Synthetic studies on glycosphingolipids from protostomia phyla: Synthesis of amphoteric glycolipid analogues containing a phosphocholine residue from the earthworm Pheretima hilgendorfi

Article abstract of DOI:10.1248/cpb.49.1464

Two kinds of amphoteric glycosphingolipid analogues from the earthworm Pheretima hilgendorfi were synthesized as follows: The key reaction is a coupling of a phosphocholine group at the position C-6 of 1 and 6 which was attempted using 2-chloro-2-oxo-1,3,

Full text of DOI:10.1248/cpb.49.1464

1464
Chem. Pharm. Bull. 49(11) 1464—1467 (2001)
Vol. 49, No. 11
Synthetic Studies on Glycosphingolipids from Protostomia Phyla:
Synthesis of Amphoteric Glycolipid Analogues Containing a
Phosphocholine Residue from the Earthworm Pheretima hilgendorfi
Noriyasu HADA,a Koji SATO,a Jun-ichiro SAKUSHIMA,b Yukihiro GODA,b Mutsumi SUGITA,c and
,a
*
Tadahiro TAKEDA
Kyoritsu College of Pharmacy,a 1–5–30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan, National Institute of Health
Sciences,b 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–8501, Japan, and Department of Chemistry, Faculty of Liberal Arts
and Education, Shiga University,c 2–5–1 Hiratsu, Otsu, Shiga 520–0862, Japan.
Received July 26, 2001; accepted August 29, 2001
Two kinds of amphoteric glycosphingolipid analogues from the earthworm Pheretima hilgendorfi were syn-
thesized as follows: The key reaction is a coupling of a phosphocholine group at the position C-6 of 1 and 6 which
was attempted using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by reaction of the resulting cyclic phos-
phate intermediate with anhydrous trimethylamine to give 2 and 7. Subsequent debenzylation afforded target
compounds (3, 8). Their ability to inhibit the histamine release in vitro was examined.
Key words amphoteric glycosphingolipid; Pheretima hilgendorfi; chemical synthesis; phosphocholine
We have been interested in structure–activity relationships amphoteric glycosphingolipid analogues containing fatty
of glycosphingolipids derived from invertebrate animal alkyl residue in place of ceramide. Furthermore, final com-
species and have synthesized oligosaccharides from various pounds were examined for their ability to inhibit the hista-
protostomia phyla.1) Sugita et al.2) found and characterized a mine release in vitro.
new neogala series of glycosphingolipids, of which core
structure was a b-D-Galp-(1→6)-b-D-Galp- containing a man- Results and Discussion
nose, glucose and phosphocholine residue from the earth-
Synthesis of Monosaccharide Containing Phospho-
worm Pheretima (P. ) hilgendorfi. In the previous paper1e) we choline Octyl 2,3,4-tri-O-benzyl-b-D-galactopyranoside
reported the synthesis of neogala series glycosphingolipids (1)8) was chosen as an acceptor prepared from octyl b-D-
containing a mannose residue from P. hilgendorfi. In the pre- galactopyranoside. Introduction of a phosphocholine group
sent study, we attempted the synthesis of the novel neogala at the position C-6 of 1 was attempted using 2-chloro-2-oxo-
series of glycosphingolipids containing the phosphocholine 1,3,2-dioxaphospholane, followed by reaction of the resulting
residue. These compounds have a phosphocholine group at cyclic phosphate intermediate with anhydrous trimethyl-
the C-6 position of the galactose moiety to form a zwitter- amine at 65 °C in a sealed vessel to give 2 in 68% yield for
ionic structure A and B (Fig. 1). Furthermore Noda and his two steps. Compound 2 revealed an [MϩH]ϩ ion peak at m/z
co-workers also isolated3) similar compounds and examined 728 in the time of flight mass spectrometer (TOF-MS) spec-
the structure–bioactivity relationships about fruiting-induc- trum. Removal of the benzyl groups from 2 by catalytic hy-
ing effect.4) As part of our program of oligosaccharide syn- drogenolysis over 10% Pd–C gave the target compound 3
thesis, the preparation of neogala series containing a phos- (Chart 1).
phocholine group at the C-6 position of the galactose moiety,
Synthesis of Disaccharide Containing Phosphocholine
from P. hilgendorfi, became of interest. Toward that end, the The disaccharide derivative 5 was obtained by condensation
key reaction is a coupling of a phosphocholine group at the of 1 with phenyl 1-thio-2,3,4-tri-O-benzyl-6-O-tert-butyl-
C-6 position of the galactose moiety. Concerning that, an- dimethylsilyl-b-D-galactopyranoside (4) in the presence of N-
other novel phosphocholine-containing glycosphingolipids iodosuccinimide (NIS) and trifluoromethanesulfonic acid
called GGPL-I and GGPL-III were found from the main cell (TfOH).9) Stereochemical control was achieved by using the
membrane lipid components of Mycoplasma fermentans and solvent effect of nitrile10) to give the desired b-glycoside 5 in
these compounds were synthesized by Kobayashi and his co- 91% yield and the a-glycoside was not detected. The
workers.5) They used a phosphorodiamidite method at the C- anomeric hydrogen atom of the nonreducing-end galactose
6 position of glucose unit, p-nitrophenyl ( pNP) 6-O-phos- unit appeared as a signal at d 4.22 (d, Jϭ7.9 Hz). The b-D
phocholine a-D-glucopyranoside. Up to the present, coupling
incorporation of phosphocholine was performed as follows:
A) phosphorylation with 2-bromoethyl phosphoryl dichloride
and trimethylamine6); B) a phosphorodiamidite method5); C)
phosphorylation with 2-chloro-2-oxo-1,3,2-dioxaphospho-
lane7) in place of 2-bromoethyl phosphoryl dichloride. How-
ever, for the phosphorylation of carbohydrate hydroxy group,
either A) or B)5b,6b) was used. In the present study, we investi-
gated mono- and disaccharides phosphorylation by means of
method C) using 2-chloro-2-oxo-1,3,2-dioxaphospholane and
Fig. 1
trimethylamine and attempted the synthesis of these novel
To whom correspondence should be addressed. e-mail: takeda-td@kyoritsu-ph.ac.jp
© 2001 Pharmaceutical Society of Japan

November 2001
1465
Chart 1
Chart 2
configuration of the newly formed glycosidic bond was also
supported by the JC,H value of 159.7 Hz in the 13C-NMR
spectrum.11) Removal of the tert-butyldimethylsilyl (TBDMS)
group from 5 by Bu4NF gave the disaccharide intermediate 6
in 86% yield. Coupling of a phosphocholine group with 6 as
described for 2 gave the desired compound 7 (91%), and re-
moval of the benzyl groups from 7 by catalytic hydrogenoly-
sis over 10% Pd–C gave the target compound 8 (Chart 2).
Biological Activities The biological activity of com-
pound 3, 8 and phosphocholine free compound 9 were as-
sayed in vitro by histamine release-inhibition test. The
method using rat basophilic leukemia cells (RBL-2H3) was
reported by Teshima et al.12) As shown in Fig. 2, compounds
3 and 8 showed a better activity for histamine release-inhibi-
tion from RBL-2H3 cell than compound 9. The IC50 values
were 2.6 mM (3), 2.9 mM (8) and Ͼ10 mM (9). On the other
hand, octyl b-D-galactopyranoside could not assay in order to
show a potent cytotoxicity.

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Vol. 49, No. 11
–Si(CH3)2). MALDI-TOF-MS: Calcd for C68H88O11Si m/z: 1109. Found:
1132 [MϩNa]ϩ.
Octyl 2,3,4-Tri-O-benzyl-b-D-galactopyranosyl-(1→6)-2,3,4-tri-O-ben-
zyl-b-D-galactopyranoside (6)
A solution of compound 5 (340 mg,
0.31 mmol) in 2 ml of tetrahydrofuran (THF) was treated with 1 M Bu4NF
(1 ml) in THF solution at 0 °C. The reaction mixture was stirred at room
temperature for 1.5 h, and then diluted with CHCl3 and washed with water,
dried (Na2SO4), and concentrated. The residue was chromatographed on sil-
ica gel using 4 : 1 hexane–ethyl acetate as an eluent to give compound 6
1
(261 mg, 86%). [a]D24 Ϫ26.1° (cϭ0.5 CHCl3), H-NMR (CDCl3) d: 7.33—
7.16 (30H, m, 6ϫPh), 4.92—4.57 (12H, m, 6ϫbenzyl methylene), 4.40
(1H, d, Jϭ8.0 Hz, H-1), 4.29 (1H, d, Jϭ7.9 Hz, H-1Ј). MALDI-TOF-MS:
Calcd for C62H74O11 m/z: 995. Found: 1018 [MϩNa]ϩ.
Octyl 2,3,4-Tri-O-benzyl-6-O-phosphocholine-b-D-galactopyranosyl-
(1→6)-2,3,4-tri-O-benzyl-b-D-galactopyranoside (7) To a solution of 6
(105 mg, 0.11 mmol) and triethylamine (21 ml, 0.15 mmol) in dry benzene
(5 ml) was added 2-chloro-2-oxo-1,3,2-dioxaphospholane (46 ml, 0.50 mmol)
at room temperature. After stirring for 12 h at room temperature, the reac-
tion mixture was filtered to remove the precipitated salts. The filtrate and
washings were combined and concentrated. This compound was transferred
into a sealed vessel as a solution in 7 ml of dry acetonitrile. To the solution
was added 95 ml of trimethylamine. The bottle was sealed and then heated in
Fig. 2. Inhibitory Effect of 3, 8 and 9 on Histamine Release from RBL-
2H3
In summary, we have synthesized for the first time the
oligosaccharides including phosphocholine using 2-chloro-2-
oxo-1,3,2-dioxaphospholane in good yield and a phospho-
choline group seemed to act as a inhibitor to a histamine re- an oil bath at 65 °C for 48 h. Resulting mixture was concentrated, and the
lease.
residue was purified by Iatrobeads chromatography, eluting with CHCl3–
MeOH (1 : 1) to give the desired compound (7) (116 mg, 91%): [a]D24
Experimental
ϩ26.6° (cϭ1.9, CHCl3). 1H-NMR (CDCl3) d: 4.44 (1H, d, Jϭ7.9 Hz, H-1Ј),
1
Optical rotations were determined with a JASCO digital polarimeter. H- 4.27 (1H, d, Jϭ7.3 Hz, H-1), 4.15 (2H, m, OCH2CH2), 3.49 (2H, m,
and 13C-NMR spectra were recorded on a JNM A 500 FT NMR spectrome-
CH2CH2N), 3.02 (9H, s, N(CH3)3). MALDI-TOF-MS: Calcd for C67H86NO14P
ter in CDCl3 with Me4Si as the internal standard. Matrix-assisted laser de- m/z: 1160. Found m/z: 1161 [MϩH]ϩ.
sorption ionization (MALDI-TOF-MS) was recorded on a Perceptive Voy-
Octyl 6-O-Phosphocholine-b-D-galactopyranosyl-(1→6)-b-D-galac-
ager RP mass spectrometer. TLC was performed on Silica gel 60 F254 (E. topyranoside (8) A solution of 7 (74.3 mg, 0.06 mmol) in MeOH (4 ml)
Merck) with detection by quenching of UV fluorescence and by spraying containing conc.HCl (0.1 ml) was hydrogenated over 10% Pd–C (54 mg) for
with 10% H2SO4. Column chromatography was carried out on Silica Gel 60 12 h at room temperature, then filtered through Celite and the filter cake was
(E. Merck). Octyl 2,3,4-tri-O-benzyl-b-D-galactopyranoside (1) was pre-
washed with methanol. Combined filtrate and washing were concentrated.
Column chromatography (MeOH : H2Oϭ1 : 1) of the residue on Sephadex
LH-20 to give 8 (36.4 mg, 92%), [a]D24 Ϫ9.7° (cϭ0.4, H2O), 1H-NMR
(D2O); d: 4.31 (1H, d, Jϭ7.9 Hz, H-1Ј), 4.22 (1H, d, Jϭ7.9 Hz, H-1), 4.14
pared by a literature method.8)
Octyl 2,3,4-Tri-O-benzyl-6-O-phosphocholine-b-D-galactopyranoside
(2) To a solution of 1 (102 mg, 0.18 mmol) and triethylamine (50 mg,
0.36 mmol) in dry benzene (5 ml) was added 2-chloro-2-oxo-1,3,2- (2H, m, OCH2CH2), 3.49 (2H, m, CH2CH2N), 3.05 (9H, s, N(CH3)3).
dioxaphospholane (50 ml, 0.54 mmol) at room temperature. After stirring for MALDI-TOF-MS: Calcd for C25H50NO14P m/z: 619. Found: 620 [MϩH]ϩ.
3 h at room temperature, the reaction mixture was filtered to remove the pre-
Octyl b-D-Galactopyranosyl-(1→6)-b-D-galactopyranoside (9) A so-
cipitated salts. The filtrate and washing were combined and concentrated. lution of 6 (15 mg, 15.1 mmol) in MeOH (2 ml) was hydrogenated over 10%
This compound was transferred into a sealed vessel as a solution in 10 ml of Pd–C (20 mg) for 12 h at room temperature, then filtered through Celite and
dry acetonitrile. To the solution was added 100 ml of trimethylamine and the the residue was washed with methanol and concentrated. Column chro-
bottle was sealed and then heated in an oil bath at 65 °C for 48 h. Resulting matography (1 : 1 CHCl3–MeOH) of the residue on Sephadex LH-20 gave 9
mixture was concentrated and the residue was purified by Iatrobeads chro- (6.7 mg, 98%); [a]D24 Ϫ24.6° (cϭ0.5, 1 : 1 CHCl3–MeOH); 1H-NMR
matography, eluting with CHCl3–MeOH–H2O (8 : 4 : 1) to give the desired (CD3OD): d: 4.30 (d, 1H, Jϭ8.0 Hz, H-1Ј), 4.19 (d, 1H, Jϭ7.5 Hz, H-1).
compound (2) (89.1 mg, 67.5%): [a]D24 ϩ17.5° (cϭ1.2, CHCl3). 1H-NMR
(CDCl3) d: 4.34 (1H, d, Jϭ7.9 Hz, H-1), 4.22 (2H, m, OCH2CH2), 3.64
(2H, m, CH2CH2N), 3.16 (9H, s, N(CH3)3). MALDI-TOF-MS: Calcd for
C40H58NO9P m/z: 727. Found m/z: 728 [MϩH]ϩ.
MOLDI-TOF-MS: Calcd for C20H38O11: m/z 454. Found: m/z 477 [MϩNa]ϩ.
Acknowledgements This work was supported by a Grant-in-Aid for
Scientific Research (No. 12672062) from the Ministry of Education, Sci-
ence, Sports and Culture of Japan. We gratefully acknowledge the financial
Octyl 6-O-Phosphocholine-b-D-galactopyranoside (3) A solution of 2
(81.5 mg, 0.11 mmol) in MeOH (4 ml) containing conc. HCl (0.1 ml) was hy- support of Uehara Memorial Foundation. The authors are grateful to Ms. J.
drogenated over 10% Pd–C (57 mg) for 12 h at room temperature, then fil- Hada for providing NMR and MS data.
tered through Celite and the filter cake was washed with methanol. Com-
bined filtrate and washings were concentrated. Column chromatography References
(MeOH : H2Oϭ1 : 1) of the residue on Sephadex LH-20 to give 3 (52.0 mg,
quant.), [a]D24 Ϫ3.8° (cϭ0.4, H2O), 1H-NMR (D2O); d 4.23 (1H, d,
Jϭ8.6 Hz, H-1), 4.14 (2H, m, OCH2CH2), 3.49 (2H, m, CH2CH2N), 3.05
(9H, s, N(CH3)3). MALDI-TOF-MS; Calcd for C19H40NO9P m/z: 457. Found:
458 [MϩH]ϩ.
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