Design, Synthesis, and Biological Evaluation of Ganglioside Hp-s1 Analogues Varying at Glucosyl Moiety

Article abstract of DOI:10.1021/acschemneuro.6b00069

Ganglioside Hp-s1 is isolated from the ovary of sea urchin Diadema setosum. It exhibited better neuritogenic activity than GM1 in pheochromocytoma 12 cells. To explore the roles of glucosyl moiety of Hp-s1 in contributing to the neurogenic activity, we developed feasible procedures for synthesis of Hp-s1 analogues (2a-2f). The glucosyl moiety of Hp-s1 was replaced with α-glucose, α-galactose, β-galactose, α-mannose, and β-mannose, and their biological activities on SH-SY5Y cells and natural killer T (NKT) cells were evaluated. We found that the orientation of C-2 hydroxyl group at glucosyl moiety of Hp-s1 plays an important role to induce neurite outgrowth of SH-SY5Y cells. Surprisingly, compound 2d could activate NKT cells to produce interleukin 2, although it did not show great activity on neurite outgrowth of SH-SY5Y cells. In general, the Hp-s1 might be considered as a lead compound for the development of novel drugs aimed at modulating the activity of neuronal cells.

Full text of DOI:10.1021/acschemneuro.6b00069

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Article
Design, synthesis, and biological evaluation of
ganglioside Hp-s1 analogues varying at glucosyl moiety
Jung-Tung Hung, Chun-Hong Yeh, Shih-An Yang, Chiu-Ya Lin, Hung-Ju Tai,
Ganesh B. Shelke, Daggula Mallikarjuna Reddy, Alice L Yu, and Shun-Yuan Luo
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00069 • Publication Date (Web): 08 Jun 2016
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Design, synthesis, and biological evaluation of ganglioside Hp-s1 ana-
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Jung-Tung Hung,1 Chun-Hong Yeh,2 Shih-An Yang,2 ChiuꢀYa Lin,2 Hung-Ju Tai,2 Ganesh B. Shel-
ke,2 Daggula Mallikarjuna Reddy,2 Alice L. Yu,1 and Shun-Yuan Luo2,*
1Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linkou, Taoyuan 333,
Taiwan
2Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan
Supporting Information Placeholder
ABSTRACT: Ganglioside Hp-s1 is isolated from ovary of sea urchin Diadema setosum. It exhibited better neuritogenic
activity than GM1 in pheochromocytoma 12 cells. To explore the roles of glucosyl moiety of Hp-s1 in contributing to the
neurogenic activity, we developed feasible procedures for synthesis of Hp-s1 analogues (2a-2f). The glucosyl moiety of Hp-
s1 was replaced with α-glucose, α-galactose, β-galactose, α-mannose, β-mannose, and their biological activities on SH-
SY5Y cells and natural killer T (NKT) cells were evaluated. We found that the orientation of C-2 hydroxyl group at gluco-
syl moiety of Hp-s1 plays an important role to induce neurite outgrowth of SH-SY5Y cells. Surprisingly, compound 2d
could activate NKT cells to produce interleukin 2, although it did not show great activity on neurite outgrowth of SH-
SY5Y cells. In general, the Hp-s1 might be considered as a lead compound for the development of novel drugs aimed at
modulating the activity of neuronal cells.
Keywords:
Ganglioside Hp-s1, neurogenic activity, SH-SY5Y cells, glucosyl moiety of Hp-s1
Introduction: Glycosphingolipids play an important role
in cell recognition and signaling.1 Gangliosides, sialic acid-
containing glycosphingolipids, are abundant in the cen-
tral and peripheral nervous systems and are critical to
stabilize the various brain functions.2,3 On the other hand,
exogenously administered gangliosides showed neu-
rotrophic and neuritogenic effects on neuronal cells in
vitro and in experimental animal models.4 For example,
ganglioside GQ1b might trigger the activation of protein
kinase on cell surface to increase proliferation5 and neu-
rite outgrowth of human neuroblastoma cell lines, GOTO
and NB-1.6 GM3 induced neuritogenesis of mouse neuro-
blastoma Neuro2a cells by decreasing Csk/c-Src ratio in
glycosphingolipid-enriched domain and then leading to
activation of the c-Src.7 Treatment of GM1 ganglioside
improved the survival of lesioned nigral dopamine neu-
rons in rats.8 Furthermore, it also ameliorated the 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced par-
kinsonian-like symptoms in nonhuman primates9 and
showed neurologic improvement in patients with acute
stroke at the first few days.10 These evidences suggest that
treatment of exogenous neuritogenic gangliosides might
serve as therapeutic strategy for neurological diseases.
Although many gangliosides have been extracted and
identified from echinoderms, such as GP-2, LG-2 and
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GAA-7 from starfish, SJG-2, HPG-1 and HLG-3 from sea
logues 2a-2f (Figure 2) were synthesized as described be-
low. The glucosyl moiety of Hp-s1 was replaced with α-
cucumber, and CJP2, CJP3 and CJP4 from feather star,11
only a few of them had been reported to have biological
activities in neuronal cells. GP-2, which was isolated from
starfish Asterina pectinifera, showed better activity than
GM1 to support the survival of cultured cerebral cortex
cells. GAA-7, which is the main oligosaccharide moiety in
Asterias amurensis versicolor, had neuritogenic and
growth-inhibitory activities on mouse neuroblastoma cell
line Neuro 2a.12 Neuritogenic activity of SJG-2, LLG-3 and
GAA-7 on rat pheochromocytoma cell PC-12 was en-
hanced in the presence of nerve growth factor (NGF)
when compared with GM1.2,13 In addition, DSG-A, which
was extracted from ovary of sea urchin Diadema setosum,
displayed higher neuritogenic activity than GM1 in the
presence of NGF on PC-12 cells.14 Because of limited natu-
ral resources and difficulties in extraction procedures,
chemical synthesis of these neuritogenic gangliosides,
such as GAA-715 and DSG-A,16 has drawn attentions of
chemists. We were interested in ganglioside Hp-s1 (Figure
1), which was discovered in the sperm of sea urchin Hemi-
1
2
3
4
5
6
7
8
glucopyranoside,
galactopyranoside,
α-galactopyranoside,
α-mannopyranoside
β-
β-
and
mannopyranoside. The sialic acid moiety of Hp-s1 was
removed to generate 2a. The compound 2b was prepared
by coupling a glucosyl moiety with phytosphingosine in
α-configuration, which is different from Hp-s1 in β-
configuration. To evaluate the role of 4-OH position of
glucosyl moiety, we synthesized compound 2c (β-
configuration) and 2d (α-configuration) by using galacto-
syl moiety. To reveal the character of 2-OH position of
glucosyl moiety, compound 2e and 2f synthesized via a
coupling reaction between mannosyl group and phyto-
sphinogosine at α-configuration and β-configuration re-
spectivly.
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By following the literature procedure, the phytosphingo-
sine derived acceptor 319b and glycosyl donors 4-820 were
synthesized.
centrotus pulcherrimus and ovary of sea urchin Diadema
17
→
→
setosum. Hp-s1 (Neu5Acα2 6Glcβ1 1Cer) is composed of
three major components, including phytosphingolipid,
glucosyl moiety, and sialic acid. An Hp-s1 analogue with
shorter phytosphingosine chain was synthesized and
showed neuritogenic activity on human neuroblastoma
cell line SH-SY5Y without NGF,18 suggesting that the neu-
ritogenic activity of Hp-s1 might have been enhanced
through modification of its glycan moiety or ceramide
part. Recently, we have shown a straightforward protocol
for the synthesis of Hp-s1.19b To further explore the role of
glucosyl moiety of Hp-s1 on neuritogenic activity, in this
study, we synthesized Hp-s1 analogues 2a-2e with substi-
tution of the glucosyl moiety with other hexopyranoses
and evaluated their neuritogenic activity in vitro.
Figure 2. Structures of ganglioside Hp-s1 analogues 2a–2f.
Then, various anomeric leaving groups, such as iodo, ace-
tate, imidate, and STol, were conjugated to glycosyl do-
nors. The compound 3 was glycosylated with donor 4,
o
promoted by TMSOTf in CH2Cl2:CH3CN solvent at -30 C
o
to room temperature (25 C) and followed by hydrolysis
to afford β-glucosyl azido compound 919b in 82% yield
(α/β = 1/3.2, Entry 1, table 1). The glycosylation product of
acceptor 3 and donor 4 showed similar polarity with start-
ing reagents and α-isomer. Separation of the compound 9
was easily attained through column chromatography by
hydrolyzing O-6 acetyl group of glucosyl moiety immedi-
ately after the first glycosylation. The α-glucosyl azido
compound 10 was synthesized by coupling imidate donor
4 with acceptor 3 in CH2Cl2 and TMSOTf as promoter,
followed by hydrolysis to get 83% yield (α/β = 3.8/1, Entry
2, table 1). β-Galactosyl azido compound 11 was synthe-
sized by treating imidate donor 5 with acceptor 3 in the
presence of TMSOTf in mixed solvent CH2Cl2:CH3CN (1:2)
and hydrolyzed with NaOMe in MeOH to get 90% yield,
with improvement in α/β ratio to 1/5 (Entry 3, Table 1).
Best condition for synthesis of α-galactosyl azido com-
pound 12, (Entries 4-6, Table 1), was rendered when ac-
Figure 1. Structures of ganglioside α-galactosyl ceramide 1
and Hp-s1 2
Result and Discussion: Recently, we have developed a
concise synthesis of α-galactosyl ceramide from
D-lyxose
in five steps19a and a straightforward method for the syn-
thesis of ganglioside Hp-s1 1 starting from phytosphingo-
sine or D-lyxose in ten steps.19b In this study, the gangli-
oside Hp-s1 2 was served as a lead molecule and its ana-
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1
4a
TMSOTf
CH2Cl2/CH3CN
(1/2)
-30 to
rt
9
ceptor 3 was treated with 2.0 equiv. of iodide donor 6 un-
82%, 1/3.2
1
2
3
4
5
6
7
8
der the influence of TBAI, DIPEA, metallic Cu in toluene
at 80 °C and successive removal of O-6 acetyl group to
afford 85% yield (α/β = 7.5/1, Entry 6, Table 1).
2
3
4a
5a
TMSOTf
TMSOTf
CH2Cl2
rt
10
83%, 3.8/1
CH2Cl2/CH3CN
(1/2)
-30 to
rt
11
When acetate donor 7 was treated with acceptor 3 in the
presence of BF3·OEt2 in CH2Cl2 as solvent at 25 °C and
hydrolysed with NaOMe to provide α-mannosyl azido
compound 13 in 53% yield. Compound 14 was synthesized
in 62% by reacting acceptor 3 with donor 8 in CH2Cl2 in
presence of BSP, DTBMP and Tf2O reagents followed by
hydrolysis (Entries 8-9, Table 1)20. All synthons 9-14 were
purified with flash chromatography to separate α-isomer
and β-isomer. The intermediate 9 was reduced to amine
via Staudinger reaction22 followed by coupling of stearic
acid in EDC, HOBt reagents to provide the expected
compound 15 in 39% yield. Then, it underwent deben-
zylation by Pd/C, H2 and removal of acetonide group by
1N HCl to get β-glucosyl ceramide 2a in 30% yield
(Scheme 1).
90%, 1/5.0
4
5
6
7
8
9
6a
6b
6b
7a
TBAI,
DIPEA,
Cu
TBAI,
DIPEA,
Cu
TBAI,
DIPEA,
Cu
BF3‧
OEt2
Toluene
Toluene
Toluene
CH2Cl2
CH2Cl2
CH2Cl2
65 oC
65 oC
80 oC
rt
12
45%, 2.0/1
9
12
10
11
12
13
14
15
16
17
18
19
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21
22
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68%, 1.8/1
12
85%, 7.5/1
13
53%, α
only
8 b
BSP,
DTBMP,
Tf2O
BSP,
DTBMP,
Tf2O
-60 to
rt
14
65%, 1/1.0
8 c
-60 to
rt
14
62%, 1/1.5
Synthesis of intermediates 17-21 is shown in Table 2 by
following our previously developed procedures.19b,23 In
brief, glycosylation of acceptor 10 with sialyl donor 16 in
CH2Cl2:CH3CN (1/2) at -30oC in the presence of NIS,
TfOH, 3Å MS afforded the disaccharide 17 in 65% yield
with α/β ratio = 3.6/1 (Entry 1, Table 2). The intermediate
compounds 11, 12, 13, and 14 were reacted in a similar
fashion with 16 to afford 18 (56% yield, α/β = 1.6/1), 19
(62% yield, α/β = 3/1), 20 (79% yield, α/β = 2.6/1), and 21
(85% yield, α/β = 2.9/1) respectively. The mixtures of α-
and β-configuration of compounds 15-18 were separated
by column chromatography and only α isomers were tak-
en for the next step (Entries 2-5, Table 2).
a acceptor/donor = 1 equiv./1.5 equiv.
b acceptor/donor = 1 equiv./2.0 equiv.
c acceptor/donor = 3.0 equiv./1 equiv.
Azides were reduced by Staudinger reaction to produce
free amines and solvent was distilled off under reduced
pressure to obtain crude product, and then subsequently
dried under high vacuum to remove water. No purifica-
tion was carried out at this stage and the crudes directly
taken for acid-amine coupling with commercially availa-
ble stearic acid by using EDC, HOBt in anhydrous CH2Cl2.
Scheme 1. Preparation of ganglioside Hp-s1 analogues 2a
C13H27
OH
O
Table 1. First glycosylation reactions between acceptor 3
and donors 4-7.
1. PPh3, THF, H2O, 50 o
C
N3
BnO
BnO
O
O
2. EDC, HOBt, C17H35COOH,
DCM, 39% in 2 steps
BnO
O
9
O
C17H35
C13H27
1. H2 (balloon), Pd/C,
MeOH/CH2Cl2
OH
O
NH
BnO
BnO
2a
O
2. 1 N HCl, MeOH/DCM
30% in 2 steps
O
BnO
O
15
Table 2. Glycosylation of acceptors 10-14 with donor 16
.
Entry Donor Promoter
Solvent
T (oC)
Product
yield, α/β
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OAc
acceptor 10-14 ,
AcO
CO2Me
STol
mean of three experiments ± SEM. * P values < 0.05, ** P
value < 0.01 and *** p < 0.001 were considered significant.
NIS, TfOH, MS 3Å
AcO
AcHN
O
product 17-21
1
2
CH2Cl2/CH3CN (1/2),
-30 oC
AcO
16
OAc
3
4
5
6
AcO
CO2Me
O
OAc
AcO
CO2Me
O
AcO
AcHN
O
AcO
AcHN
O
AcO
BnO
BnO
O
AcO
BnO
O
C13H27
C13H27
N3
N3
BnO
O
BnO
O
O
O
BnO
O
O
7
17
18
8
9
OAc
AcO
CO2Me
O
OAc
AcO
AcHN
O
AcO
CO2Me
10
11
12
13
14
15
16
17
18
19
20
21
22
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AcO
AcO
AcHN
O
BnO
BnO
O
OBn
O
O
AcO
BnO
BnO
C13H27
C13H27
N3
N3
BnO
O
O
O
O
O
O
19
20
OAc
AcO
CO2Me
AcO
O
C13H27
O
OBn
O
AcHN
N3
AcO
BnO
BnO
O
O
O
Figure 4. Induction of IL-2 by Hp-s1 analogues in mouse
NK1.2 cells. CD1d-expressing A20 cells were loaded with
0.1 or 1 uM of alpha-galactosylceramide (C1) and Hp-s1
analogues, and then cultured with mouse NK1.2 cells for
three days. After culture, supernatant was collected to
determine the secretion of IL-2 by ELISA. Data were pre-
sented as mean ± SD.
21
Entry Acceptor Product
Yield (α/β)
65% (3.6/1)
56% (1.6/1)
62% (3.0/1)
79% (2.6/1)
85% (2.9/1)
1
2a
3
4
5
10
11
12
13
14
17
18
19
20
21
a
As shown in scheme 2, the azide group of 17-21 were re-
duced to amine, and then resulting amine was coupled
with acid to get amides 22-26 with yields of 57%, 58%,
64%, 66%, and 67% respectively. These compounds 22-26
were treated with 1N NaOH in MeOH for deacetylation
and debenzylation was achieved by using Pd(OH)2,
AcOH, CHCl3, MeOH under 60 psi H2 pressure to furnish
the target molecules 2b, 2c, 2d, 2e, and 2f in 38%, 49%,
63%, 53%, and 54% yields respectively.
α/β ratio was confirmed by NMR.
Scheme 2. Finalizing synthesis of ganglioside Hp-s1 ana-
logues 2b-2f.
OAc
AcO
CO2Me
O
AcO
AcHN
17,
18,
O
22 α
,
-glc, 57%
1. PPh3, THF,
H2O, 50 oC
O
C17H35
C13H27
23, β-gal, 58%
, α-gal, 64%
α
-man, 66%
26, β-man, 67%
AcO
19
NH
24
25
,
O
2. C17H35COOH,
EDC, HOBt,
DCM
Biological activity of Hp-s1 analogues
20,
21
O
,
(BnO)3
O
The effects of Hp-s1 2 and its analogues 2a-2f on neurite
outgrowth of human neuroblastoma cell SH-SY5Y were
determined. SH-SY5Y cells were treated with Hp-s1 and
its analogues for 72 hours. After treatment, the neurite-
bearing cells and branch point were counted. As shown in
figure 3A, the percentage of neurite-bearing cell was sig-
nificantly higher in Hp-s1 2 treated cells (37.1 ± 0.8) than
in untreated (15.6 ± 1.6, p=0.0013) and DMSO-treated cells
(17.1 ± 2.4, p=0.0028). Compound 2b (27 ± 4.9, p=0.3) and
2c (28.2 ± 2.8, p=0.44) showed decreased percentage of
neurite-bearing cells but not reached the statistic signifi-
cant when compared with Hp-s1, indicating that the α-
conformation and orientation of C4-OH of glucose might
have minor negative effects on the activity of Hp-s1 to
induce neurite outgrowth. Notably, the percentage of
neurite-bearing cells was significantly lower than the Hp-
s1 if both the C4-OH and β-conformation were modified
as compound 2d (19.6 ± 3.1, p=0.01). Surprisingly, treat-
ment of compound 2e (20.4 ± 1.9, p=0.015) or 2f (22.5 ±
3.81, p=0.042) led to a significant decrease in neurite out-
growth, suggesting that the C2-OH of glucose in Hp-s1
plays an important role in the stimulation of neuronal
cells. Interestingly, percentage of neurite-bearing cell only
O
2b, 38%
1. 1N NaOH, MeOH
2c
, 49%
2d
, 63%
2e, 53%
2f
2. Pd(OH)2, 60 psi H2, AcOH,
CHCl3, MeOH, 5 h
,
54%
Figure 3. Effect of Hp-s1 analogues on neurite outgrowth
of SH-SY5Y cells. Cells were incubated with indicated Hp-
s1 analogues (10 ꢀM) for 72 hours, and then percentage of
neurite bearing cells (A) and percentage of branch point
count (B) were determined. Three independent experi-
ments were conducted and the results were presented as
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were measured (40 cells/field). One-way analysis of vari-
slightly decreased in 2a-treated cells (28.4 ± 2, p=0.48)
1
2
3
4
5
6
7
8
compared to Hp-s1-treated cells, implying that the sialic
acid group might not be a pivotal functional group in Hp-
s1 to provoke neurite formation. Additionally, compound
2a-2c increased the percentage of neurite-bearing cells
when compared with untreated and DMSO-treated cells,
although they did not reach the statistically significant.
Similar results were obtained for the percentage of branch
point count (Figure 3B).
ance (ANOVA) and Tukey’s multiple comparison post hoc
test was used for statistical analysis.
Mouse CD1d-overexpressing A20 cells were loaded with
the indicated Hp-s1 analogues (0.1 and 1 μM) and then
cultured with mNK1.2 cells. Three days later, supernatants
were harvested and the level of IL-2 was determined by
ELISA.
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On the other hand, the structure of compound 2b and 2d
is similar to α-galactosyl ceramide (α-GalCer) (Figure 1),
which was originally extracted from marine sponge. α-
GalCer is a well-known glycolipid, which could stimulate
invariant natural kill T cells (NKT) to secrete potent cyto-
kines to regulate immune response. To examine whether
Hp-s1 analogues 2a-2f could stimulate mouse NK1.2 cells,
CD1d-expressing A20 cells were loaded with Hp-s1 2a-2f
analogues (0.1 uM and 1 uM) and then incubated with
mouse NK1.2 cells for three days. Supernatant was collect-
ed from the cultured cells and production of interleukin 2
(IL-2) was determined by ELISA. As shown in figure 4,
only compound 2d activated the mouse NK1.2 cells to
produce IL-2. Surprisingly, the mouse NK1.2 cells were not
activated by compound 2b, which is only difference in the
orientation of C4 hydroxyl group of galactosyl moiety
when compared with compound 2d. None of β-
conformation compounds (2a, 2c, and 2f) displayed the
ability to stimulate the mouse NK1.2 cells, especially the
2c which was changed 2d from α-conformation to β-
conformation, indicating that the α-conformation is im-
portant for compound 2d to activate the NKT cells.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
*E-mail: syluo@dragon.nchu.edu.tw. Phone: 886-4-22840411.
Fax: 886-4-22862547.
Funding
This work has been supported by the Ministry of Sci-
ence and Technology (MOST) in Taiwan (104-2113-M-
005-001) and the Chang Gung Memorial Hospital at
Linkou (CMRPG3D1492).
ACKNOWLEDGMENT
S. Y. Luo and A. L. Yu designed the research. J. T. Hung ana-
lyzed the data. C. H. Yeh, S. A. Yang and H. J. Tai prepared
the compounds 2a-2f. J. T. Hung wrote the manuscript with
the help of G. B. Shelke and D. M. Reddy.
Conclusion
We have achieved the total synthesis of various Hp-s1
analogues 2a-2f with replacing the glucosyl moiety of Hp-
s1 with α-glucose, α- and β-galactose, α- and β-mannose.
The effects of orientation of hydroxyl group at C2 and C4
position of glucosyl moiety of Hp-s1 on neurite outgrowth
of SH-SY5Y cells and activation of NKT cells were investi-
gated. We found that C2 hydroxyl group of glucosyl moie-
ty in Hp-s1 plays an important role in the stimulation of
neurite outgrowth of SH-SY5Y cells. In addition, the com-
pound 2d showed the ability to activate NKT cells, alt-
hough it did not have any effect on neurite outgrowth of
SH-SY5Y cells.
Abbreviations
Abbreviations are used as usual meanings.
REFERENCES
(1) a. Hakomori, S., and Igarashi, Y. (1995) Functional Role of Glyco-
sphingolipids in Cell Recognition and Signaling. J. Biochem. 118, 1091–
103. b. Morita, M., Motoki, K., Akimoto, K., Natori, T., Sakai, T.,
Sawa, E., Yamaji, K., Koezuka, Y., Kobayashi, E., and Fukushima, H.
(1995) Structure-activity relationship of alpha-galactosylceramides
against B16-bearing mice. J. Med. Chem. 38, 2176–2187.
(2) Jennifer, M. R., Gregory E. R., Murray D. M. (2013) The role of
gangliosides in brain development and the potential benefits of peri-
natal supplementation. Nutrition Res. 33, 877-887.
(3) Kolter, Thomas. (2012) Ganglioside biochemistry. ISRN Bio-
chem. Article ID 506160.
(4) Nobile, O. E., Carpo, M., and Scarlato, G. (1994) Gangliosides
their role and clinical neurology. Drugs. 47, 576–585.
(5) Tsuji, S., Yamashita, T., and Nagai, Y. (1988) A novel, carbohy-
drate signal-mediated cell surface protein phosphorylation: gangli-
oside GQ1b stimulates ecto-protein kinase activity on the cell surface
of a human neuroblastoma cell line, GOTO. J. Biochem. (Japan). 104,
498–503.
(6) Tsuji, S., Arita, M., and Nagai, Y. (1983) GQ1b, a bioactive gangli-
oside that exhibits novel nerve growth factor (NGF)-like activities in
the two neuroblastoma cell lines. J. Biochem. 94, 303–306.
(7) Prinetti, A., Iwabuchi, K., and Hakomori, S. (1999) Glycosphinꢀ
golipidꢀenriched signaling domain in mouse neuroblastoma neuro2a cells.
J. Biol. Chem. 274, 20916–20924.
Materials and Methods
Neurite outgrowth and IL-2 production
Human neuroblastoma SH-SY5Y cells (ATCC CRL-2266)
were obtained from American type cell culture (ATCC,
Manassas, VA) and maintained in DMEM:Ham's F12 (1:1)
medium supplemented with 10% fetal bovine serum at
37°C in 5% CO2. For analysis of neurite outgrowth, cells
(1×104) were seeded into each well of six-well plate and
then incubated with indicated compounds (10 ꢀM) for 72
hours. Neurite-bearing cells and neurite branch point
were counted as described [17]. Triplicate wells for each
tested compound and three random fields in each well
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(8) Agnati, L. F., Fuxe, K., Calza, L., Benfenati, F., Cavicchioli,
(18) Tsai, Y. F., Shih, C. H., Su, Y. T., Yao, C. H., Lian, J. F., Liao, C. C.,
L., Toffano, G., and Goldstein, M. (1983) Gangliosides increase the
survival of lesioned nigral dopamine neurons and favour the recov-
ery of dopaminergic synaptic function in striatum of rats by collat-
eral sprouting. Acta. Physiol. Scand. 119, 347–363.
(9) Schneider, J. S., Pope, A., Simpson, K., Taggart, J., Smith, M. G.,
and DiStefano, L. (1992) Recovery from experimental parkinsonism
in primates with GM1 ganglioside treatment. Science 256, 843–846.
(10) Argentino, C., and Sacchetti, M. L. (1989) Ganglioside therapy in
acute ischemic stroke. Stroke 20, 1143–1149.
Hsia, C. W., Shui H. A., and Rani, R. (2012) The total synthesis of
a ganglioside Hp-s1 analogue possessing neuritogenic activity by
chemoselective activation glycosylation. Org. Biomol. Chem. 10, 931–
934.
1
2
3
4
5
6
7
8
(19) a. Yen, Y. F., Kulkarni, S. S., Chang, C. W., and Luo, S. Y. (2013)
Concise synthesis of a-galactosyl ceramide from
D-galactosyl iodide
and -lyxose Carbohydr. Res. 368, 35-39. b. Chen, W. S., Sawant, R.
D
C., Yang, S.A., Liao, Y. J., Liao, J. W., Badsara S. S., and Luo, S. Y.
(2014) Synthesis of ganglioside Hp-s1. RSC Adv. 4, 47752-47761. c. Yu,
R. K., Tsai Y. T., and Arigo, T. (2012) Functional roles of gangliosides
in neurodevelopment-An overview of recent advances. Neurochem.
Res. 37, 1230–1244.
(11) Higuchi, R., Inagaki, M., Yamada, K., and Miyamoto, T. (2007)
Biologically active gangliosides from echinoderms. J. Nat.
Med. 61, 367–370.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(12) Higuchi, R., Inukai, K., Jhou, J. X., Honda, M., Komori, T., Tsuji,
S., and Nagai, Y. (1993) Structure and biological activity of gangli-
oside molecular species. Liebigs Annalen der Chemie. 4, 359–366.
(13) Yamagishi, M., Hosoda, Y. R., Tamai, H., Konishi, M., Imamura,
A., Ishida, H., Yabe, T., Ando, H., and Kiso, M. (2015) Structure‐
activity relationship study of the neuritogenic potential of the glycan
of starfish ganglioside LLG‐3. Mar. Drugs. 13, 7250–7274.
(14) Yamada, K., Tanabe, K., Miyamoto, T., Kusumoto, T., Inagaki,
M., and Higuchi, R. (2008) Isolation and structure of a monomethyl-
ated ganglioside possessing neuritogenic activity from the ovary of
the sea urchin diadema setosum. Chem. Pharm. Bull. 56, 734–737.
(15) Tamai, H., Imamura, A., Ogawa, J., Ando, H., Ishida, H., and Kiso,
M. (2015) First total synthesis of ganglioside GAAꢀ7 from starꢀ
fish asterias amurensis versicolor. Eur. J. Org. Chem. 23, 5199–5211.
(16) Wu, Y. F., Tsai, Y. F., Guo, J. R., Yu, C. P., Yu, H. M., and Liao, C.
C. (2014) First total synthesis of ganglioside DSG-A possessing neuri-
togenic activity. Org. Biomol. Chem. 12, 9345–9349.
(17) a. Yamada, K., Tanabe, K., Miyamoto, T., Kusumoto, T., Inagaki,
M., and Higuchi, R. (2008) Isolation and structure of a monomethyl-
ated ganglioside possessing neuritogenic activity from the ovary of
the sea urchin diadema setosum. Chem. Pharm. Bull. 56, 734–737.
b. Ijuin, T., Kitajima, K., Song, Y., Kitazume, S., Inoue, S., Haslam,
S. M., Morris, H. R., Dell, A., Inoue, Y. (1996) Isolation and identifi-
cation of novel sulfated and nonsulfated oligosialyl glycosphin-
golipids from sea urchin sperm. Glycoconjugate J. 13, 401–413.
(20) David C. and Qingjia Y. (2004) Benzylidene Acetal Fragmentation
Route to 6ꢀDeoxy Sugars: Direct Reductive Cleavage in the Presence of
Ether Protecting Groups, Permitting the Efficient, Highly Stereocontrolled
Synthesis of βꢀDꢀRhamnosides from DꢀMannosyl Glycosyl Donors. Total
Synthesis of αꢀDꢀGalꢀ(1ꢀ>3)ꢀαꢀDꢀRhaꢀ(1ꢀ>3)ꢀ βꢀDꢀRhaꢀ(1ꢀ>4)ꢀβꢀDꢀGluꢀ
OMe, the Repeating Unit of the Antigenic Lipopolysaccharide from Eschꢀ
erichia hermannii ATCC 33650 and 33652. J. AM. CHEM. SOC. 126,
8232ꢀ8236.
(21) a. Mukhopadhyay, B. (2006) Sulfuric acid immobilized on silica:
an efficient promoter for one-pot acetalation–acetylation of sugar
derivativest. Tetrahedron Lett. 47, 4337–4341. b. Xu, P., Chen, X. Z.,
Liu, L., Jin, Z. P.,
and Lei, P.S. (2010) A new series of macrolide derivatives with 400-
O-saccharide substituents. Bioorg. Med. Chem. Lett. 20, 5527–5531. c.
Wang, C. C., Lee, J. C., Luo, S. Y., Kulkarni, S. S., Huang, Y. W., Lee,
C. C., Chang K. L., and Hung, S. C. (2007) Regioselective one-pot
protection of carbohydrates. Nature. 446, 896–899. d. Chi, F. C.,
Kulkarni, S. S., Zulueta M. M. L., and Hung, S. C. (2009) Synthesis of
Alginate Oligosaccharides Containing L-Guluronic Acids. Chem.
Asian J. 3, 386–390.
(22) Liu, S., and Edgar, K. J. (2015) Staudinger Reactions for Selective
Functionalization of Polysaccharides: A Review. Biomacromolecules.
16, 2556−2571.
(23) Hung, J. T., Sawant, R. C., Chen, J. C., Yen, Y. F., Chen, W. S.,
Alice, L. Y., and Luo S. Y. (2014) Design and synthesis of galactose-6-
OH-modified α-galactosyl ceramide analogues with Th2-biased im-
mune responses. RSC Adv. 4, 47341–47356.
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