First total synthesis of ganglioside DSG-A possessing neuritogenic activity

Article abstract of DOI:10.1039/c4ob01882f

The first total synthesis of ganglioside DSG-A (1) is achieved via chemoselective glycosylation and a [1 + 1 + 2] synthetic strategy. We have also developed an efficient method that can be handled on large scale (50 g) for the synthesis of the phytosphingosine. This journal is

Full text of DOI:10.1039/c4ob01882f

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First total synthesis of ganglioside DSG-A
possessing neuritogenic activity†
Cite this: DOI: 10.1039/c4ob01882f
Yu-Fa Wu,a Yow-Fu Tsai,*a Jhe-Ruei Guo,a Cheng-Ping Yu,a Hui-Ming Yu*b and
Received 3rd September 2014,
Accepted 8th October 2014
Chun-Chen Liaoa
DOI: 10.1039/c4ob01882f
www.rsc.org/obc
The first total synthesis of ganglioside DSG-A (1) is achieved via Neu5Acα(2→6)Glcβ(1→1)Cer possessing a 2-hydroxy octadeca-
chemoselective glycosylation and a [1 + 1 + 2] synthetic strategy. noyl moiety), than the other gangliosides. However, to-date,
We have also developed an efficient method that can be handled DSG-A has not yet been synthesized and its mechanism of sti-
on large scale (50 g) for the synthesis of the phytosphingosine.
mulating neurogenesis remains unclear because of the
difficulty in obtaining a sufficient amount of it in pure form
from natural resources. Thus it is important to develop a
powerful synthetic strategy for obtaining sufficient DSG-A, not
only for efficacious validation but also for further structural
modifications to examine the structure–activity relationships
(SAR). Herein, we wish to report a total synthesis of DSG-A with
an efficient methodology for a gram-scale (50 g) synthesis of
the phytosphingosine moiety.
The retrosynthetic analysis of DSG-A (1) is outlined in
Scheme 1. We intend adopting a [1 + 1 + 2] strategy to assem-
ble the four building blocks, including the sialyl donor 4 or 5,
the glucose derivative 6, the succinimidyl acetoxyester 7, and
the phytosphingosine derivative 8. The glucosyl acceptor 6 was
first sialylated with sialyl donor 4 or 5 to give the disaccharide
2α which was then glycosylated with the phytoceramide 3,
readily prepared by the amidization of amine 8 with the
succinimidyl ester 7, to generate the target molecule DSG-A. It
is noteworthy that in order to achieve highly chemoselective
glycosylations, the BoxS and EtS groups were introduced at
the anomeric carbons of 4 (or 5) and 6, respectively. For
the β-stereoselective glycosylation of disaccharide 2α with
Gangliosides, sialic acid-containing glycosphingolipids, are
most abundant in the brain and nervous system where they
comprise up to 6% of the total lipids. However, they are also
ubiquitous in other tissues.1 Many studies have indicated that
gangliosides play a pivotal role not only in cell–cell and
cell–matrix interactions,2 but also in the development and the
functions of the nervous system.3 For example, mice lacking
complex gangliosides, such as GD1a and GT1b, display pro-
gressive symptoms similar to those of axon degeneration and
gross dysmyelination seen in neurodegenerative diseases.4 Fur-
thermore, numerous studies have demonstrated that adminis-
tration of ganglioside GM1 to patients with Parkinson’s
disease or a number of different types of central nervous
system lesions results in significant symptom reduction, as
well as biochemical and behavioral recovery.5 These results
suggest that gangliosides may serve as potential candidates for
treating neurodegenerative diseases.
Many gangliosides extracted from marine invertebrates
show neuritogenic activity against the rat pheochromocytoma
cell line PC-12 in the presence of NGF.6 Among these ganglio-
sides, SJG-2, LLG-3, GAA-7, LLG-5 and DSG-A (1) were found to
be more effective than the mammalian ganglioside GM1,
suggesting that these echinodermatous gangliosides can be
considered as lead compounds for the development of chemo-
therapeutic agents for the treatment of neurodegenerative
diseases. DSG-A, isolated from the ovary of the sea urchin
Diadema setosum,7 has
a
simpler structure, (9-O-Me-
aDepartment of Chemistry, Chung Yuan Christian University, 200 Chung Pei Road,
Chung Li, 32023, Taiwan. E-mail: tsaiyofu@cycu.edu.tw
bGenomics Research Center, Academia Sinica, 128 Academia Road, Section 2,
Nankang, Taipei 11529, Taiwan
†Electronic supplementary information (ESI) available: The detailed experi-
mental procedures for the preparation and characterization of all compounds.
See DOI: 10.1039/c4ob01882f
Scheme 1 Retrosynthesis of the ganglioside DSG-A (1).
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Scheme 4 Synthesis of the optically pure (R)-2-hydroxyoctadecanoic
acid derivative 7.
Scheme 2 Synthesis of the sialyl donors 4 and 5.
glucose 15, which was employed as a starting material, was
transformed into the orthoester 16 by a known method.12 To
simplify the reaction procedures, we developed a one-pot
thioethylation and desilylation of orthoester 16 by treatment
with EtSH in the presence of HgBr2 at 60 °C followed by
addition of H2O at room temperature to observe the thioethyl
glycoside 6 as a single β-stereoisomer with a free C6-OH in
83% yield.13 Alternatively, a stepwise strategy was also used to
prepare 6, wherein 16 was thioethylated with EtSH in the pres-
ence of TMSOTf to generate the ethylthioglycoside 17 in 65%
yield,14 which was then treated with TBAF to generate the desi-
lylated 6 in 89% yield.15 However, the resulting overall yield
(58%) through a stepwise strategy was not better than that
(83%) obtained by one-pot two-step reactions.
phytoceramide 3, a group displaying a neighboring group
effect to control the stereoselectivity was added to the C2-OH
of glucose 6. Moreover, to enhance the reactivity of glycosyla-
tion electron-donating groups were added to the C3- and
C4-OH in 6.
The preparative methods used to obtain 4 and 5 are shown
in Scheme 2. Peracetyl phenylthioglycoside 9 8 was first de-
acetylated with MeONa–MeOH to give tetraol 10, which was used
without further purification for the chemoselective methyl-
ation on C9-OH with Meerwein reagent9 in MeCN in the pres-
ence of DTBMP at −10 °C to generate monomethylated 11 in
82% yield over two steps. To avoid affecting the subsequent
conversion of the PhS group in compound 11 to BoxS, the free
hydroxyl group of triol 11 was protected with Ac2O/pyridine at
room temperature to produce the acetate 12 in 95% yield. The
PhS group was then converted into the BoxS group by chlori-
nation of 12 with iodine chloride, followed by substitution of
the resulting chloride with 2-mercaptobenzoxazole (HSBox) in
the presence of Hunig’s base.10 The desired sialyl donor 4 was
obtained in 70% overall yield in two steps. For the synthesis of
the sialyl donor 5, the oxazolidinone 13 was synthesized using
a documented protocol.11 The subsequent procedure for the
methylation of C9-OH in 13 and conversion of the PhS group
in compound 14 to BoxS was the same as that of the conver-
sion of compound 11 to compound 4.
Optically pure (R)-2-hydroxyoctadecanoic acid derivative 7
was synthesized (Scheme 4) from benzyl (+)-malate 18, pre-
pared by the known method.16 In order to convert the carb-
oxylic group to the aldehyde group via the thioester and to
obtain a high yield of thioesterification, the hydroxyl group of
18 was first converted to 19 with the silyloxyl group by treat-
ment with TBSCl in 74% yield. Thioesterification of 19 with
EtSH in the presence of EDC/DMAP in dry CH2Cl2 generated
the thioester 20 in 93% yield, which was then transformed
into aldehyde 21 with Fukuyama’s method in 91% yield.17 To
establish the long-carbon side chain, Wittig olefination was
carried out using aldehyde 21 as a substrate to generate the
protected hydroxyalkenoate 22 in 82% yield as a single (E)-
stereoisomer, fully characterized by its 1H–1H homodecoupling
1
After preparing the desired sialyl donors 4 and 5, we then
focused on the synthesis of a suitable glucose derivative
such as 6 that could function as both glycosyl acceptor and
donor (Scheme 3). The commercially available pentaacetylated
in the H NMR spectrum (δ(double bond H) 5.47 (d, J = 15.6 Hz)).
Treatment of 22 with H2/10% Pd/C produced hydroxyalkanoic
acid 23 as a white solid in 91% yield ([α]2D5 +16.6 (c 0.10,
MeOH)) by debenzylation, saturation of the double bond and
removal of the TBS group; the desilylation occurred presumably
due to the presence of trace acid and water. The hydroxyl group
in 23 was protected with Ac2O/pyridine to form the acetate fol-
lowed by esterification with N-hydroxysuccinimide to provide the
desired succinimidyl acetoxyester 7 in 86% yield in two steps.
The preparations of the phytosphingosine derivative 8 and
phytoceramide 3 are illustrated in Scheme 5. The primary
hydroxyl group of substrate 2412 was first chemoselectively sily-
lated with TIPSCl, and the resulted silyl ether 25 gave enol 26
by Wittig olefination in 73% overall yield in two steps. Enol 26
Scheme 3 Preparation of the glucose derivative 6.
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Scheme 6 Examination of the glycosylation of disaccharide 2α with
phytoceramide 3.
Before completing the synthesis of ganglioside DSG-A,
glycosylation of the sialyl donors with a glucosyl acceptor
was first evaluated (Table 1). The best result was obtained by
glycosylating donor 5 with acceptor 6, which was carried out in
the presence of activator AgOTf in CH2Cl2 to give disaccharide
32α as a single α-stereoisomer in excellent yield (93%) (entry
8).20 To glycosylate the donor 4 with the acceptor 6, eight sets
of reaction conditions (promoter, solvent, and temperature)
were used to generate glycal 33 and the disaccharide 2 as a
mixture of the α- and β-stereoisomers; among the tested con-
ditions, only that in entry 5 resulted in a good yield and stereo-
selectivity for disaccharide 2. For example, in Et2O and in
CPME (promoter AgOTf), the yields of the disaccharide were
Scheme 5 Construction of the protected phytoceramide 3.
was converted into mesylate 27 (97% yield), which was then 54% and 46%, respectively, and the β-stereoisomer was the
desilylated with TBAF to afford the hydroxymesylate 28 in 96% major product (entries 1 and 3). Although activation by
yield. To introduce the required amino group at C-2 in phyto- Cu(OTf)2 in THF resulted in a greater stereoselectivity (α : β =
sphingosine, tandem addition and intramolecular cyclization 8 : 1), the yield of the desired disaccharide was very poor (entry
of hydroxymesylate 28 with benzylisocyanate in the presence of 7, 33%).
NaH was employed to generate the 2-oxazolidone 29 in 81%
Next, we turned our attention to the coupling of the disac-
yield,18 which was then subjected to a cross-metathesis reac- charide 2α with the protected phytoceramide 3. As shown in
tion with 1-tetradecene in the presence of Grubbs’ catalyst to Scheme 6, an excellent stereoselectivity (β only) and a higher
form phytosphingosine derivative 30 as a ca. 16 : 1 mixture of yield (82%) for the synthesis of the protected DSG-A analogue
E- and Z-stereoisomers in 94% yield.19 Upon exposure to 34 were achieved using NIS/AgOTf in CH2Cl2 at −70 °C. In con-
NaOH in MeOH–H2O, oxazolidone 30 was converted to the trast, activator MeOTf reduced the yield dramatically (55%)
benzylaminoenol 31 in 94% yield. Hydrogenation of 31 fol- though an excellent stereoselectivity (β only) was maintained.
lowed by amidization of the corresponding aminoalcohol 8
Finally, the target molecule DSG-A was accomplished as
(99%) with succinimidyl acetoxyester 7 generated the required shown in Scheme 7. The disaccharide 32α was first converted
phytoceramide 3 in 94% yield.
into 2α in 82% yield by a three-step sequence, involving
Table 1 Glycosylation of sialyl donors 4 and 5 with glucosyl acceptor 6
Temp
(°C)
Time
(h)
Yield of 2 or 32
(α/β ratio)a
Yield
Entry
Donor
Promoter
Solvent
of 33a
1
2
3
4
5
6
7
8
4
4
4
4
4
4
4
5
AgOTf
AgOTf
AgOTf
AgOTf
AgOTf
Bi(OTf)3
Cu(OTf)2
AgOTf
Et2O
−40 to rt
−40 to rt
−40
11
7
7
7
7
7
9
18
54 (1.0 : 1.4)
—
43
—
52
30
30
52
52
0
c
CH3CN
CPME
THF–CH2Cl2
THF
THF
THF
46 (1.0 : 1.5)
60 (1.5 : 1.0)
61 (3.0 : 1.0)
37 (4.0 : 1.0)
33 (8.0 : 1.0)
93 (α only)
b
−40
−40
−40
−40 to rt
−40
CH2Cl2
a Isolated yield. b 3 : 1 (v/v). c No reaction.
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Notes and references
1 (a) S. Sonnino and V. Chigorno, Biochim. Biophys. Acta,
2000, 1469, 63–77; (b) M. Hadjiconstantinou and
N. H. Neff, J. Neurochem., 1998, 70, 1335–1345.
2 T. Angata and A. Varki, Chem. Rev., 2002, 102, 439–
470.
3 (a) H. E. Murrey, C. I. Gama, S. A. Kalovidouris, W. I. Luo,
E. M. Driggers, B. Porton and L. C. Hsieh-Wilson, Proc.
Natl. Acad. Sci. U. S. A., 2006, 103, 21–26; (b) S. A. Kalovidouris,
C. I. Gama, L. W. Lee and L. C. Hsieh-Wilson, J. Am. Chem.
Soc., 2005, 127, 1340–1341; (c) R. Kleene and M. Schachner,
Nat. Rev. Neurosci., 2004, 5, 195–208; (d) J. S. Schneider and
L. DiStefano, Neurology, 1994, 44, 748–750; (e) C. B. Zeller
and R. B. Marchase, Am. J. Physiol., 1992, 262, C1341–
C1355; (f) H. Manev, E. Costa, J. T. Wroblewski and
A. Guidotti, FASEB J., 1990, 4, 2789–2797; (g) C. L. Schengrund,
Brain Res. Bull., 1990, 24, 131–141; (h) A. C. Cuello, Adv.
Pharmacol., 1990, 21, 1–50.
Scheme 7 Completion of the total synthesis of the ganglioside DSG-A (1).
4 (a) S. Chiavegatto, J. Sun, R. J. Nelson and R. L. Schnaar,
Exp. Neurol., 2000, 166, 227–234; (b) K. A. Sheikh, J. Sun,
Y. J. Liu, H. Kawai, T. O. Crawford, R. L. Proia, J. W. Griffin
and R. L. Schnaar, Proc. Natl. Acad. Sci. U. S. A., 1999, 96,
7532–7537.
5 (a) G. Wu, Z.-H. Lu, Z. Kulkarni and R. W. Ledeen, J. Neuro-
sci. Res., 2012, 90, 1997–2008; (b) J. S. Schneider, S. Sendek,
C. Daskalakis and F. Cambi, J. Neurol. Sci., 2010, 292,
45–51.
amidization, deacetylation, decarbonylation and acetylation.
The protected phytoceramide 3 was glycosylated with 2α in the
presence of the promoter NIS/AgOTf in anhydrous CH2Cl2 at
−70 °C to generate the protected DSG-A analogue 34 in 82%
yield with the glucose moiety in β-configuration, as demon-
1
strated with its H NMR spectrum (δ(anomeric H) 4.31 (d, J = 8.0
Hz)). Deisopropylidenation was then performed by treatment
of 34 with 80% AcOH at 85 °C to give the diol 35 in 90% yield.
Debenzylation of 35 using H2/20% Pd(OH)2 followed by de-
acetylation generated DSG-A (1) as a white solid compound in
96% yield in two steps.
6 M. Kaneko, K. Yamada, T. Miyamoto, M. Inagaki and
R. Higuchi, Chem. Pharm. Bull., 2007, 55, 1051–1052.
7 K. Yamada, K. Tanabe, T. Miyamoto, T. Kusumoto,
M. Inagaki and R. Higuchi, Chem. Pharm. Bull., 2008, 56,
734–737.
8 A. Marra and P. Sinay, Carbohydr. Res., 1989, 187, 35–42.
9 M. J. Diem, D. F. Burow and J. L. Fry, J. Org. Chem., 1977,
42, 1801–1802.
10 B. N. Harris, P. P. Patel, C. P. Gobble, M. J. Stark and C. De
Meo, Eur. J. Org. Chem., 2011, 4023–4027.
11 D. Crich and W. Li, J. Org. Chem., 2007, 72, 2387–
2391.
12 Y.-F. Tsai, C.-H. Shih, Y.-T. Su, C.-H. Yao, J.-F. Lian,
C.-C. Liao, C.-W. Hsia, H.-A. Shui and R. Rani, Org. Biomol.
Chem., 2012, 10, 931–934.
13 Z.-G. Wang, J. D. Warren, V. Y. Dudkin, X. Zhang,
U. Iserloh, M. Visser, M. Eckhardt, P. H. Seeberger and
S. J. Danishefsky, Tetrahedron, 2006, 62, 4954–4978.
14 (a) P. Pornsuriyasak and A. V. Demchenko, Chem. – Eur. J.,
2006, 12, 6630–6646; (b) P. H. Seeberger, M. Eckhardt,
C. E. Gutteridge and S. J. Danishefsky, J. Am. Chem. Soc.,
1997, 119, 10064–10072; (c) M. L. Sznaidman, S. C. Johnson,
C. Crasto and S. M. Hecht, J. Org. Chem., 1995, 60, 3942–
3943.
Conclusions
In summary, we have achieved the first total synthesis of
ganglioside DSG-A by employing a [1 + 1 + 2] synthetic strategy
and chemoselective glycosylation. The glycosylation of sialyl
donor 5 with glucosyl acceptor 6 and the conjugation of the
synthesized disaccharide 2α with the protected phytoceramide
3 resulted in both excellent yield and stereoselectivity. In
addition, we have also developed an efficient method that can
be applied to a large scale synthesis of phytosphingosine
(50 g) in an efficient manner. Currently, the application of
one-pot two-step glycosylations to prepare a variety of DSG-A
analogues for various biological tests, including neuritogenic
activity, is under active investigation.
Acknowledgements
15 P. J. Garegg, L. Olsson and S. Oscarson, J. Org. Chem., 1995,
60, 2200–2204.
We gratefully acknowledge financial support from the Ministry
of Science and Technology, Taiwan (NSC 102-2113-M-033-003, 16 (a) K. Danielmeier and E. Steckhan, Tetrahedron: Asym-
NSC 102-2113-M-033-007, and NSC 101-2120-M-033-001).
metry, 1995, 6, 1181–1190; (b) S. Cammas, I. Renard,
Org. Biomol. Chem.
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K. Boutault and P. Guérin, Tetrahedron: Asymmetry, 1993, 4, 19 G. C. Vougioukalakis and R. H. Grubbs, Chem. Rev., 2010,
1925–1930. 110, 1746–1787.
17 T. Fukuyama, S.-C. Lin and L. Li, J. Am. Chem. Soc., 1990, 20 (a) H. Tanaka, Y. Tateno, Y. Nishiura and T. Takahashi,
112, 7050–7051.
Org. Lett., 2008, 10, 5597–5600; (b) M. N. Kamat, N. P. Rath
and A. V. Demchenko, J. Org. Chem., 2007, 72, 6938–6946;
(c) A. V. Demchenko, M. N. Kamat and C. De Meo, Synlett,
2003, 1287–1290.
18 (a) S.-g. Lee, C. W. Lim, D. C. Kim and J. K. Lee, Tetrahe-
dron: Asymmetry, 2000, 11, 1455–1458; (b) W. R. Roush and
M. A. Adam, J. Org. Chem., 1985, 50, 3752–3757.
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