Synthesis and biological activities of glycosphingolipid analogues from marine sponge Aplysinella rhax

Article abstract of DOI:10.1016/j.bmcl.2007.07.108

A novel glycosphingolipid, β-d-GalNAcp(1 → 4)[α-d- Fucp(1 → 3)]-β-d-GlcNAcp(1→)Cer (1), isolated from the marine sponge Aplysinella rhax, has a unique structure, with d-fucose and N-acetyl-d-galactosamine attached to a reducing-end N-acetyl-d-glucosamine

Full text of DOI:10.1016/j.bmcl.2007.07.108

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5912–5915
Synthesis and biological activities of glycosphingolipid
analogues from marine sponge Aplysinella rhax
Noriyasu Hada,* Taishi Nakashima, Suraj Prakash Shrestha, Ryo Masui,
Yuji Narukawa, Kayoko Tani and Tadahiro Takeda
Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
Received 8 June 2007; revised 24 July 2007; accepted 26 July 2007
Available online 29 August 2007
Abstract—A novel glycosphingolipid, b-D-GalNAcp(1 ! 4)[a-D- Fucp(1 ! 3)]-b-D-GlcNAcp(1!)Cer (1), isolated from the marine
sponge Aplysinella rhax, has a unique structure, with D-fucose and N-acetyl-D-galactosamine attached to a reducing-end N-acetyl-D-
glucosamine through an a1 ! 3 and b1 ! 4 linkage, respectively. We synthesized glycolipid analogues carrying a 2-branched fatty
alkyl residue or a 2-trimethylsilyl ethyl residue in place of ceramide (2 and 3), non-natural type trisaccharide analogue containing an
L-fucose residue (4), and other analogues (5 and 6). Among these prepared compounds, 2 showed the most potent nitric oxide (NO)
production inhibitory activity against LPS-activated J774.1 cells. In addition, their structure–activity relationships were established.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The structure and the biological functions of many gan-
gliosides have been widely investigated and are reported
in various reviews.1,2 However, in our continuing sys-
tematic studies of the role and biological functions of
glycolipids in various invertebrate animal species that
do not have gangliosides, we have synthesized some no-
vel glycolipid derivatives found in these invertebrates.3–8
Recently, Zollo et al.9 isolated and characterized a novel
neutral glycosphingolipid (1, Fig. 1) from the marine
sponge Aplysinella rhax, the carbohydrate structure of
which features a D-fucose and an N-acetyl-D-galactos-
amine attached to a reducing-end N-acetyl-D-glucosa-
mine through an a1 ! 3 and a b1 ! 4 linkage,
respectively. This was the first report on glycolipids con-
activity on LPS-induced NO release by J774.1 macro-
phages.9 Therefore, in order to investigate the strength
of these compounds, inhibitory against nitric oxide re-
lease, we synthesized the trisaccharide analogues 2 and
3, containing a 2-branched fatty alkyl residue and a
2-(trimethylsilyl)ethyl (TMS-Et) residue, respectively,
in place of ceramide; a non-natural-type trisaccharide
analogue with an L-fucose residue (4); and other ana-
logues 5 and 6 (Fig. 1).
The target glycolipid analogues were prepared by a
conventional synthetic pathway (Scheme 1). Glycosyl
acceptor 7 was prepared from the known compound 2-
(trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranoside11
by deacetylation, benzylidenation, chloroacetylation,
and reductive ring-opening of the benzylidene acetal to
afford compound 7. Glycosylation of 7 with 8, which
was prepared from 1,3,4,6,tetra-O-acetyl-2-deoxy-2-
(2,2,2- trichloroethoxycarbonylamino)-b-D-galactopyra-
nose,11 was carried out in the presence of TMSOTf and
NAcp(1 ! D4)-[fau-cDo-sFeu.cp(1 ! 3)]-b-D-GlcNAcp(1!)Cer (1),
has structural variations at the ceramide parts.
Glycosphingolipids found in nature are classified into
many types on the basis of their basic carbohydrate
structure; these types include the globo-, lacto-, gan-
glio-, mollu-, arthro-, and gala series.10 However, to
the best of our knowledge, the carbohydrate structure,
GalNAcb1 ! 4GlcNAcb1!, represents a new type of
glycolipid core structure. Furthermore, these glycolipids
have been also found to exhibit significant inhibitory
taining
This
compound,
b-D-Gal-
˚
4A molecular sieves (4A MS) in CH2Cl2. The reaction
produced the desired disaccharide 9 in 80% yield, as evi-
1
denced by H NMR spectroscopy (H-10, 4.25 ppm, J
8.5 Hz).12 The chloroacetyl group in 9 was removed by
the action of thiourea to afford 10, which was used
in the subsequent coupling reaction. The D-fucopyrano-
syl donor 1113 was prepared in the same way as the
L-fucopyranosyl donor phenyl 2,3,4-tri-O-benzyl-1-
thio-b-L-fucopyranoside, as reported by Hasegawa
Keywords: Glycosphingolipid; Aplysinella rhax; D-fucose; Nitric oxide.
*
Corresponding author. Tel.: +81 3 5400 2666; fax: +81 3 5400
2556; e-mail: hada-nr@kyoritsu-ph.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.108

N. Hada et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5912–5915
5913
OH
(CH ) -CH
3
O
2 m
OH
OH
HO
NH
OH
CH
3
O
HO
O
O
O
(CH ) -CH
2 n
O
CH
3
NHAc
Me
OH
NHAc
HO
O
HO
OH
-GalNAcp(1-4)-[
β-D
α-
D
-Fucp(1-3)]-
β-
D-GlcNAcp(1-)Cer
1
natural products
OH
OH
HO
OH
OH
HO
HO
O
NHAc
HO
O
NHAc
O
O
(CH ) CH
O
O
2 13
3
3
O
O
TMS
O
O
(CH ) CH
2 13
NHAc
Me
NHAc
Me
O
O
HO
HO
HO
3
HO
2
OH
OH
OH
OH
HO
OH
OH
HO
OH
OH
HO
HO
O
NHAc
HO
O
(CH ) CH
2 13
O
O
3
3
(CH ) CH
2 13
HO
O
O
3
3
O
O
O
O
O
O
(CH ) CH
2 13
O
O
(CH ) CH
2 13
TMS
HO
NHAc
NHAc
OH
NHAc
NHAc
O
O
Me
HO
OH
OH
Me
HO
OH
OH
5
6
4
Figure 1. Target analogue compounds.
PhS
BnO
O
Me
BnO
O
OAc
OAc
OAc
OAc
O
HO
4
BnO
TMS
R O
CAO
BnO
O
O
OBn
11
O
O
1
NHTroc
R
O
AcO
O
c
AcO
O
7
TMS
O
a
RO
3
3
2
R HN
NHR
NHTroc
R
NHTroc
+
O
Me
4
R O
OAc
OAc
AcO
9 R = CA
10 R = H
4
R O
b
4
O
OR
3
CCl
1
2
4
O
3
R
R
R
R
d,f
NHTroc
12 OSE
13 OSE
H
H
Troc Bn
6
8
NH
d
e
Ac
Ac
H
Ac
Ac
H
14
3
H OC(NH)CCl
3
f
OSE
H
OR
OR
RO
RO
O
(CH ) CH
(CH ) CH
O
O
2 13
3
3
a
2 13
3
3
HO
O
+
14
(CH ) CH
2 13
O
(CH ) CH
2 13
NHAc
Me
NHAc
15
O
16 R = Ac
2 R = H
RO
f
RO
OR
Scheme 1. Reagents: (a) TMSOTf, 4A MS, CH2Cl2, 9: 80%, 16: 12%; (b) thiourea, Pyr.–EtOH, 77%; (c) NIS, TfOH, MS4A CH2Cl2, 89%; (d) i—Zn–
AcOH; ii—Ac2O–Pyr.; iii—H2, Pd–C, MeOH–AcOH; iv—Ac2O–Pyr., 71% over four steps; (e) i—TFA, CH2Cl2; ii—CCl3CN, DBU, CH2Cl2, 64%;
(f) NaOMe, 1,4-dioxane–MeOH,2:86%, 3: 94%.
et al.14 Fucosyl donor 11 was then allowed to react with
disaccharide acceptor 10 in the presence of N-iodosuc-
cinimide (NIS), trifluoromethanesulfonic acid (TfOH),15
and 4A MS in dichloromethane at ꢀ60 ꢁC to afford the
desired trisaccharide 12 (89%), which contains a newly
introduced a-glycosidic linkage; this was confirmed by
1H NMR spectroscopy (H-100, 4.97 ppm, J 3.7 Hz).16
Removal of the NTroc-protecting group in 12 was car-
ried out via treatment with zinc powder in acetic acid,
and the resulting crude amine was N-acetylated with
acetic anhydride. Hydrogenation of the N-acetylated
compound over a catalyst of 10% palladium on charcoal
in methanol-acetic acid and subsequent acetylation gave
the per-O-acetylated trisaccharide 13. Complete depro-
tection of disaccharide derivative 10 and trisaccharide
13 were carried out to give analogue compounds 6 (with-
out fucose) and 3 (with TMS-Et spacer), respectively.
Next, compound 13 was converted into the trichloroace-
timidate derivative 14, which was then glycosylated with
2-tetradecylhexadecan-1-ol (15)17 in the presence of
trimethylsilyltrifrate (TMSOTf)18 to give the desired
b-glycoside 16 (12%). Finally, removal of all acetylated
groups under basic conditions gave the target molecule
2. Unfortunately, due to the formation of several side
products, the imidate donor 14 gave a poor yield on
coupling with 15. Therefore, 2-Troc-protected imidate
donor 18 was used instead of 14 (Scheme 2). Hydrogen-
olytic removal of the benzyl group of 12 over Pd–C in
MeOH–AcOH, followed by removal of the TMS-Et
group, yielded imidate donor 18. Coupling of 18 with
15 in the presence of TMSOTf afforded the desired
b-glycoside 19 in 40% yield. Treatment of 19 with zinc

5914
N. Hada et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5912–5915
OAc
OAc
AcO
4
R O
O
O
1
1
R
1
OR
OR
O
O
1
3
R O
3
2
(CH ) CH
2 13
O
O
R HN
O
3
3
NHR
R
15
c
1
O
R O
O
Me
4
O
(CH ) CH
2 13
R O
2
2
NHR
NHR
4
18
R O
4
1
2
O
Me
OR
3
1
R
R
R O
1
2
4
1
R
R
R
R
R O
19 Ac
16 Ac
Troc
Ac
H
1
d
e
OR
12 OSE
17 OSE
H
H
Troc Bn
Troc Ac
Troc Ac
a
b
2
H
18
H OC(NH)CCl
3
Scheme 2. Reagents: (a) i—H2, Pd–C, MeOH–AcOH; ii—Ac2O–Pyr., 60%,; (b) i—TFA, CH2Cl2; ii—CCl3CN, DBU, CH2Cl2, 92%; (c) TMSOTf,
MS4A CH2Cl2, 40%; (d) i—Zn–AcOH; (ii) Ac2O-Pyr., 58%; (e) NaOMe, 1,4-dioxane–MeOH, 94%.
Table 1. Inhibitory effects of 2, 3, 4, 5, and 6 on NO production in LPS-activated J774.1 cells
Each data represents the mean SD for quadruplet experiments.
powder in acetic acid cleaved the Troc group and subse-
quent N-acetylation gave 20. Finally, removal of the
O-acetyl groups of 20 under basic conditions followed
by column chromatography using a Sephadex LH-20
gave the target glycolipid analogue 2 (Scheme 2). The
as a host defense by damaging pathogenic DNA and
as a regulatory molecule with homeostatic activity.22
However, excessive production has detrimental effects
on many organ systems of the body, which can lead to
tissue damage and even to fatal development (septic
shock).23
1
structure of 2, 3, and 6 was demonstrated by H NMR
and MALDI-TOF-MS spectrometry.19 Compound 4
was prepared by an analogous method using an L-fucose
derivative instead of D-fucose.20 Compound 5, with a
LeX glycan structure, was also synthesized in a same
manner, replacing N-acetyl-D-galactosamine with D-
galactose.21
The inhibitory effect of glycosphingolipid (1) has been
evaluated and reported in previous papers.9 However,
in order to reveal the effect of differences in carbohy-
drate structure on biological function, we examined
ꢀ
inhibition of LPS-induced NO2 release by the synthe-
sized compounds 2–6 (Table 1). The inhibition assays
for 2, 3, and 6 suggested that the presence of fucose en-
hances the inhibitory effect, and that the aglycon moiety
is not important in this process. In addition, comparison
of the results obtained for 2, 4, and 5 revealed that the
presence of D-fucose and GalNAc enhances inhibitory
activity.
Nitric oxide (NO), which is naturally synthesized by a
family of enzymes known as NO-synthase (NOS), is
an important signaling molecule that acts in many tis-
sues to regulate a diverse range of physiological pro-
cesses. Two forms of NOS are recognized: constitutive
isoforms (endothelial NOS and neuronal NOS) and an
inducible isoform for which mRNA translation and pro-
tein synthesis are required. When certain cells are acti-
vated by specific proinflammatory agents such as
endotoxin, tumor necrosis factor (TNF), interferon-
gamma (IFN-c), or interleukin-1 (IL-1), NO is produced
by inducible NOS (iNOS). The NO thus produced acts
Acknowledgments
This work was supported by a Grant-in-Aid for Scien-
tific Research (No. 19590011) from the Ministry of Edu-

N. Hada et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5912–5915
5915
MALDI-TOF-MS: m/z [M+Na]+ calcd for C33H34O4SNa:
549.2; found: 549.4.
14. Komba, S.; Ishida, H.; Kiso, M.; Hasegawa, A. Bioorg.
Med. Chem. 1996, 4, 1833.
cation, Culture, Sports, Science and Technology of Ja-
pan (MEXT), and the (MEXT) High-Tech Research
Center project.
15. Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetra-
hedron Lett. 1990, 31, 4313.
23
References and notes
16. Analytical data for the compound 12: ½aꢁD +15.2ꢁ (c 1.0,
CHCl3); 1H NMR (500 MHz, CDCl3): d 4.97 (d, 1H,
J1,2 = 3.7 Hz, H-1 of fuc), 4.52 (br. d, 1H, H-1 of
GlcNAc), 4.46 (br. s, 1H, H-1, of GalNAc). 13C NMR
(125 MHz, CDCl3): d: 170.5, 170.3, 170.2, 170.1, 154.2,
138.8, 138.7, 138.5, 138.4, 138.33, 138.28, 138.2, 129.0,
128.7, 128.6, 128.4, 128.3, 128.2, 128.16, 128.13, 128.0,
127.93, 127.85, 127.7, 127.6, 127.5, 127.3, 127.2, 127.1,
125.2, 100.2, 99.6, 98.8, 95.8, 95.3, 79.4, 77.6, 76.4, 76.2,
75.2, 74.8, 74.5, 74.4, 73.5, 73.4, 73.31, 73.25, 73.2, 72.8,
70.8, 70.6, 70.3, 69.6, 67.3, 66.7, 66.2, 61.3, 61.1, 54.2, 52.8,
52.5, 21.4, 20.62, 20.59, 20.5, 18.1, 17.9, 17.2, 16.6, 16.5,
ꢀ0.1, ꢀ1.4. MALDI-TOF-MS: m/z [M+Na]+ calcd for
C63H78Cl6N2O20SiNa: 1443.3; found: 1443.1.
1. Angata, T.; Varki, A. Chem. Rev. 2002, 102, 439.
2. Varki, A. Glycobiology 1992, 2, 25.
3. Hada, N.; Sonoda, Y.; Takeda, T. Carbohydr. Res. 2006,
341, 1341.
4. Yamamura, T.; Hada, N.; Kaburaki, A.; Yamano, K.;
Takeda, T. Carbohydr. Res. 2004, 339, 2749.
5. Ohtsuka, I.; Hada, N.; Sugita, M.; Takeda, T. Carbohydr.
Res. 2003, 337, 2037.
6. Ohtsuka, I.; Hada, N.; Ohtaka, H.; Sugita, M.; Takeda, T.
Chem. Pharm. Bull. 2002, 50, 600.
7. Hada, N.; Sato, K.; Sakushima, J.-I.; Goda, Y.; Sugita,
M.; Takeda, T. Chem. Pharm. Bull. 2001, 49, 1464.
8. Hada, N.; Ohtsuka, I.; Sugita, M.; Takeda, T. Tetrahedo-
ron Lett. 2000, 41, 9065.
17. Hasegawa, A.; Suzuki, N.; Ishida, H.; Kiso, M. J.
Carbohydr. Chem. 1996, 15, 623.
18. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25,
212.
9. Borbone, N.; Marino, S. D.; Iorizzi, M.; Zollo, F.;
Debitus, C.; Ianaro, A.; Pisano, B. Eur. J. Org. Chem.
2001, 4651.
23
19. Analytical data for the compound 2: ½aꢁD ꢀ21.4ꢁ (c 0.1,
1:1 CHCl3-MeOH); 1H NMR (500 MHz, CDCl3-
CD3OD): d 5.15 (d, 1H, J = 3.6 Hz, H-1 of Fuc), 4.59
(d, 1H, J = 8.3 Hz, H-1 of GlcNAc), 4.32 (d, 1H, d, 1H,
J = 8.0 Hz, H-1 of GalNAc). 13C NMR (125 MHz,
CDCl3-CD3OD): d 173.5, 172.6, 102.7, 101.8, 101.3,
80.7, 76.4, 76.1, 75.3, 73.6, 73.0, 72.3, 71.1, 70.3, 69.6,
68.0, 62.4, 61.3, 55.7, 54.2, 49.6, 38.9, 32.6, 31.8, 31.5, 30.8,
30.7, 30.3, 30.0, 27.5, 27.3, 23.4, 23.3, 23.0, 16.5, 14.4.
MALDI-TOF-MS: m/z [M+Na]+ calcd for C52H98N2O15-
Na: 1014.7; found: 1014.4. Analytical data for the com-
10. Glycolipid; Wiegandt, H., Ed.; Elsevier: New York, 1985.
11. Ellervik, U.; Magnusson, G. Carbohydr. Res. 1996, 280,
251.
23
12. Analytical data for the compound 9: ½aꢁD ꢀ19.4ꢁ (c 2.0,
CHCl3); 1H NMR (500 MHz, CDCl3): d 7.37–7.49 (m, 5H,
Ph), 5.23 (d, 1H, J3,4 = 3.7 Hz, H-40), 5.19 (dd, 1H,
J = 9.2 Hz, 10.4 Hz, H-3), 5.13 (d, 1H, J2,NH = 10.4 Hz,
NH), 4.83 (d, 1H, J = 12.2 Hz, benzyl methylene), 4.63–
4.76 (m, 6H, H-30, CH2CCl3 · 2), 4.51 (d, 1H,
J1,2 = 7.9 Hz, H-1), 4.37 (d, 1H, J = 12.2 Hz, benzyl
23
1
methylene), 4.25 (d, 1H, J1 ,2 = 8.5 Hz, H-10), 4.08 (dd,
2H, J = 14.7 Hz, J = 19.9 Hz, ClCH2CO), 4.05–4.12 (m,
1H, H-60a), 3.89–4.00 (m, 3H, H-60b, H-4,
CH2CH2Si(CH3)3), 3.74 (br. t, 1H, H-6a), 3.50 ꢂ 3.66
(m, 5H, H-2, H-6b, H-20, H-50, CH2CH2Si(CH3)3), 3.47
(br. d, 1H, H-5), 2.11, 2.08, 1.95 (each s, 3H, Ac), 0.87–
0.95 (m, 2H, CH2CH2Si(CH3)3), ꢀ0.012 (s, 9H, Si(CH3)3).
13C NMR (CDCl3): d 170.4 (NHCO), 170.1 (NHCO),
167.1 (COCH2Cl), 153.8, 137.4, 129.3, 129.1, 128.8, 100.4
(C-1), 100.2 (C-10), 95.5 (CCl3 · 2), 74.5 (CH2CCl3 · 2),
74.2 (C-5), 74.0 (C-3), 73.7 (benzyl methylene), 70.5 (C-50),
69.8 (C-30), 67.5 (C-40), 67.0 (C-6), 66.2 (C-40), 61.2 (C-60),
56.2 (C-2), 52.4 (C-20), 40.8 (CH2Cl), 20.7, 20.7, 20.6, 18.1,
ꢀ1.38. MALDI-TOF-MS: m/z [M+Na]+ calcd for
C38H52Cl7N2O17Si: 1104.1; found: m/z 1104.3.
pound 3: ½aꢁD +8.5ꢁ (c 0.3, MeOH); H NMR (500 MHz,
CDCl3-CD3OD): d 5.25 (d, 1H, J = 3.7 Hz, H-1 of Fuc),
4.58 (d, 1H, J = 8.0 Hz, H-1 of GlcNAc), 4.38 (d, 1H, d,
1H, J = 8.0 Hz, H-1 of GalNAc). MALDI-TOF-MS: m/z
[M+Na]+ calcd for C27H50N2O15SiNa: 693.3; found:
693.5. Analytical data for the compound 6: 1H NMR
(500 MHz, CD3OD): d 4.45 (d, 1H, J = 8.5 Hz, H-1 of
GlcNAc), 4.37 (d, 1H, d, 1H, J = 8.5 Hz, H-1 of Gal-
NAc).). MALDI-TOF-MS: m/z [M+Na]+ calcd for
C21H40N2O11SiNa: 547.2; found: 547.7.
0
0
23
20. Analytical data for the compound 4: ½aꢁD ꢀ54.3ꢁ (c 0.1,
1:1 CHCl3-MeOH); 1H NMR (500 MHz, CDCl3-
CD3OD): MALDI-TOF-MS: m/z [M+Na]+ calcd for
C52H98N2O15Na: 1014.7; found: 1014.4.
23
21. Analytical data for the compound 5: ½aꢁD ¼ ꢀ33:9ꢃ (c
0.3, 1:1 CHCl3-MeOH); 1H NMR (500 MHz, CDCl3-
CD3OD): d 5.01 (d, 1H, J = 3.6 Hz, H-1 of Fuc),
4.46 (d, 1H, J = 8.3 Hz, H-1 of Gal), 4.44 (d, 1H, d,
1H, J = 8.0 Hz, H-1 of GlcNAc). MALDI-TOF-MS:
m/z [M+Na]+ calcd for C50H95NO15Na: 972.7; found:
972.8.
23
13. Analytical data for the compound 11: ½aꢁD +5.8ꢁ (c 1.0,
CHCl3); 1H NMR (500 MHz, CDCl3): d 7.53–7.18 (m,
20H, Ph · 4), 5.02–4.66 (m, 6H, benzyl methylene · 3),
4.60 (d, 1H, J1,2 = 9.8 Hz, H-1), 3.93 (t, 1H, J2,3 = 9.7 Hz,
H-2), 3.63 (d, 1H, J3,4 = 2.4 Hz, H-4), 3.59 (m, dd, H-3),
3.52 (q, 1H, H-5), 1.26 (d, 3H, J5,6 = 6.1 Hz, H-6); 13C
NMR (125 MHz, CDCl3): d 138.7, 138.4, 138.3, 134.3,
131.5, 128.7, 128.4, 128.3, 128.1, 127.9, 127.6, 127.5, 127.4,
126.9, 87.5, 84.5, 77.1, 76.6, 75.5, 74.6, 72.8, 17.3;
22. Kuo, P. C.; Schroeder, R. A. Ann. Surg. 1995, 221, 220.
23. Vincent, J. L.; Zhang, H.; Szabo, C.; Preiser, J. C. Am. J.
Respir. Crit. Care. Med. 2000, 161, 1781.