
N,N,N0,N0-tetraisopropylphosphorodiamidite), but also the former
route represents a more convenient procedure for synthesis of
phosphocholine glycolipids. This phosphorylation approach has
not heretofore been used for a carbohydrate hydroxyl group. It is
hoped that this method may be widely applied to coupling reac-
tions of phosphocholines with carbohydrates in the future.
3. Conclusions
In summary, we have succeeded for the first time in carrying
out total syntheses, in good yield, of phosphocholine-containing
OBz
OClAc
OR2
OR1
O
O
SPh
OR2
BzO
R2O
O
OBz
OH
OBz
OR2
8
O
O
OR2
OBz
R2O
O
O
b
BzO
a
OR2
9
OBz
BzO
O
O
R2O
O
O
OR2
TMS
TMS
OBz
15
R1 = ClAc R2 = Bz
R1 = H R2 = Bz
1 = PC R2 = Bz
1 = PC R2 = H
16
a
e
f
17
18
4
R
R
O
OR2
OR1
OR2
C15H31
O
HN
OBz
O
OClAc
O
(CH2)12CH3
R2O
HO
OR2
R2O
O
O
OR2
OBz
O
OBz
11
O
BzO
OR2
R2O
OBz
BzO
HN
O
C15H31
O
OBz
d
(CH2)12CH3
O
O
OR2
OBz
BzO
O
OR2
R1
20 R1 = ClAc R2 = Bz
BzO
R2
a
e
f
R1 = H
R1 = PC R2 = Bz
1 = PC R2 = H
R2 = Bz
21
22
2
R1 = OSE R2 = H
16
19
c
R
1 = H
R2 = OC(NH)CCl3
R
Scheme 2. Reagents: (a) thiourea, 5:3 EtOH–Pyr, 15: 88%, 17: 85%, 21: 99%; (b) NIS, TfOH, CH2Cl2, 4 Å MS, 60%; (c) (i) CF3CO2H, CH2Cl2; (ii) CCl3CN, DBU, CH2Cl2, 99%; (d)
TMSOTf, CH2Cl2, 4 Å MS, 60%; (e) (i) 2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphorodiamidite, 1H-tetrazole, CH2Cl2, 3 Å MS; (ii) 1H-tetrazole, choline tosylate; (iii) m-CPBA,
MeOH; (iv) aq NH3, 18: 68%, 22: 75%; (f) NaOMe, MeOH, 4: 69%, 2: 81%.

2224
N. Hada et al. / Carbohydrate Research 343 (2008) 2221–2228
OBz
OBz
OPC
O
1) POCl3, Et3N, 3 Å MS, CH2Cl2
2) choline tosylate.Pyr.
3) H2O
OH
O
OBz
OBz
O
O
BzO
BzO
O
O
OBz
OBz
OCer
OCer
BzO
BzO
79%
OBz
OBz
14
13
Scheme 3.
at ꢀ60 °C for 2 h, and then the mixture was neutralized with Et3N.
The reaction mixture was filtered, and the filtrate was washed with
aq Na2S2O3, dried (MgSO4), and concentrated. The product was
purified by silica gel column chromatography using 5:1 hexane–
90
80
70
60
50
40
30
20
10
0
***
* * *
***
***
***
***
24
EtOAc as the eluent to give 9 (715 mg, 74%): ½aꢂD +123.6 (c 1.6,
***
**
CHCl3). 1H NMR (500 MHz, CDCl3): d 8.08–7.14 (m, 30H, 6 ꢃ Ph),
0
*
5.89 (d, 1H, J3,4 3.7 Hz, H-4), 5.79 (d, 1H, J3 ,4 3.7 Hz, H-40), 5.78–
0
0
6.25
12.5
25
5.71 (m, 2H, H-2, 20), 5.55–5.51 (m, 2H, H-3, 30), 4.87 (d, 1H, J1 ,2
0
0
7.9 Hz, H-10), 4.72 (d, 1H, J1,2 7.9 Hz, H-1), 4.23–4.11 (m, 5H,
H-6a, 6b, 60a, 60b, –OCH2CH2), 3.96–3.89 (m, 4H, H-5, 50, –OCH2Cl),
3.51–3.45 (m, 1H, –OCH2CH2), 0.87–0.70 (m, 2H, –OCH2CH2), ꢀ0.11
(s, 9H, Si(CH3)3). 13C NMR (125 MHz, CDCl3): d 166.7, 165.5, 165.13
165.05, 133.6, 133.4, 133.3, 133.0, 130.0, 129.9, 129.6, 129.2, 129.1,
129.0, 128.7, 128.6, 128.5, 128.3, 128.21, 128.15, 125.2, 101.0,
100.8, 72.9, 71.9, 71.5, 71.0, 69.8, 69.6, 68.5, 67.8, 67.7, 67.5,
63.1, 40.4, 17.7, ꢀ1.5. MALDI-TOFMS m/z: [M+Na]+ calcd for
1
2
3
4
5
6
Compound
Figure 2. Effects of six compounds on IL-8 production in TNFa-stimulated gran-
ulocytic cells. ATRA-differentiated HL-60 cells (5 ꢃ 105 cell/mL) were stimulated
with each of the six compounds (final 6.25 lM, 12.5 lM, 25 lM) for 24 h. After that,
TNFa (10 ng/mL) was added and incubated for 48 h. The IL-8 contents of culture
supernatant were determined by ELISA. The values represent the mean SD of
C61H59ClO18SiNa, 1165.3; found, 1164.4.
4.3. 2,3,4-Tri-O-benzoyl-6-O-chloroacetyl-b-
D
-
*
**
***
three independent experiments. P <0.05,
the control.
P <0.01,
P <0.005 compared with
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-a-D-
galactopyranosyl trichloroacetimidate (10)
glycosphingolipids found in invertebrate species. The presence of
phosphocholine and ceramide groups resulted in the enhancement
of IL-8 production in a TNFa-stimulated granulocytic HL-60 cells.
These target molecules lie in the class of easily accessible glycolip-
ids in the field of carbohydrate chemistry; however, as these
molecules exhibited immunomodulatory activity, they may be
important and interesting for their other biological activities.
To a solution of 9 (100 mg, 87 lmol) in CH2Cl2 (2.0 mL), cooled
to 0 °C, was added CF3CO2H (2 mL), and the mixture was stirred for
30 min at room temperature and concentrated. EtOAc and toluene
(1:2) were added and evaporated to give the 1-hydroxy compound.
To a solution of the residue in CH2Cl2 (2 mL) cooled at 0 °C were
added trichloroacetonitrile (130 lL, 1.30 mmol) and DBU (12.5 lL,
84 lmol). The reaction mixture was stirred for 1 h at 0 °C. After
completion of the reaction, the mixture was concentrated. Column
chromatography of the residue on silica gel (20:1 toluene–EtOAc)
4. Experimental
25
gave 10 (100 mg, 97%): ½aꢂD +127.0 (c 0.5, CHCl3); 1H NMR (CDCl3):
4.1. General methods
d 8.33 (s, 1H, NH), 8.08–7.14 (m, 30H, 6 ꢃ Ph), 6.77 (d, 1H, J1,2
3.7 Hz, H-1), 6.08 (d, 1H, J3,4 3.1 Hz, H-4), 5.87 (dd, 1H, J2,3
10.9 Hz, H-3), 5.81 (dd, 1H, H-2), 5.81 (d, 1H, J3 ,4 3.7 Hz, H-40),
0
0
Optical rotations were measured with a Jasco P-1020 digital
polarimeter. 1H NMR and 13C NMR spectra were recorded with a
JMN A500 FT NMR spectrometer with Me4Si as the internal stan-
dard for solutions in CDCl3. MALDI-TOFMS was recorded on a
Perseptive Voyager RP mass spectrometer. High-resolution mass
spectra were recorded on a JEOL JMS-700 under FAB conditions
(HRFABMS). TLC was performed on Silica Gel 60 F254 (E. Merck)
with detection by quenching of UV fluorescence and by charring
with 10% H2SO4. Column chromatography was carried out on
Silica Gel 60 (E. Merck). Ceramide 6 was purchased from Acros
Organics Chemical Co. 2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-
5.72 (dd, 1H, J1 ,2 7.9 Hz, J2 ,3 10.9 Hz, H-20), 5.48 (dd, 1H, H-30),
4.87 (d, 1H, H-10), 4.71 (t, 1H, H-6a), 4.16–4.08 (m, 4H, H-6b, 60a,
60b, H-5), 3.96–3.87 (m, 4H, H-50, –OCH2Cl). 13C NMR (125 MHz,
CDCl3): d 166.7, 165.6, 165.5 165.4, 165.2, 165.1, 133.6, 133.5,
133.3, 133.2, 130.1, 129.9, 129.8, 129.7, 129.3, 129.1, 129.0,
128.74, 128.69, 128.6, 128.34, 128.25, 128.2, 125.2, 101.0, 93.7,
90.7, 71.6, 71.2, 71.0, 69.4, 68.3, 68.0, 67.8, 67.5, 63.0, 40.5. MAL-
DI-TOFMS m/z: [M+Na]+ calcd for C58H48Cl4NO18Na, 1209.2; found,
1208.3.
0
0
0
0
b-
D
-galactopyranoside (7)2h and phenyl 2,3,4-tri-O-benzoyl-6-O-
-galactopyranoside (8) were prepared as reported.9
4.4. 2,3,4-Tri-O-benzoyl-6-O-chloroacetyl-b-
D-
chloroacetyl-b-
D
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-
galactopyranosyl-(1?1)-(2S,3R,4E)-3-O-benzoyl-2-
4.2. 2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-6-O-
chloroacetyl-b- -galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-
b- -galactopyranoside (9)
hexadecanamido-4-octadecene-1,3-diol (12)
D
D
To a solution of 10 (65 mg, 55 lmol) and (2S,3R,4E)-3-O-
benzoyl-2-hexadecanamido-4-octadecene-1,3-diol 11 (38 mg,
59 lmol) in dry CH2Cl2 (0.8 mL) was added 4 Å MS (400 mg), and
the mixture was stirred for 2 h at room temperature, then cooled
to 0 °C. TMSOTf (8 lL, 44 lmol) was added, and the mixture was
stirred for 2 h at 0 °C, then neutralized with Et3N. The solids were
filtrated off and washed with CHCl3. The combined filtrate and
A solution of compounds 7 (500 mg, 0.84 mmol) and 8 (669 mg,
1.01 mmol) containing activated 4 Å MS (1 g) in dry CH2Cl2 (2 mL)
was stirred under an atmosphere of argon for 2 h at room temper-
ature. After cooling to 0 °C, successively NIS (285 mg, 1.27 mmol)
and TfOH (22.3 lL, 0.25 mmol) were added, stirring was continued

N. Hada et al. / Carbohydrate Research 343 (2008) 2221–2228
2225
washings were successively washed with water, dried (MgSO4),
and concentrated. The product was purified by silica gel column
chromatography using 10:1 toluene–EtOAc as eluent to give 12
tion was stirred for 1.5 h at the room temperature, and pyridine
(1 mL) and then choline tosylate (12.7 mg, 46 lmol) were added
at room temperature. This solution was stirred for 10 h at room
temperature, and H2O (1 mL) was added with stirring for 1 h at
the same temperature. After that time, the solution was filtered
and concentrated. The product was purified by Iatrobeads column
chromatography using 4:5:1 CHCl3–MeOH–H2O as eluent to give
25
(57 mg, 62%): ½aꢂD +71.0 (c 1.2, CHCl3); 1H NMR (CDCl3): d 8.12–
7.21 (m, 35H, 7 ꢃ Ph), 5.87–5.82 (m, 3H, H-40, ˜C@CH, –NH), 5.75
(d, 1H, H-4), 5.66–5.62 (m, 2H, H-2, 20), 5.57–5.44 (m, 4H, H-3, 30,
˜C@CH, Bz-CH), 4.60 (1H, d, J1 ,2 7.9 Hz, H-10), 4.48 (1H, d, J1,2
7.3 Hz, H-1), 4.30 (1H, b, N–CH), 4.12–3.89 (m, 6H, H-6, 60, 5, 50,
COCH2Cl), 3.44 (dd, 1H, O–CH2), 3.30 (dd, 1H, O–CH2), 13C NMR
(125 MHz, CDCl3): d 172.5, 166.7, 165.5, 137.3, 133.6, 130.6,
130.1, 129.7, 129.6, 129.2, 128.6, 128.5, 128.4, 128.3, 125.0, 101.0
(C-10), 100.7 (C-1), 74.0, 72.8, 71.3, 70.8, 70.5, 69.6, 68.2, 67.8,
67.4, 63.0, 50.4, 40.4, 36.5, 32.3, 31.9, 29.7, 29.5, 29.3, 29.2, 29.0,
25.5, 22.7, 14.1. MALDI-TOFMS m/z: [M+Na]+ calcd for
0
0
25
14 (32.1 mg, 79%): ½aꢂD +40.3 (c 0.7, CHCl3); 1H NMR (CDCl3): d
8.00–6.70 (m, 35H, 7 ꢃ Ph), 5.82 (d, 1H, –NH), 5.75–5.72 (m, 2H,
H-40, ˜C@CH), 5.54–5.33 (m, 7H, H-2, 20, 3, 30, 4, ˜C@CH, Bz-CH),
4.65 (d, 1H, J1 ,2 7.3 Hz, H-10), 4.52–4.50 (br, 1H, H-1), 4.16–4.03
(m, 4H, H-6a, N–CH, N–CH2), 3.85–3.83 (m, 2H, H-6), 3.77–3.76
(q, 1H, H-6), 3.47–3.45 (br d, 2H, PO–CH2) 3.33–3.24 (m, 2H, H-5,
50), 3.07 (br s, 10H, N–(CH3)3, O–CH2). 13C NMR (125 MHz, CDCl3):
0
0
C97H116ClNO21Na, 1688.8; found, 1688.9.
d
173.5, 100.0 (C-10), 99.7 (C-1), 74.1, 73.0, 72.2, 71.7, 70.1, 69.5,
69.4, 69.0, 67.8, 67.3, 66.7, 65.8, 65.5. MALDI-TOFMS m/z: [M+H]+
4.5. 2,3,4-Tri-O-benzoyl-b-
D-galactopyranosyl-(1?6)-2,3,4-tri-
calcd for C100H128N2O23P, 1755.9; found, 1756.0.
O-benzoyl-b- -galactopyranosyl-(1?1)-(2S,3R,4E)-3-O-benzoyl-
D
2-hexadecanamido-4-octadecene-1,3-diol (13)
4.7. 6-O-Phosphocholine-b-
galactopyranosyl-(1?6)-b- -galactopyranosyl-(1?1)-
(2S,3R,4E)-2-hexadecanamido-4-octadecene-1,3-diol (1)
D-galactopyranosyl-(1?6)-b-D-
D
To a solution of 12 (133 mg, 80 lmol) in 3:5 pyridine–EtOH
(1.6 mL) was added thiourea (31 mg, 0.40 mmol), and the mixture
was stirred for 2 h at 80 °C. The mixture was diluted with CHCl3,
washed with NaHCO3 and water, dried (MgSO4), and concentrated.
The product was purified by silica gel column chromatography
To a solution of 14 (59.8 mg, 34.0 lmol) in MeOH (2.0 mL) was
added NaOMe (10 mg) at 45 °C. The mixture was stirred for 1 h and
then neutralized with Amberlite IR 120 [H+]. The mixture was fil-
tered and concentrated. The product was purified by Sephadex
LH-20 column chromatography in MeOH to give 1 (21.6 mg,
24
using 5:1 toluene–EtOAc as eluent to give 13 (82 mg, 65%): ½aꢂD
+87.7 (c 2.0, CHCl3); 1H NMR (CDCl3): d 8.10–7.21 (m, 35H,
7 ꢃ Ph), 5.91 (d,1H, –NH), 5.87–5.82 (m, 1H, H-40), 5.71–5.67 (m,
2H, H-4, 20), 5.64–5.56 (m, 2H, H-2, ˜C@CH), 5.54–5.44 (m, 4H,
24
62%): ½aꢂD +12.0 (c 0.3, MeOH); 1H NMR (19:1 DMSO-d6–D2O): d
4.17 (d, 1H, J1 ,2 7.3 Hz, H-10), 4.07 (m, 3H, H-1, PO–CH2). MALDI-
TOFMS m/z: [M+H]+ calcd for C51H100N2O16P, 1027.7; found,
1028.2. HRFABMS m/z: [M+Na]+ calcd for C51H99N2O16PNa,
1049.6630; found, 1049.6583.
0
0
H-3, 30, ˜C@CH, Bz-CH), 4.60 (d, 1H, J1 ,2 7.9 Hz, H-10), 4.47 (d,
1H, J1,2 7.9 Hz, H-1), 4.34–4.30 (br t, 1H, N–CH), 4.01–4.00 (m,
2H, H-60), 3.84–3.78 (m, 2H, H-6), 3.53–3.49 (q, 1H, O–CH2),
3.41–3.34 (m, 3H, H-5, 50, O–CH2). 13C NMR(125 MHz, CDCl3): d
172.5, 166.6, 165.5, 165.1, 143.2, 137.3, 133.7, 133.6, 133.4,
133.3, 130.2, 130.1, 129.7, 129.6, 129.2, 129.0, 128.7, 128.6,
128.5, 128.4, 128.3, 128.2, 125.0, 101.1 (C-10), 100.7(C-1), 74.1,
73.9, 72.6, 71.6, 71.3, 70.5, 70.0, 68.8, 68.2, 67.4, 67.0, 60.7, 50.4,
36.5, 32.3, 31.9, 29.7, 29.5, 29.3, 29.2, 29.0, 25.5, 22.7, 14.1. MAL-
DI-TOFMS m/z: [M+Na]+ calcd for C95H115NO20Na, 1612.8; found,
1612.6.
0
0
4.8. b-D-Galactopyranosyl-(1?6)-b-D-galactopyranosyl-(1?1)-
(2S,3R,4E)-2-hexadecanamido-4-octadecene-1,3-diol (5)
To a solution of 12 (8.4 mg, 5.0 lmol) in dioxane–MeOH
(2.0 mL) was added NaOMe (10 mg) at 45 °C. The mixture was stir-
red for 1 h and then neutralized with Amberlite IR 120 [H+]. The
mixture was filtered and concentrated. The product was purified
by Sephadex LH-20 column chromatography in MeOH to give 5
24
4.6. 2,3,4-Tri-O-benzoyl-6-O-phosphocholine-b-
D
-
(3.8 mg, 88%): ½aꢂD ꢀ0.42 (c 0.1, 1:1 CHCl3–MeOH); 1H NMR
(19:1 DMSO-d6–D2O): d 4.15 (d, 1H, J1 ,2 7.3 Hz, H-10), 4.06 (d,
1H, J1,2 7.3 Hz, H-1). MALDI-TOFMS m/z: [M+H]+ calcd for
0
0
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
D
-
galactopyranosyl-(1?1)-(2S,3R,4E)-3-O-benzoyl-2-
hexadecanamido-4-octadecene-1,3-diol (14)
C
46H88NO13, 861.6; found, 861.9. HRFABMS m/z: [M+Na]+ calcd
for C46H87NO13Na, 884.6075; found, 884.6058.
4.6.1. Method A
To a solution of 13 (82.1 mg, 52 lmol) and 3 Å MS (300 mg) in
dry CH2Cl2 (2.0 mL) were added 2-cyanoethyl-N,N,N0,N0-tetraiso-
propylphosphorodiamidite (24.6 lL, 77 lmol) and 1H-tetrazole
(4.0 mg, 57 lmol) at room temperature under Ar. The solution
was stirred for 0.5 h at the room temperature. To this was added
1H-tetrazole (10.9 mg, 0.16 mmol), followed by choline tosylate
(56.9 mg, 0.21 mmol) at room temperature. This solution was stir-
red for 4 h at room temperature, and MeOH (1 mL) and m-CPBA
(14.6 mg, 59 lmol) were then added, with stirring for 1 h at the
room temperature. After that time, 30% aq NH3 was added to mix-
ture, and it was stirred for 1 h at room temperature. The solution
was filtered and concentrated. The product was purified by Iat-
robeads column chromatography using 4:5:1 CHCl3–MeOH–H2O
as eluent to give 14 (59.8 mg, 66%).
4.9. 2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-b-D-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-
galactopyranoside (15)
To a solution of 9 (1.33 g, 1.2 mmol) in 3:5 pyridine–EtOH
(6.0 mL) was added thiourea (446 mg, 5.9 mmol), and the mixture
was stirred for 2.5 h at 80 °C. The mixture was diluted with CHCl3,
washed with aq NaHCO3 and water, dried (MgSO4), and concen-
trated. The product was purified by silica gel column chromato-
graphy using 5:1 toluene–EtOAc as eluent to give 15 (1.09 g,
24
88%): ½aꢂD +151.0 (c 1.4, CHCl3); 1H NMR (CDCl3): d 8.23–7.31
(30H, m, Ar-H), 6.08 (1H, d, H-40), 5.92–5.87 (2H, m, H-4, H-20),
5.81 (1H, dd, H-2), 5.65–5.61 (2H, m, H-3, 30), 4.94 (1H, d, J1 ,2
0
0
7.9 Hz, H-10), 4.81(1H, d, J1,2 7.9 Hz, H-1), 4.26–4.19 (2H, m, H-50,
H-60a), 4.07–4.01 (3H, m, H-60b, H-5, H-6a), 3.75–3.62 (1H, m, O–
CH2), 3.61–3.53 (2H, m, H-6b, O–CH2). 13C NMR (125 MHz, CDCl3):
4.6.2. Method B
To a solution of 13 (36.7 mg, 23 lmol) and 3 Å MS (130 mg) in
dry CH2Cl2 (1.0 mL) were added phosphoryl chloride (2.2 lL,
23 lmol) and Et3N (3.5 lL, 25 lmol) at ꢀ10 °C under Ar. The solu-
d 166.6, 165.6, 165.5, 165.21, 165.16, 133.7, 133.5, 133.2, 133.1,
130.1, 130.0, 129.7, 129.6, 129.4, 129.2, 128.9, 128.8, 128.6,
128.5, 128.4, 128.3, 128.23, 128.16, 101.3, 100.9, 74.2, 72.9, 72.0,

2226
N. Hada et al. / Carbohydrate Research 343 (2008) 2221–2228
71.8, 70.0, 69.9, 68.8, 68.5, 67.8, 67.5, 60.7, 17.8, ꢀ1.5. MALDI-TOF-
MS m/z: [M+Na]+ calcd for C59H58ClO17SiNa, 1089.3; found, 1089.8.
(4.5 mg, 64 lmol) at room temperature under Ar. The solution
was stirred for 0.5 h at room temperature, and 1H-tetrazole
(13.6 mg, 0.19 mmol) and choline tosylate (71.5 mg, 0.26 mmol)
were added at room temperature. This solution was stirred for an
additional 4 h at room temperature, and MeOH (1 mL) and m-CPBA
(19.2 mg, 78 lmol) were added with stirring for 1 h at the same
temperature. After that, 30% aq NH3 was added and the mixture
was stirred for 1 h at room temperature. The solution was filtered
through a pad of Celite and concentrated. The product was purified
by Iatrobeads column chromatography using 4:5:1 CHCl3–MeOH–
4.10. 2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-6-O-
chloroacetyl-b-
b- -galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranoside (16)
D-galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-
D
D-
A
solution of compounds 15 (300 mg, 0.28 mmol) and 8
(223 mg, 0.34 mmol) containing activated 4 Å MS (400 mg) in dry
CH2Cl2 (1.5 mL) was stirred under an atmosphere of Ar for 1.5 h
at room temperature. After cooling to 0 °C, successively NIS
(127 mg, 0.56 mmol) and TfOH (14.9 lL, 0.17 mmol) were added,
stirring was continued at 0 °C for 2 h, and then the mixture was
neutralized with Et3N. The reaction mixture was filtered, and the
filtrate was washed with Na2S2O3 and water, dried (MgSO4), and
concentrated. The product was purified by silica gel column chro-
matography using 5:1 toluene–EtOAc as the eluent to give 16
25
H2O as eluent to give 8 (75.5 mg, 68%): ½aꢂD +40.3 (c 0.7, CHCl3); 1H
NMR (CDCl3): d 8.02–6.95 (45H, m, 9 ꢃ Ph), 5.87 (1H, d, H-4), 5.80
(1H, d, H-40) 5.76 (1H, d, H-400) 5.69–5.45 (6H, m, H-2, 20, 200, 3, 30,
300), 5.00 (2H, d, J1,2 1 ,2 8.6 Hz, H-1, 10), 4.84 (1H, d, J1 ,2 7.9 Hz,
H-100), 4.40–4.39 (1H, t, H-6a), 4.30–4.20 (4H, m, H-6b, 60a,
PO–CH2), 4.11–4.06 (1H, m, H-60b), 4.00–3.93 (5H, m, H-600, 5, 500,
O–CH2), 3.60–3.50 (4H, m H-500, O–CH2, N–CH2), 3.08 (9H, s,
0
0
00 00
13
N(CH3)). C NMR (125 MHz, CDCl3): 100.7 (C-1), 100.3 (C-10),
24
(273 mg, 60%): ½aꢂD +67.0 (c 6.6, CHCl3). 1H NMR (500 MHz, CDCl3):
100.3 (C-100), 73.3, 72.2, 72.0, 71.8, 71.6, 71.5, 71.1, 70.0, 69.7,
68.3, 68.1, 67.9, 67.8, 67.2, 65.9, 62.5, 58.8, 53.3. MALDI-TOFMS
m/z: [M+H]+ calcd for C91H93NO28PSi, 1706.5; found, 1706.8.
d 8.16–7.30 (45H, m, 9 ꢃ Ph), 6.01–6.00 (2H, d, H-4, 40), 5.88–5.87
(1H, d, H-400), 5.82–5.72 (3H, m, H-2, 20, 200), 5.63–5.56 (3H, m, H-3,
30, 300), 4.87 (1H, d, J1,2 7.9 Hz, H-1), 4.82 (1H, d, J1 ,2 7.9 Hz, H-10),
0
0
4.71 (1H, d, J1 ,2 7.9 Hz, H-100), 4.24–4.15 (4H, m, H-6, 60), 4.09–
4.13. 2-(Trimethylsilyl)ethyl 6-O-phosphocholine-b-D-
00 00
3.90 (7H, m, H-600, 5, 50, O–CH2, C–CH2–Cl), 3.67 (1H, q, O–CH2)
3.58–3.55 (1H, m, H-500), 13C NMR (125 MHz, CDCl3): d 166.6,
165.5, 165.4, 165.2, 165.1, 165.0, 164.9, 133.5, 133.3, 133.2,
133.1, 133.0, 130.04, 129.96, 129.7, 129.64, 129.55, 129.38,
129.35, 129.2, 129.0, 128.7, 128.64, 128.56, 128.5, 128.39, 128.36,
128.3, 128.2, 128.1, 100.8 (C-1,10), 100.7 (C-100), 72.7, 72.5, 71.9,
71.6, 71.5, 70.9, 69.9, 69.6, 68.4, 67.9, 67.7, 67.4, 67.2, 66.5, 62.9,
40.4, 17.7, ꢀ1.5. MALDI-TOFMS m/z: [M+Na]+ calcd for
galactopyranosyl-(1?6)-b-D-galactopyranosyl-(1?6)-b-D-
galactopyranoside (4)
To a solution of 18 (75.5 mg, 44 lmol) in MeOH (3.0 mL) was
added NaOMe (15 mg) at 45 °C, and the mixture was stirred for
1 h, then neutralized with Amberlite IR 120 [H+]. The mixture
was filtered and concentrated. The product was purified by Sepha-
dex LH-20 column chromatography in MeOH to give 4 (23.6 mg,
24
C88H81ClO26SiNa, 1639.5; found, 1639.5.
69%): ½aꢂD ꢀ17.4 (c 0.6, MeOH); 1H NMR (CD3OD): d 4.41–4.27
(2H, m, H-1, 100), 4.20–4.18 (1H, t, H-100). 13C NMR (125 MHz,
CD3OD): d 105.4 (C-1), 105.2 (C-10), 104.4 (C-100), 75.34, 75.27,
75.0, 74.83, 74.76, 74.7, 72.5, 72.4, 70.1, 70.01, 69.98, 69.8, 69.7,
68.2, 67.5, 66.0, 60.1, 54.8, 19.2, ꢀ1.3. MALDI-TOFMS m/z: [M+H]+
calcd for C28H57NO19PSi, 770.3; found, 770.0. HRFABMS m/z:
[M+Na]+ calcd for C28H56NO19PSiNa, 792.2851; found, 792.2871.
4.11. 2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-b-
D
-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranoside (17)
D
-
-
D
To a solution of 16 (273 mg, 0.17 mmol) in 3:5 pyridine–EtOH
(3.2 mL) was added thiourea (64.2 mg, 0.84 mmol), and the mixture
was stirred for 2.5 h at 80 °C. The mixture was diluted with CHCl3,
washed with NaHCO3 and water, dried (MgSO4), and concentrated.
The product was purified by silica gel column chromatography
using 10:1 toluene–acetone as eluent to give 17 (222 mg, 85%):
4.14. 2,3,4-Tri-O-benzoyl-6-O-chloroacetyl-b-
D
-
D
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-a-
galactopyranosyl trichloroacetimidate (19)
-
-
D
24
½aꢂD +123.7 (c 3.9, CHCl3); 1H NMR (CDCl3): d 8.10–7.13 (45H, m,
To a solution of 16 (100 mg, 62 lmol) in CH2Cl2 (2.0 mL), cooled
to 0 °C, was added CF3CO2H (2 mL), and the mixture was stirred for
30 min at room temperature and concentrated. EtOAc and toluene
(1:2) were added and evaporated to give the 1-hydroxy compound.
To a solution of the residue in CH2Cl2 (2 mL) cooled at 0 °C were
added trichloroacetonitrile (62.4 lL, 0.62 mmol) and DBU (18.5 lL,
124 lmol). The reaction mixture was stirred for 1 h at 0 °C. After
completion of the reaction, the mixture was concentrated. Column
Ar-H), 5.95 (1H, d, H-400), 5.91 (1H, d, H-40), 5.73–5.65 (4H, m,
H-4, H-200, 20, 2), 5.53–5.47 (3H, m, H-300, 30, 3), 4.76 (1H, d, J1 ,2
00 00
7.9 Hz, H-100), 4.71 (1H, d, J1 ,2 7.9 Hz, H-10), 4.58 (1H, d, J1,2
7.9 Hz, H-1), 4.13–4.02 (3H, m, H-500, 50 H-600a), 3.95–3.90 (1H, m,
O–CH2), 3.86–3.81 (3H, m, H-600b, H-5, H-60a), 3.55–3.45 (3H, m,
H-60b H-6a, O–CH2), 3.35–3.31 (1H, m, H-6b). 13C NMR (125 MHz,
CDCl3): d 166.5, 165.4, 165.3, 165.18, 165.15, 165.1, 133.5, 133.4,
133.2, 133.0, 130.1, 130.0, 129.71, 129.66, 129.6, 129.4, 129.2,
129.0, 128.9, 128.7, 128.5, 128.41, 128.36, 128.3, 128.2, 128.1,
101.0, 100.8, 74.1, 72.7, 72.3, 71.9, 71.74, 71.69, 69.9, 68.7, 68.4,
67.9, 67.4, 67.0, 66.7, 60.7, 17.7, ꢀ1.5. MALDI-TOFMS m/z:
[M+Na]+ calcd for C86H80O25SiNa, 1563.5; found, 1564.0.
0
0
chromatography of the residue on silica gel (20:1 toluene–EtOAc)
25
gave 19 (102.3 mg, 99%): ½aꢂ +100.9 (c 3.3, CHCl3); 1H NMR
D
(CDCl3): d 8.36 (s, 1H, NH), 6.76 (d, 1H, J1,2 3.7 Hz, H-1), 4.74 (d,
1H, J1 ,2 7.9 Hz, H-10), 4.57 (d, 1H, J1 ,2 7.9 Hz, H-100). The product
0
0
00 00
was used directly in the next step (Section 4.15).
4.12. 2-(Trimethylsilyl)ethyl 2,3,4-tri-O-benzoyl-6-O-
4.15. 2,3,4-Tri-O-benzoyl-6-O-chloroacetyl-b-
D
-
phosphocholine-b-
benzoyl-b- -galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranoside (18)
D
-galactopyranosyl-(1?6)-2,3,4-tri-O-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranosyl-(1?1)-(2S,3R,4E)-3-O-benzoyl-2-
D
-
-
D
D-
D
hexadecanamido-4-octadecene-1,3-diol (20)
To a solution of 13 (100 mg, 65 lmol) and 3 Å MS (300 mg) in
dry CH2Cl2 (2.0 mL) were added 2-cyanoethyl-N,N,N0,N0-tetraiso-
propylphosphorodiamidite (30.9 lL, 97 lmol) and 1H-tetrazole
To a solution of 19 (82.0 mg, 49 lmol) and (2S,3R,4E)-3-O-
benzoyl-2-hexadecanamido-4-octadecene-1,3-diol (11, 47.5 mg,

N. Hada et al. / Carbohydrate Research 343 (2008) 2221–2228
2227
74 lmol) in dry CH2Cl2 (1 mL) was added 4 Å MS (400 mg), and the
mixture was stirred for 2 h at room temperature, then cooled to
0 °C. TMSOTf (10.8 mg, 59 lmol) was added, and the mixture was
stirred for 2.5 h at 0 °C, then neutralized with Et3N. The solids were
filtrated off and washed with CHCl3. The combined filtrate and
washings were successively washed with water, dried (MgSO4),
and concentrated. The product was purified by silica gel column
chromatography using 6:1 toluene–EtOAc as eluent to give 20
99.9 (C-100), 73.3, 71.8, 71.6, 71.3, 71.1, 70.1, 69.6, 69.5, 67.9, 67.7,
67.6, 66.8, 65.81, 65.76, 65.1, 58.6, 53.3, 42.0 35.7, 31.7, 31.3,
29.0, 28.9, 28.7, 28.5, 28.3, 25.2, 22.0, 20.2, 13.2. MALDI-TOFMS
m/z: [M+H]+ calcd for C127H150N2O31P, 2229.9; found, 2230.4.
4.18. 6-O-Phosphocholine-b-
galactopyranosyl-(1?6)-b- -galactopyranosyl-(1?1)-(2S,
3R,4E)-2-hexadecanamido-4-octadecene-1,3-diol (2)
D-galactopyranosyl-(1?6)-b-D-
D
25
(63.0 mg, 60%): ½aꢂD +73.8 (c 2.9, CHCl3); 1H NMR (CDCl3): d
8.08–7.21 (50H, m, 10 ꢃ Ph), 5.88–5.76 (4H, m, H-4, 40, 400, –NH),
5.65–5.53 (5H, m, H-2, 20, 200, C@CH), 5.48–5.42 (4H, m, H-3, 30,
Compound 2 was prepared from 22 (41.9 mg) as described for
24
preparation of 1, yielding 18.0 mg (81%). ½aꢂD +24.7 (c 0.1, MeOH);
300, Bz-CH), 4.60 (1H, dd, J1,2 7.3 Hz, H-1), 4.58 (2H, dd, J1 2 7.9 Hz,
1H NMR (19:1 DMSO-d6–D2O): d 4.18 (d, 1H, J1 ,2 7.9 Hz, H-100),
0
0
00 00
H-10), 4.37 (1H, d, J1 ,2 7.9 Hz, H-100), 4.26 (1H, br s, N–CH), 4.04-
00 00
4.17 (d, 1H, J1 ,2 7.3 Hz, H-10), 4.07 (d, 1H, J1,2 7.3 Hz, H-1). MAL-
DI-TOFMS m/z: [M+H]+ calcd for C57H110N2O21P, 1189.7; found,
1190.5. HRFABMS m/z: [M+Na]+ calcd for C57H109N2O21PNa,
1211.7158; found, 1211.7140.
0
0
3.76 (10H, m, H-6, 60, 600, 5, 50, O–CH2), 3.48-3.45 (1H, q, O–CH2),
3.35–3.31 (1H, q, H-50), 3.28–3.26 (1H, q, H-500). 13C NMR
(125 MHz, CDCl3): d 172.4, 166.7, 165.4, 165.2, 165.0, 137.2,
133.3, 130.6, 130.1, 130.0, 129.8, 129.7, 129.3, 129.0, 128.6,
128.54, 128.46, 128.4, 128.3, 125.0, 100.9 (C-1), 100.8 (C-10),
100.7 (C-100), 73.8, 72.6, 72.3, 71.6, 71.4, 70.9, 70.5, 69.9,
69.8, 68.2, 67.7, 67.2, 66.2, 62.9, 50.6, 50.4, 40.4, 36.5, 32.3,
31.9, 29.70, 29.65, 29.5, 29.3, 29.2, 29.0, 25.5, 22.7, 14.1. MALDI-
TOFMS m/z: [M+Na]+ calcd for C124H138ClNO29Na, 2162.9; found,
2162.7.
4.19. Biological activities
HL-60 cells were suspended in complete medium (RPMI1640
containing 5% FBS) (FBS, Nippon Bio Supply Center, Tokyo). Expo-
nentially growing cells were incubated in fresh media at a concen-
tration at a 5 ꢃ 105/mL in the presence of ATRA (1 lM) for 2 days.
Each test compound was added at various doses. After 1 day of
addition of test compounds, TNFa (10 ng/mL) was added, and the
mixture was incubated for further 2 days. The supernatants were
obtained, and IL-8 was quantitated using an ELISA method.8
4.16. 2,3,4-Tri-O-benzoyl-b-
D-galactopyranosyl-(1?6)-2,3,4-tri-
O-benzoyl-b- -galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-
D
galactopyranosyl-(1?1)-(2S,3R,4E)-3-O-benzoyl-2-
hexadecanamido-4-octadecene-1,3-diol (21)
Acknowledgments
Compound 21 was prepared from 20 (132.1 mg) as described
24
for preparation of 13, yielding 100.5 mg (79%): ½aꢂD +79.5 (c 2.5,
This work was supported by a Grant-in-Aid for Scientific
Research (No. 19590011) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (MEXT), and Open
Research Center project of the MEXT. The authors are grateful to
Ms. J. Hada for providing HR-FAB MS data.
CHCl3); 1H NMR (CDCl3): d 8.10–7.20 (75H, m, Ar-H), 5.91–5.82
(4H, m, H-4, 40, 400, –NH), 5.71–5.43 (9H, m, H-2, 20, 200, 3, 30 300,
0
0
˜C@CH, Bz-CH), 4.59 (1H, d, J1,2 7.3 Hz, H-1), 4.55 (1H, d, J1 ,2
7.9 Hz, H-10), 4.36 (1H, d, J1 ,2 7.3 Hz, H-100), 4.31 (1H, br s, N–
CH), 4.00–3.94 (2H, m, H-6), 3.87–3.78 (4H, m, H-60,600), 3.69 (1H,
t, O–CH2) 3.34–3.28 (4H, m, H-5, 50, 500, O–CH2). 13C NMR
(125 MHz, CDCl3): d 172.4, 166.5, 165.5, 165.4, 165.3, 165.1,
165.0, 164.9, 137.2, 133.4, 133.2, 133.1, 132.8, 130.6, 130.2,
130.0, 129.74, 129.69, 129.6, 129.5, 129.3, 129.2, 129.00, 128.95,
128.80, 128.76, 128.6, 128.5, 128.4, 128.3, 128.20, 128.15, 125.0,
101.0 (C-1), 100.8 (C-10), 100.7 (C-100), 74.2, 73.8, 72.5, 72.0, 71.8,
71.5, 71.3, 70.5, 69.9, 68.7, 68.0, 67.8, 67.0, 66.9, 66.4, 60.7, 50.4,
36.5, 32.3, 31.9, 29.7, 29.5, 29.32, 292.26, 29.2, 29.0, 25.5, 22.7,
14.1. MALDI-TOFMS m/z: [M+Na]+ calcd for C122H137NO28Na,
2086.9; found, 2087.5.
00 00
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Compound 22 was prepared from 21 (100.5 mg) as described
25
for preparation of 14, yielding 81 mg (75%): ½aꢂD +53.8 (c 2.0,
CHCl3); 1H NMR (CDCl3): d 8.84–8.02 (50H, m, 10;ꢃ Ph), 5.86–
5.64 (5H, m, H-2, 4, 40, 400, –NH), 5.60–5.47 (7H, m, H-20, 200, 3, 30
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