A first total synthesis of a hybrid-type ganglioside associated with amyotrophic lateral sclerosis-like disorder

Article abstract of DOI:10.1002/chem.201002184

The hybrid ganglioside X1, which was identified in the bovine brain, was synthesized for the first time. Ganglioside X1 is believed to be involved in the development of amyotrophic lateral sclerosis-like disorders in patients with neurological disorders a

Full text of DOI:10.1002/chem.201002184

DOI: 10.1002/chem.201002184
A First Total Synthesis of a Hybrid-Type Ganglioside Associated with
Amyotrophic Lateral Sclerosis-Like Disorder**
Shinya Nakashima,[a, b] Hiromune Ando,*[a, b] Akihiro Imamura,[a, b] Nobuhiro Yuki,[c]
Hideharu Ishida,[a] and Makoto Kiso*[a, b]
Abstract: The hybrid ganglioside X1,
which was identified in the bovine
brain, was synthesized for the first
time. Ganglioside X1 is believed to be
involved in the development of amyo-
trophic lateral sclerosis-like disorders
in patients with neurological disorders
after treatment with bovine brain gan-
gliosides. A convergent approach using
two branched glycan units, the GM2-
core trisaccharide and the lacto-ganglio
tetrasaccharide, efficiently provided the
highly branched heptasaccharide part
of ganglioside X1, which was conjugat-
ed with the ceramide part to produce
the protected ganglioside X1. Global
deprotection delivered homogenous
ganglioside X1, with which serum from
the patient was reacted.
Keywords: gangliosides
sides glycosylation
·
glyco-
·
· immuno-
assays · natural products · total
synthesis
Introduction
riched in nervous tissues. The patients improved after plas-
mapheresis and immunosuppressant treatment, suggesting
that the condition is autoimmune mediated.[1] In contrast,
gangliosides extracted from bovine brain, which have been
used as a therapeutic agent for many neurological disorders,
might have caused the patient to display ALS-like disorder[2]
The patientꢀs IgM reacted strongly with GM2 and GalNAc-
GD1a, which are minor gangliosides in bovine brain, but
not with asialo-GM2, GM1, or GD1a, indicating that their
Amyotrophic lateral sclerosis (ALS) is characterized by a
progressive and selective loss of motor neurons in the brain
and spinal cord, and is a devastating disorder of still un-
known etiology and pathogenesis. Some patients who had
been misdiagnosed as having ALS carried IgM autoantibod-
ies against the GM1 ganglioside, which is a member of a
group of sialic acid containing glycosphingolipids that is en-
terminal trisaccharides [GalNAcbACHTGNUTREN(NNUG 1,4)ACHTUNRTGENGN(UN NeuAcaCAHTNUGTREN(GUNN 2,3)Gal)]
(GM2-core) are the epitope.[2,3] The patient who underwent
plasmapheresis improved quickly, and the serum possessed
killing activity to GM2-containing cells, suggesting that IgM
antibodies with anti-GM2 reactivity function in the develop-
ment of the ALS-like disorder.[2,4] By using the patientꢀs
IgM antibodies, further, novel GM2-core-containing ganglio-
sides, X1 and X2 were identified in bovine brain. They were
characterized as hybrid-type gangliosides, lacto-ganglio-
series gangliosides, in which the core sequence of lacto-
[a] S. Nakashima, Dr. H. Ando, Dr. A. Imamura, Dr. H. Ishida,
Dr. M. Kiso
Department of Applied Bioorganic Chemistry
Gifu University, 1-1 Yanagido
Gifu-shi, Gifu 501-1193 (Japan)
Fax : (+81)58-293-3452
hando@ gifu-u.ac.jp
[b] S. Nakashima, Dr. H. Ando, Dr. A. Imamura, Dr. M. Kiso
Department of Applied Bioorganic Chemistry
Institute for Integrated Cell-Material Sciences (WPI program)
Kyoto University, Yoshida Ushinomiya-cho
series (GalbACHUTGTNERNNUG(1,3)GlcNAcbHCATUNGTRENN(UGN 1,3)Gal) and ganglio-series
Sakyo-ku, Kyoto 606-8501 (Japan)
(GalNAcb
AHCTUNGTRENNUNG
[c] Dr. N. Yuki
Their unusual structures may be immunogenic in humans
to induce the pathogenic antibodies with anti-GM2 reactivi-
ty. Otherwise, X1 (1) and X2 (2) may be present in humans
and may be target molecules for autoantibodies in some pa-
tients who are misjudged to have ALS. The novel GM2-
core-containing gangliosides are required to identify such
treatable patients with immunotherapy. Herein, we report a
first total synthesis of 1 in detail.
Departments of Microbiology & Medicine
National University of Singapore
5 Science Drive 2, Blk MD4A, Level 5
Singapore 117597
[**] Synthetic studies on sialoglycoconjugates, Part 151; for Part 150, see:
T. Komori, A. Imamura, H. Ando, H. Ishida, M. Kiso, Carbohydr.
Res. 2009, 344, 1453–1463.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002184.
588
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Chem. Eur. J. 2011, 17, 588 – 597

FULL PAPER
As shown in Scheme 2, the
terminal GM2-core unit 5 could
be readily synthesized accord-
ing to a previously reported
method from N-Troc galactosa-
minyl (GalN) donor 8 and N-
Troc sialyl galactose 10,[6,9]
which can be rapidly produced
on a large scale because of its
high tendency to crystallize.
The synthesis of the tetrasac-
charide
counterpart
com-
menced with the preparation of
lactose acceptor 9. The 2-O-piv-
To build the entire ganglio-
side X1 structure, the glycan
unit needed to be accessed
through a highly efficient syn-
thetic route and be designed to
have
a
form suitable for
straightforward introduction of
the ceramide unit. The glycan
structure of 1 has two galactos-
amine residues at the C4 hy-
droxyl groups of the external
and internal galactose residues
of sialyl lactotetraose. At first
glance, direct and double galac-
tosaminylation of the sialyl lac-
totetraose appeared to be a fea-
sible approach to the assembly
of the glycan part of target 1.
However, we also presumed
that the glycan structure would
defy this approach because the
reactivity of the C4 hydroxyl
group of galactose, flanked by
the sialic acid residue at C-3, is
markedly low. Similarly, this
Scheme 1. Retrosynthetic analysis of target compound 1. P=protecting group, LG=leaving group, Bz=benzo-
yl, Piv=pivaloyl, Troc=2,2,2-trichloroethoxycarbonyl, Bn=benzyl.
low reactivity could impede the selective protection–depro-
tection of the C4 hydroxyl group. Based on these conjec-
tures, we devised a convergent approach to synthesize the
target molecule (Scheme 1). First, the target (1) was divided
into the glycan unit 3 and ceramide unit 4. Then, the glycan
unit was disassembled at the bACTHNUGRTNEUNG(1,3)-linkage between Gal
and GlcNAc, following a previous report on sialyl Lewis
A,[5] thus providing two branched fragments, 5 and 6. The
left fragment, containing the GM2-core sequence, was de-
signed as a trichloroacetimidate donor, which was developed
by our research group.[6] The right fragment, containing the
lacto- and ganglio-series glycan sequence, was further disas-
sembled into glucosaminyl donor 7, galactosaminyl donor
8,[7] and lactose acceptor 9.[8] For the final connection of the
full-length glycan unit and ceramide, the lactose unit was de-
signed with pivaloate at C2 to prevent orthoester formation.
Scheme 2. Synthesis of terminal trisaccharide unit 5. MP=p-methoxy-
phenyl.
aloyl derivative of lactoside was successfully prepared from
1-O-silylated lactose 11 through 2-O-silylated derivative 12
according to the procedure reported by Schmidt and co-
workers (Scheme 3).[8b]
The glucosaminyl (GlcN) donor was designed in a 4,6-
benzylidenated form to further incorporate the GM2-core
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589

H. Ando, M. Kiso et al.
toside 18 (21%). The position of GlcN within 16 was con-
firmed by comparison of the chemical shifts in 1H NMR
spectra of 16 and its acetylated derivative. Upon acetylation
(Ac2O, Pyr), the H-3 of Gal shifted downfield from d=3.8
to 4.9 ppm, indicating that the C3 hydroxyl group was free
in compound 16. The structures of compounds 17 and 18
1
were confirmed by H NMR spectroscopic analysis and by
mass spectrometry. In contrast, the desired product 15 was
not obtained from this reaction. Disappointingly, replacing
GlcN 7 with GalN donor 8 in this reaction resulted in un-
regioselective glycosylation, generating
bACHTNGUTERN(UNG 1,4’)-linked
Scheme 3. Synthesis of lactosyl acceptor 9 bearing a pivaloate at the C2
position. TDS=thexyldimethylsilyl.
(35%), (1,3’)-linked (42%), and double glycosylated
bACHTUNGTRENNUNG
(21%) products (Table 1).
unit at the C3 hydroxyl group after assembly of the tetrasac-
charide. The Troc group was chosen as the directing func-
tionality for stereoselective b-glucosaminylation and also as
a temporary protecting group for the C3 hydroxyl group,
thus leading to structure 7, which was synthesized from the
reported compound 13[10] through benzylidanation and intro-
duction of the Troc group at the C3 hydroxyl group of 14
(Scheme 4).[11]
Table 1. Glycosylation of 9 with glycosaminyl donors.
Entry
Donor
Yield of products [%]
(1,3’)-linked (1,4’)-linked
G
U
bis-linked
21
1
2
42
35
35
22
39
20
Scheme 4. Synthesis of glucosaminyl donor 7. a) BDA, CSA/CH3CN-
THF, 61% (crystalline); b) TrocCl/Pyr, 94%. BDA=benzaldehyde di-
methyl acetal, CSA=(ꢁ)-camphor-10-sulfonic acid, Pyr=pyridine.
3
63
15
To establish the hybrid branches stemming from the gal-
actose residue, the GlcN donor 7 was first reacted with the
3’,4’-diol lactose acceptor 9, aiming for regioselective glyco-
sylation at the more reactive C3 hydroxyl group rather than
at the C4 hydroxyl group.
Although there are many examples of specific glycosyla-
tion of the C3 hydroxyl group of the 3,4-diol of galactose
moiety by using glycosyl donors, especially sialic acid
donors,[6,8,9,12] the GlcN donor 7 preferentially provided the
Furthermore, triacetyl GlcN donor 19 and benzylidenated
GalN donor 20[14] were investigated. It was likely that the
triacetyl GalN donor 8 and the triacetyl GlcN donor 19
were unselective (Table 1, entry 2). However, in the case of
the benzylidenated GalN donor 20, the C3 hydroxyl group
was preferentially glycosylated to afford the (1,3’)-linked
(63%), (1,4’)-linked (15%), and double glycosylated (20%)
products as anomeric mixtures, showing a regioselectivity
opposite to that observed with benzylidenated GlcN donor
7 (Table 1, entry 3). This unexpected regioselectivity also
contrasts with the reaction of N-phthaloyl GlcN donor 21
and lactose acceptor 22 reported by Ogawa and co-work-
bACHTUNGTRENNUNG(1,4’)-linked product (Scheme 5). Thus, the reaction of 7
(1.2 equiv) and 9 (1.0 equiv), promoted by NIS-TfOH[13] in
CH2Cl2 at ꢀ408C, yielded trisaccharide 16 (58%) with
double glycosylated 17 (19%) and 3’,4’-benzylidenated lac-
ers,[15] in which the corresponding b
ACTHNUTRGNE(UNG 1,3’)glucosaminyl prod-
uct 23 was obtained as the predominant product
(Scheme 6).
Although the unexpected results are interesting, the prin-
ciple underlying these phenomena is still unclear. Because
the regioselectivity is incongruous with the targeted struc-
ture, we redesigned the lactosyl acceptor as a monoalcohol
derivative.
The C3 hydroxyl group of 9 was capped with the p-me-
thoxybenzyl (MBn) group by stannylation and etherification
(dibutyltin(IV) oxide (DBTO), MBnCl, and TBAB)[16] to
Scheme 5. Unexpected regioselectivity in the glycosylation of 9 with 7.
NIS=N-iodosuccinimide, TfOH=trifluoromethanesulfonic acid, MS=
molecular sieves, Bzdn=benzylidene.
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Chem. Eur. J. 2011, 17, 588 – 597

Synthesis of a Hybrid Ganglioside X1
FULL PAPER
C135H160N4O52 [1/2M+Na]+ 1357.4892; found 1357.4893 [1/2
M+Na]+). However, the stereochemistry of the new glycosi-
dic bond between Gal and GlcN could not be defined as the
b form because the signals from the H-1 and H-2 protons of
1
Gal observed by H NMR spectroscopic analysis overlapped
with other signals such as the H-1 proton from the inner
GalN, H-3 from the terminal GalN, H-4 from the terminal
Neu5Ac, and the CH2 protons from the benzyl group. The
protecting groups of heptasaccharide 29 were then manipu-
lated to generate the corresponding imidate donor. This pro-
cess began with sequential hydrogenolysis of the benzyl
groups catalyzed by Pd(OH)2/C and acetylation to provide
1-O-acetyl intermediate 30, which was then advanced by hy-
drazinolysis of the anomer acetate and trichloroacetimidate
formation by treatment with trichloroacetonitrile (CCl3CN)
Scheme 6. The C3-selective glycosylation of 3’,4’-diol lactosyl acceptor 22
with glucosaminyl donor 21 developed by Ogawa et al.[15] Phth=phthalo-
yl.
provide 24 in 93% yield (Scheme 7). As expected, glycosyla-
tion of the lactose acceptor 24 (1.0 equiv) and GalN donor 8
(1.5 equiv) proceeded smoothly at ꢀ208C to yield the gan-
gliotriose (Gg3) sequence 25 in 87% yield as a single
and 1,8-diazabicycloACTHNUTRGNEUNG
[5.4.0]undec-7-ene (DBU),[18] to gener-
ate the imidate donor 31 in 76% over four steps. At this
stage, the anomeric configuration of the terminal Gal was
1
assigned as the b form from H NMR spectroscopic analysis,
whereby the H-1 proton was observed at d=4.88 ppm as a
doublet with a coupling constant of 7.5 Hz, and the H-2
proton was observed at d=5.29 ppm as a double doublet
(J2,3 =9.6 Hz).
Finally, the obtained full-length glycan donor 31 was gly-
cosidated with the known ceramide acceptor 4.[19] The best
result was obtained when the reaction was conducted in
CHCl3 at room temperature upon activation by BF3·OEt2
(1.2 equiv for the donor), producing the protected ganglio-
side X1 (32) in 26% yield.[8a,20] In other approaches, use of
trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a cat-
alyst diminished the coupling yield to around 5 to 10%.
Under the optimized reaction conditions, 61% of the glyco-
syl donor was recovered as the hemiacetal, which could
again be converted into the donor 31 to be used for the con-
jugation of ceramide. Formation of the orthoester was not
observed. To approach ganglioside X1, the acyl protecting
groups were removed by applying Zemplꢂnꢀs method, fol-
lowed by saponification of the methyl ester on the sialic
acid residue, to successfully yield 13.5 mg of target com-
pound 1 in pure form.
Scheme 7. Synthesis of unit 28 of the glycan part of the target compound.
a) TFA/CH2Cl2, 98%; b) i) Zn, AcOH/MeOH; ii) Ac2O/CH2Cl2-MeOH,
75%. TBAB=n-tetrabutylammonium bromide, MBn=p-methoxybenzyl,
TFA=trifluoroacetic acid.
The synthetic compound 1 was immunostained with IgM
antibodies and compared to natural X1 on a thin-layer chro-
matographic plate. Figure 1 shows that synthetic 1 had simi-
lar mobility to natural X1 from bovine brain, and that
serum IgM antibodies from the patient with ALS-like disor-
der[2] reacted with both natural and synthetic X1.
isomer. Upon treatment with trifluoroacetic acid (TFA) in
CH2Cl2, the Gg3 derivative was converted into C3-hydroxy
acceptor 26 (98%). Subsequent glycosylation with GlcN
donor 7, mediated by NIS-TfOH in CH2Cl2, was also suc-
cessful in converting Gg3 into lacto-ganglio-tetraose 27 in
84% yield. Finally, successive unmasking of the three Troc
groups by treatment with zinc in AcOH–MeOH and N-ace-
tylation, delivered fragment 28 in 75% yield over two steps.
Fortunately, the convergent assembly of the heptasacchar-
ide part proceeded in accordance with our initial expecta-
tion (Scheme 8). Thus, the left fragment, GM2-core donor 5,
was glycosidated with the right fragment 28 by using
Schmidtꢀs method[17] to provide the glycan framework of X1
29 in 86% yield. The heptasaccharide structure of 29 was
confirmed by mass spectrometry (ESI-TOF; m/z calcd for
Conclusion
We have succeeded in the total synthesis of the lacto-ganglio
series ganglioside X1. The convergent approach employing
the GM2-core unit and the lacto-ganglio tetraosyl unit al-
lowed access to the target structure with high efficiency, pro-
ducing homogenous X1 in sufficient quantity for biological
study. Furthermore, we confirmed that the patientꢀs serum
IgM bound to the synthesized 1 on a 96-well microtiter
Chem. Eur. J. 2011, 17, 588 – 597
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591

H. Ando, M. Kiso et al.
Experimental Section
General procedures: 1H and 13C NMR
spectra were recorded with JEOL
JNM-ECA600
spectrometers.
1H NMR chemical shifts (d) are ex-
pressed in ppm relative to the signal
of Me4Si as an internal standard,
except when the samples were mea-
sured in [D6]acetone, in which case the
shift was referenced against the signal
of acetone (d=2.09 ppm). 13C NMR
chemical shifts (d) are expressed in
ppm relative to the signal of the sol-
vent. High-resolution mass spectrome-
try (HRMS) was performed with
a
Bruker Daltonics micrOTOF (ESI-
TOF) mass spectrometer. Specific ro-
tations were measured with a Horiba
SEPA-300 high-sensitivity polarimeter.
Melting points were determined with
an AS ONE ATM-01 apparatus. Mo-
lecular sieves were purchased from
Wako Chemicals and dried at 3008C
for 2 h in a muffle furnace prior to
use. Reactions were carried out under
an atmosphere of argon unless other-
wise specified. Solvents as reaction
media were dried over molecular
sieves and used without further purifi-
cation. TLC analyses were performed
on Merck TLC plates (silica gel 60F254
on glass), and compounds were visual-
ized either by exposure to UV light
(254 nm), by spraying with 10%
H2SO4 solution in EtOH, or by treat-
ment with ninhydrin reagent, followed
by heating. Flash column chromatog-
Scheme 8. Assembly of glycan part 29 and final conjugation with ceramide to deliver target structure 1.
TMSOTf=trimethylsilyl trifluoromethanesulfonate, DMAP=4-dimethylaminopyridine, DMF=N,N-dimethyl-
formamide, DBU=1,8-diazabicycloACTHNUTRGNEUNG[5.4.0]undec-7-ene.
Figure 1. Reactivity of the synthesized X1 with serum that recognizes
GM2 epitope [GalNAc b1-4 (NeuAc a2-3) Gal b]. A) TLC plate stained
with the orcinol reagent for hexose. B) Immunostained chromatogram
that had been overlaid first with serum from a patient with an ALS-like
disorder[2] then with peroxidase-conjugated anti-human m-chain specific
antibodies. Lane 1: Authentic GM2, GM1, GD1a, GD1b, and GT1b.
Lane 2: GM2-epitope containing gangliosides, X1 and X2, from bovine
brain.[3] Lane 3: The synthesized X1 in the present study. Orcinol reagent
stains GM2, GM1, GD1a, GD1b, GT1b, X1, and X2 from bovine brain
and the synthesized X1. Serum IgM antibodies from the patient strongly
bind to GM2, X1, and X2 from bovine brain, and the synthesized X1.
Figure 2. Reactivity of the synthesized X1 with serum that recognizes the
GM2 epitope, developed on a microtiter plate. The patientꢀs serum IgM
antibodies react with X1 and GM2, but with neither GM1 nor GD1a.
IgM antibodies against the X1, GM2, GM1, and GD1a (5 pmol/well)
were measured in the patientꢀs serum (1:500 dilution) according to re-
ported procedure.[21] For structures of GM2, GM1, and GD1a, see the
Supporting Information.
plate (Figure 2). Using the synthesized 1, we will test serum
samples from a number of patients who were misdiagnosed
with ALS to identify those treatable with immunotherapy.
raphy on silica gel (Fuji Silysia Co., 80 mesh and 300 mesh) or Sephadex
(Pharmacia LH-20) were performed with the solvent systems (v/v) speci-
fied. Evaporation and concentration were conducted in vacuo.
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Synthesis of a Hybrid Ganglioside X1
FULL PAPER
Compound 7: 2,2,2-Trichloroethyl chloroformate (200 mL, 1.45 mmol)
was added to a solution of 14 (513 mg, 0.959 mmol) in pyridine (4.8 mL)
at 08C, and the mixture was stirred for 30 min at RT. Upon completion
of the reaction (confirmed by TLC analysis; EtOAc/hexane, 1:2), the re-
action mixture was quenched by addition of MeOH at 08C and the resid-
ual solvent was removed by coevaporation with toluene. The residue was
dissolved in EtOAc and the solution was washed with 2m aqueous HCl,
saturated aqueous NaHCO3, and brine, dried (Na2SO4), and concentrat-
ed. The residue was purified by column chromatography on silica gel
(EtOAc/hexane, 1:5) to give 7 (641 mg, 94%), which was recrystallized
from EtOAc/hexane. M.p. 158–1608C; [a]D =ꢀ26.0 (c=1.0, CHCl3);
1H NMR (600 MHz, CDCl3): d=7.51–7.33 (m, 10H; 2 Ph), 5.52 (s, 1H;
PhCH), 5.39 (t, J2,3 =J3,4 =9.7 Hz, 1H; H-3), 5.29 (d, J2,NH =8.3 Hz, 1H;
NH), 5.11 (d, J1,2 =10.3 Hz, 1H; H-1), 4.79–4.71 (m, 4H; 2 CH2), 4.41
(dd, J5,6 =5.2 Hz, Jgem =10.7 Hz, 1H; H-6), 3.82 (t, J5,6’ =10.3 Hz, 1H; H-
6’), 3.74 (t, J4,5 =9.7 Hz, 1H; H-4), 3.68–3.60 ppm (m, 2H; H-2, H-5);
13C NMR (150 MHz, CDCl3): d=153.9, 153.8, 136.6, 133.1, 131.5, 129.2,
129.2, 128.6, 128.2, 126.1, 101.4, 95.2, 94.2, 86.8, 78.4, 76.9, 74.6, 70.4, 68.4,
55.5 ppm; HRMS: m/z calcd for C25H23Cl6NO8SNa+: 729.9168 [M+Na]+;
found: 729.9168.
(m, 5H; H-3a, H-5a, H-6’a, H-2b, H-5d), 3.41 (t, J5,6 =J5,6’ =5.5 Hz, 1H;
H-5b), 3.16 (m, 1H; H-6’b), 1.11 ppm (s, 9H; tBu); 13C NMR (150 MHz,
[D6]DMSO): d=176.2, 154.6, 153.6, 153.4, 139.1, 139.1, 139.0, 138.4,
137.6, 137.4, 137.4, 129.1, 128.4, 128.2, 128.2, 128.2, 127.8, 127.8, 127.0,
127.6, 127.6, 127.5, 127.3, 127.3, 127.2, 126.4, 126.3, 102.6, 101.7, 100.5,
100.5, 100.3, 99.6, 96.4, 95.8, 95.0, 95.0, 81.9, 80.5, 78.8, 77.9, 77.0, 76.6,
76.5, 76.2, 76.1, 74.7, 74.5, 74.4, 74.2, 73.6, 73.6, 72.5, 72.4, 72.3, 72.1, 70,2,
68.9, 67.9, 67.5, 65.3, 64.7, 56.6, 55.8, 38.4, 27.0 ppm; HRMS: m/z calcd
for C90H94Cl12N2O28Na+: 2093.2148 [M+Na]+; found: 2093.2139.
Compound 18a: [a]D =ꢀ8.4 (c=1.6, CHCl3); 1H NMR (600 MHz,
CDCl3): d=7.41–7.16 (m, 30H; 6 Ph), 5.95 (s, 1H; PhCH), 5.15 (t, J1,2
=
=
=
J
2,3 =8.6 Hz, 1H; H-2a), 4.92 (d, Jgem =11.0 Hz, 1H; CH2), 4.88 (d, Jgem
12.4 Hz, 1H; CH2), 4.82 (d, Jgem =11.7 Hz, 1H; CH2), 4.74 (d, Jgem
11.0 Hz, 1H; CH2), 4.62–4.59 (m, 3H; 3 CH2), 4.48 (d, J1,2 =8.6 Hz, 1H;
H-1a), 4.48 (d, J1,2 =8.3 Hz, 1H; H-1b), 4.45 (d, Jgem =12.4 Hz, 1H; CH2),
4.39 (d, Jgem =12.4 Hz, 1H; CH2), 4.34 (t, J2,3 =J3,4 =6.2 Hz, 1H; H-3b),
4.23 (d, Jgem =12.4 Hz, 1H; CH2), 4.13 (d, J4,5 =1.4, 9.6 Hz, 1H; H-4b),
4.09 (t, J3,4 =J4,5 =9.3 Hz, 1H; H-4a), 3.90 (dd, J5,6 =4.1 Hz, Jgem =11.0 Hz,
1H; H-6a), 3.77 (dd, J5,6’ =1.4 Hz, Jgem =11.0 Hz, 1H; H-6’a), 3.67 (t,
J
1,2 =J2,3 =8.6 Hz, 1H; H-3a), 3.65–3.61 (m, 2H; H-5b, H-6b), 3.49–3.44
(m, 3H; H-5a, H-2b, H-6’b), 1.13 ppm (s, 9H; tBu); 13C NMR (150 MHz,
CDCl3): d=176.7, 138.7, 138.2, 138.2, 137.2, 129.2, 128.4, 128.4, 128.3,
128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.6, 127.6, 127.5, 127.2,
126.3, 103.3, 102.0, 99.8, 81.1, 80.3, 77.9, 76.5, 75.5, 73.9, 73.7, 73.5, 73.4,
73.3, 72.3, 72.2, 70.1, 69.0, 68.0, 38.7, 27.1 ppm; HRMS: m/z calcd for
C59H64O12Na+: 987.4290 [M+Na]+; found: 987.4290.
Compounds 16, 17, and 18: Molecular sieves (4 ꢃ, 200 mg) were added
to
a solution of compounds 7 (98 mg, 137 mmol) and 9 (101 mg,
115 mmol) in CH2Cl2 (2.50 mL). The suspension was stirred for 30 min at
ꢀ408C, whereupon NIS (51 mg, 227 mmol) and TfOH (2.0 mL, 22.6 mmol)
were added. Stirring was continued for 40 min at ꢀ408C, at which point
completion of the reaction was indicated by TLC (EtOAc/hexane 2:5, de-
veloped twice). The reaction mixture was filtered through Celite and the
removed molecular sieves were washed with CHCl3. The combined fil-
trate and washings were extracted with CHCl3, and organic layer was
washed with saturated aqueous NaHCO3, saturated aqueous Na2S2O3,
and brine, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel (EtOAc/hexane 1:4!1:3!2:5) to
give 16 (97 mg, 58%), 17 (57 mg, 19%), and 18 (30 mg, 21%) as diaste-
reoisomers (a/b=13:17).
Compound 18b: [a]D =ꢀ15.4 (c=1.3, CHCl3); 1H NMR (600 MHz,
CDCl3): d=7.43–7.16 (m, 30H; 6 Ph), 5.91 (s, 1H; PhCH), 5.13 (t, J1,2
=
=
J
2,3 =8.6 Hz, 1H; H-2a), 4.89 (d, Jgem =10.3 Hz, 1H; CH2), 4.87 (d, Jgem
11.0 Hz, 1H; CH2), 4.68 (d, Jgem =11.7 Hz, 1H; CH2), 4.60–4.55 (m, 4H;
4 CH2), 4.46–4.41 (m, 3H; H-1a, H-1b, CH2), 4.39 (d, Jgem =11.7 Hz, 1H;
CH2), 4.25 (d, Jgem =11.0 Hz, 1H; CH2), 4.19–4.16 (m, 2H; H-3b, H-4b),
4.06 (t, J3,4 =J4,5 =9.3 Hz, 1H; H-4a), 3.85 (dd, J5,6 =6.9 Hz, Jgem =9.7 Hz,
1H; H-6a), 3.76–3.72 (m, 2H; H-6’a, H-5b), 3.65 (dd, J5,6 =6.9 Hz, Jgem
=
Compound 16: [a]D =ꢀ45.5 (c=1.0, CHCl3); 1H NMR (600 MHz,
[D6]acetone): d=7.50–7.21 (m, 30H; 6 Ph), 7.18 (d, J2,NH =8.9 Hz, 1H;
NH), 5.72 (s, 1H; PhCH), 5.38 (t, J2,3 =J3,4 =9.7 Hz, 1H; H-3c), 5.36 (d,
9.7 Hz, 1H; H-6b), 3.61 (dd, J2,3 =8.6 Hz, J3,4 =9.3 Hz, 1H; H-3a), 3.45
(dd, J5,6’ =6.2 Hz, Jgem =9.7 Hz, 1H; H-6’b), 3.42 (m, 1H; H-5a), 3.37 (m,
1H; H-2b), 1.13 ppm (s, 9H; tBu); 13C NMR (150 MHz, CDCl3): d=
176.7, 138.7, 138.3, 138.2, 138.2, 137.8, 137.2, 129.2, 128.4, 128.3, 128.3,
128.3, 128.2, 128.0, 127.8, 127.8, 127.8, 127.7, 127.6, 127.5, 127.5, 127.1,
126.6, 104.3, 102.1, 99.8, 81.1, 80.9, 79.0, 76.3, 76.1, 75.4, 73.6, 73.5, 73.3,
73.3, 72.2, 71.8, 70.1, 68.8, 68.0, 38.7, 27.1 ppm; HRMS: m/z calcd for
C59H64O12Na+: 987.4290 [M+Na]+; found: 987.4290.
J
J
1,2 =8.2 Hz, 1H; H-1c), 5.21 (d, Jgem =10.3 Hz, 1H; CH2), 5.01 (t, J1,2
2,3 =8.6 Hz, 1H; H-2a), 4.99 (d, Jgem =11.0 Hz, 1H; CH2), 4.89 (d, Jgem
=
=
12.4 Hz, 1H; CH2), 4.87–4.73 (m, 6H; OH, 4 CH2), 4.66 (d, J1,2 =8.6 Hz,
1H; H-1a), 4.62 (d, Jgem =10.3 Hz, 1H; CH2), 4.57 (d, J1,2 =7.6 Hz, 1H;
H-1b), 4.54 (d, Jgem =11.0 Hz, 1H; CH2), 4.52 (d, Jgem =12.4 Hz, 1H;
CH2), 4.42 (d, Jgem =12.4 Hz, 1H; CH2), 4.39 (d, Jgem =12.3 Hz, 1H; CH2),
4.21–4.18 (m, 2H; H-6c, CH2), 4.13 (d, J3,4 =1.4 Hz, 1H; H-4b), 4.06 (t,
Compound 24: Dibutyltin(IV) oxide (37 mg, 148 mmol), 4-methoxybenzyl
chloride (18.6 mL, 137 mmol), and tetrabutylammonium bromide (45 mg,
138 mmol) were added to a solution of 9 (101 mg, 115 mmol) in toluene
(1.1 mL). The mixture was stirred for 9 h at 808C (completion of the re-
action was confirmed by TLC analysis; EtOAc/hexane, 1:1), then triethyl-
amine was added and the mixture was concentrated. The residue was pu-
rified by column chromatography on silica gel (EtOAc/hexane 1:3) to
J
3,4 =J4,5 =9.3 Hz, 1H; H-4a), 3.95 (t, J4,5 =9.7 Hz, 1H; H-4c), 3.93–3.75
(m, 5H; H-6a, H-6’a, H-3b, H-2c, H-6’c), 3.72 (t, J2,3 =J3,4 =8.6 Hz, 1H;
H-3a), 3.68–3.57 (m, 4H; H-5a, H-2b, H-6b, H-5c), 3.55 (t, J5,6 =J5,6’
=
5.5 Hz, 1H; H-5b), 3.32 (dd, J5,6ꢀ =5.5 Hz, Jgem =9.6 Hz, 1H; H-6’b),
1.16 ppm (s, 9H; tBu); 13C NMR (150 MHz, [D6]acetone): d=176.9,
155.4, 154.5, 140.3, 140.2, 139.5, 138.6, 138.5, 129.6, 129.0, 129.0, 128.9,
128.9, 128.8, 128.8, 128.6, 128.5, 128.3, 128.3, 128.2, 128.1, 128.0, 127.8,
127.7, 127.1, 103.5, 102.7, 101.9, 100.8, 96.9, 95.7, 81.9, 81.8, 79.6, 78.3,
77.4, 77.3, 77.1, 76.0, 75.1, 74.9, 74.6, 74.5, 73.6, 73.5, 73.1, 70.9, 70.0, 69.0,
68.9, 66.7, 58.0, 39.2, 27.4 ppm; HRMS: m/z calcd for C71H77Cl6NO20Na+:
1496.3062 [M+Na]+; found: 1496.3063.
Compound 17: [a]D =ꢀ37.0 (c=0.5, CHCl3); 1H NMR (600 MHz,
[D6]DMSO): d=8.24 (d, J2,NH =8.9 Hz, 1H; NHd), 7.41–7.19 (m, 35H; 7
Ph), 5.77 and 5.75 (2 s, 2H; 2 PhCH), 5.37 (t, J2,3 =J3,4 =9.6 Hz, 1H; H-
3c), 5.30–5.27 (m, 2H; H-1c, H-3d), 5.04 (d, Jgem =10.3 Hz, 1H; CH2),
1
give 24 (106 mg, 93%). [a]D =ꢀ7.0 (c=1.0, CHCl3); H NMR (600 MHz,
CDCl3): d=7.34–7.17 (m, 27H; 6 ArH), 6.84 (d, J=8.2 Hz, 2H; ArH),
5.13 (t, J1,2 =J2,3 =8.3 Hz, 1H; H-2a), 4.97 (d, Jgem =11.0 Hz, 1H; CH2),
4.87 (d, Jgem =11.7 Hz, 1H; CH2), 4.76 (s, 2H; CH2), 4.63 (d, Jgem
=
11.0 Hz, 1H; CH2), 4.62–4.57 (m, 3H; 3 CH2), 4.56 (d, Jgem =12.4 Hz,
1H; CH2), 4.46 (d, J1,2 =8.3 Hz, 1H; H-1a), 4.42–4.40 (m, 2H; H-1b,
CH2), 4.33 (d, Jgem =12.0 Hz, 1H; CH2), 4.28 (d, Jgem =11.0 Hz, 1H; CH2),
4.05 (t, J3,4 =J4,5 =9.3 Hz, 1H; H-4a), 3.96 (brs, 1H; H-4b), 3.82 (dd, J5,6
4.5 Hz, Jgem =10.3 Hz, 1H; H-6a), 3.79 (s, 3H; OCH3), 3.73 (d, Jgem
=
=
10.3 Hz, 1H; H-6’a), 3.63 (dd, J2,3 =8.3 Hz, J3,4 =9.3 Hz, 1H; H-3a), 3.57
(t, J1,2 =J2,3 =8.6 Hz, 1H; H-2b), 3.52 (dd, J5,6 =7.2 Hz, Jgem =8.6 Hz, 1H;
H-6b), 3.43 (dd, J4,5 =9.3 Hz, J5,6 =4.5 Hz, 1H; H-5a), 3.35 (dd, J2,3 =8.6
Hz, J3,4 =4.1 Hz, 1H; H-3b), 3.35 (dd, J5,6’ =4.1 Hz, Jgem =8.6 Hz, 1H; H-
6’b), 3.31 (dd, J5,6 =7.2 Hz, J5,6ꢀ =4.1 Hz, 1H; H-5b), 2.39 (s, 1H; OH),
1.13 ppm (s, 9H; tBu); 13C NMR (150 MHz, CDCl3): d=176.7, 159.3,
138.8, 138.6, 138.2, 138.0, 137.2, 129.9, 129.4, 128.3, 128.2, 128.2, 128.0,
127.8, 127.7, 127.6, 127.6, 127.6, 127.5, 127.4, 127.0, 113.8, 102.7, 99.7,
81.0, 80.7, 79.4, 76.4, 75.5, 75.3, 73.8, 73.4, 73.1, 72.8, 72.2, 71.7, 70.0, 68.5,
4.99 (d, J1,2 =8.2 Hz, 1H; H-1d), 4.98–4.94 (m, 2H; 2 CH2), 4.90 (d, Jgem
=
12.4 Hz, 1H; CH2), 4.85–4.82 (m, 3H; 3 CH2), 4.76–4.68 (m, 4H; H-1a,
H-2a, 2 CH2), 4.63 (d, Jgem =12.4 Hz, 1H; CH2), 4.58 (d, Jgem =11.7 Hz,
1H; CH2), 4.53 (d, Jgem =11.7 Hz, 1H; CH2), 4.37–4.32 (m, 4H; H-1b, H-
6d, 2 CH2), 4.26 (d, Jgem =11.7 Hz, 1H; CH2), 4.23 (d, Jgem =12.3 Hz, 1H;
CH2), 4.20 (t, J3,4 =J4,5 =9.6 Hz, 1H; H-4d), 4.15 (brs, 1H; H-4b), 4.14–
4.07 (m, 3H; H-2d, H-6c, CH2), 4.05 (d, Jgem =10.3 Hz, 1H; CH2), 3.91 (t,
J
3,4 =J4,5 =9.6 Hz, 1H; H-4c), 3.84–3.71 (m, 6H; H-4a, H-3b, H-2c, H-5c,
H-6’c, H-6’d), 3.61 (dd, J5,6 =4.5 Hz, Jgem =12.0 Hz, 1H; H-6a), 3.58–3.42
Chem. Eur. J. 2011, 17, 588 – 597
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
593

H. Ando, M. Kiso et al.
68.1, 66.2, 55.2, 38.7, 27.1 ppm; HRMS: m/z calcd for C60H68O13Na+:
1019.4552 [M+Na]+; found: 1019.4552.
Compound 27: Molecular sieves (4 ꢃ, 450 mg) were added to a solution
of 7 (130 mg, 183 mmol) and 26 (163 mg, 122 mmol) in CH2Cl2 (3.0 mL).
The suspension was stirred for 30 min at ꢀ108C, whereupon NIS (83 mg,
366 mmol) and TfOH (3.2 mL, 36.6 mmol) were added. Stirring was contin-
ued for 40 min at ꢀ108C (completion of the reaction was indicated by
TLC, EtOAc/hexane 1:1). The reaction mixture was filtered through
Celite and the removed molecular sieves were washed with CHCl3. The
combined filtrate and washings were extracted with CHCl3, and organic
layer was washed with saturated aqueous NaHCO3, saturated aqueous
Na2S2O3, and brine, dried (Na2SO4), and concentrated. The residue was
purified by column chromatography on silica gel (EtOAc/hexane 1:2!
1:1) to give 27 (198 mg, 84%). [a]Dꢀ35.0 (c=1.0, CHCl3); 1H NMR
(600 MHz, [D6] DMSO): d=8.23 (d, J2,NH =8.9 Hz, 1H; NHd), 7.42–7.17
(m, 30H; 6 Ph), 7.08 (d, J2,NH =9.6 Hz, 1H; NHc), 5.68 (s, 1H; PhCH),
5.32 (d, J3,4 =3.4 Hz, 1H; H-4c), 5.29 (t, J2,3 =J3,4 =10.0 Hz, 1H; H-3d),
5.25 (dd, J2,3 =11.7 Hz, J3,4 =3.4 Hz, 1H; H-3c), 5.16 (d, J1,2 =8.2 Hz, 1H;
H-1c), 5.00 (d, J1,2 =8.9 Hz, 1H; H-1d), 5.00 (d, Jgem =12.4 Hz, 1H; CH2),
4.98 (d, Jgem =12.4 Hz, 1H; CH2), 4.87 (d, Jgem =12.4 Hz, 1H; CH2), 4.85
(d, Jgem =12.4 Hz, 1H; CH2), 4.78 (d, Jgem =11.7 Hz, 1H; CH2), 4.74–4.69
(m, 4H; H-1a, H-2a, 2 CH2), 4.58 (d, Jgem =11.7 Hz, 1H; CH2), 4.58 (d,
Compound 25: Molecular sieves (4 ꢃ, 415 mg) were added to a solution
of 8 (125 mg, 218 mmol) and 24 (145 mg, 145 mmol) in CH2Cl2 (3.65 mL).
The suspension was stirred for 30 min at ꢀ208C, whereupon NIS (74 mg,
327 mmol) and TfOH (2.9 mL, 32.7 mmol) were added. Stirring was contin-
ued for 15 min at ꢀ208C, when completion of the reaction was indicated
by TLC (EtOAc/hexane 1:2, developed twice). The reaction mixture was
filtered through Celite and the removed molecular sieves were washed
with CHCl3. The combined filtrate and washings were extracted with
CHCl3, and organic layer was washed with saturated aqueous NaHCO3,
saturated aqueous Na2S2O3, and brine, dried (Na2SO4), and concentrated.
The residue was purified by column chromatography on silica gel
(EtOAc/hexane, 1:2!1:1!3:2, then EtOAc/Toluene, 1:5) to give 25
(184 mg, 87%). [a]D =ꢀ12.0 (c=1.0, CHCl3); 1H NMR (600 MHz,
[D6]DMSO): d=7.57 (d, J2,NH =9.0 Hz, 1H; NH), 7.43–7.16 (m, 27H; 6
ArH), 6.84 (d, J=8.2 Hz, 2H; ArH), 5.30 (d, J3,4 =2.7 Hz, 1H; H-4c),
5.18 (dd, J2,3 =11.4 Hz, J3,4 =2.7 Hz, 1H; H-3c), 5.03 (d, Jgem =10.3 Hz,
1H; CH2), 4.84 (d, J1,2 =8.3 Hz, 1H; H-1c), 4.78 (d, Jgem =11.7 Hz, 1H;
CH2), 4.76–4.68 (m, 5H; H-1a, H-2a, 3 CH2), 4.66 (d, Jgem =10.3 Hz, 1H;
CH2), 4.55 (d, Jgem =11.7 Hz, 1H; CH2), 4.54 (d, Jgem =12.3 Hz, 1H; CH2),
J
gem =11.0 Hz, 1H; CH2), 4.53 (d, Jgem =11.7 Hz, 1H; CH2), 4.36 (d, J1,2
=
7.6 Hz, 1H; H-1b), 4.36 (d, Jgem =10.3 Hz, 1H; CH2), 4.32 (d, Jgem
=
4.44–4.41 (m, 3H; 3 CH2), 4.38 (d, J1,2 =7.5 Hz, 1H; H-1b), 4.31 (d, Jgem
11.7 Hz, 1H; CH2), 4.24 (d, Jgem =12.3 Hz, 1H; CH2), 4.11 (dd, J5,6
=
12.4 Hz, 1H; CH2), 4.30–4.21 (m, 5H; H-6c, H-4d, H-6d, 2 CH2), 4.15 (d,
3,4 =2.1 Hz, 1H; H-4b), 4.13–4.02 (m, 4H; H-5c, H-6’c, H-2d, CH2), 3.95
=
J
6.2 Hz, Jgem =11.0 Hz, 1H; H-6c), 4.05 (dd, J5,6’ =6.2 Hz, Jgem =11.0 Hz,
1H; H-6’c), 4.02 (s, 1H; H-4b), 3.99 (t, J5,6 =J5,6ꢀ =6.2 Hz, 1H; H-5c), 3.96
(d, Jgem =12.3 Hz, 1H; CH2), 3.81 (t, J3,4 =J4,5 =9.3 Hz, 1H; H-4a), 3.77–
3.65 (m, 8H; H-3a, H-5a, H-6a, H-6’a, H-2c, OCH3), 3.56 (dd, J1,2 =7.5
Hz, J2,3 =8.6 Hz, 1H; H-2b), 3.53 (dd, J5,6 =4.2 Hz, Jgem =11.0 Hz, 1H; H-
6b), 3.47 (d, J2,3 =8.6 Hz, 1H; H-3b), 3.40 (dd, J5,6 =4.2 Hz, J5,6’ =5.2 Hz,
1H; H-5b), 3.28 (dd, J5,6ꢀ =5.2 Hz, Jgem =11.0 Hz, 1H; H-6’b), 2.12, 1.97
and 1.87 (3ꢄs, 9H; 3 Ac), 1.14 ppm (s, 9H; tBu); 13C NMR (150 MHz,
[D6]DMSO): d=176.2, 170.2, 170.1, 169.6, 158.9, 154.3, 139.0, 138.9,
138.8, 138.3, 137.5, 130.8, 129.6, 128.8, 128.4, 128.3, 128.3, 128.2, 127.8,
127.8, 127.7, 127.5, 127.5, 127.4, 127.2, 113.7, 102.1, 101.5, 99.6, 96.4, 80.8,
80.3, 79.5, 79.3, 76.4, 74.7, 74.4, 74.3, 73.5, 73.5, 73.4, 72.5, 72.3, 72.3, 71.0,
70.3, 70.2, 69.8, 69.7, 67.8, 66.7, 61.6, 55.1, 52.5, 40.2, 38.4, 27.0, 20.6,
20.6, 20.5 ppm; HRMS: m/z calcd for C75H86Cl3NO22Na+: 1480.4599
[M+Na]+; found: 1480.4599.
(m, 1H; H-2c), 3.93 (d, Jgem =12.4 Hz, 1H; CH2), 3.83 (t, J5,6’ =Jgem
10.0 Hz, 1H; H-6’d), 3.77–3.73 (m, 2H; H-4a, H-3b), 3.62 (dd, J5,6
=
=
3.9 Hz, Jgem =10.7 Hz, 1H; H-6a), 3.60–3.43 (m, 7H; H-3a, H-5a, H-6’a,
H-2b, H-5b, H-6b, H-5d), 3.18 (dd, J5,6’ =7.9 Hz, Jgem =12.3 Hz, 1H; H-
6’b), 2.19, 1.98 and 1.91 (3ꢄs, 9H; 3ꢄAc), 1.13 ppm (s, 9H; tBu);
13C NMR (150 MHz, [D6]DMSO): d=176.2, 170.2, 170.0, 169.8, 154.6,
154.5, 153.4, 139.1, 139.0, 138.8, 138.4, 137.6, 137.3, 129.1, 128.7, 128.3,
128.2, 128.1, 127.7, 127.7, 127.7, 127.6, 127.5, 127.3, 127.2, 126.3, 102.5,
101.6, 100.6, 100.2, 99.6, 96.6, 95.7, 95.0, 82.3, 80.4, 78.7, 77.9, 76.5, 76.3,
76.1, 74.7, 74.6, 74.2, 73.7, 73.5, 73.0, 72.5, 72.3, 72.1, 71.0, 70.2, 69.8, 69.5,
67.9, 67.5, 67.2, 65.2, 61.7, 55.9, 52.5, 38.4, 27.0, 20.7, 20.6 ppm; HRMS:
m/z calcd for C86H95Cl9N2O29Na+: 1957.3109 [M+Na]+; found: 1957.3103.
Compound 28: Zinc powder (3.00 g) was added to a solution of 27
(204 mg, 105 mmol) in a mixture of MeOH (5.2 mL) and AcOH (5.2 mL),
and the mixture was stirred for 30 min at RT (completion of the reaction
was confirmed by TLC analysis; CHCl3/MeOH, 20:1). The reaction mix-
ture was filtered through Celite and the removed zinc powder was
washed with CHCl3. The combined filtrate and washings were concen-
trated and the residue was extracted with CHCl3. The organic layer was
washed with saturated aqueous NaHCO3 and brine, dried (Na2SO4), con-
centrated, and exposed to high vacuum for 4 h. The residue was treated
with a solution of Ac2O (24.0 mL, 254 mmol) in MeOH (600 mL) and
CH2Cl2 (600 mL) for 40 min (completion of the reaction was confirmed
by TLC analysis; CHCl3/MeOH, 15:1). The reaction mixture was extract-
ed with EtOAc and this solution was washed with saturated aqueous
NaHCO3 and brine, dried (Na2SO4), and concentrated. The residue was
purified by column chromatography on silica gel (CHCl3/MeOH, 50:1!
40:1!30:1) to give 28 (118 mg, 75%). [a]D =ꢀ41.5 (c=1.0, CHCl3);
1H NMR (600 MHz, CD3CN): d=7.52–7.20 (m, 30H; 6 Ph), 7.16 (d,
Compound 26: TFA (41.0 mL, 552 mmol) was added to a solution of 25
(101 mg, 69.0 mmol) in CH2Cl2 (1.4 mL) at 08C, and the mixture was
stirred for 7.5 h at RT (completion of the reaction was confirmed by
TLC; EtOAc/hexane, 1:1), then the reaction mixture was quenched by
the addition of triethylamine at 08C. The residue was extracted with
CHCl3 and the solution was washed with saturated aqueous NaHCO3
and brine, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel (EtOAc/hexane 2:3) to give 26
(90 mg, 98%). [a]D =ꢀ23.5 (c=1.0, CHCl3); 1H NMR (600 MHz,
[D6]DMSO): d=7.65 (d, J2,NH =8.9 Hz, 1H; NH), 7.44–7.18 (m, 25H; 5
Ph), 5.30 (d, J3,OH =5.5 Hz, 1H; OH), 5.29 (d, J3,4 =3.5 Hz, 1H; H-4c),
5.12 (dd, J2,3 =11.0 Hz, J3,4 =3.5 Hz, 1H; H-3c), 5.03 (d, Jgem =10.3 Hz,
1H; CH2), 4.99 (d, J1,2 =8.3 Hz, 1H; H-1c), 4.85 (d, Jgem =11.7 Hz, 1H;
CH2), 4.83 (d, Jgem =10.3 Hz, 1H; CH2), 4.78–4.73 (m, 3H; H-2a, 2 CH2),
4.70 (d, J1,2 =8.3 Hz, 1H; H-1a), 4.60 (d, Jgem =11.7 Hz, 1H; CH2), 4.55
(d, Jgem =12.4 Hz, 1H; CH2), 4.44–4.40 (m, 3H; H-1b, 2 CH2), 4.35 (d,
J
2,NH =10.3 Hz, 1H; NHc), 6.75 (d, J2,NH =9.7 Hz, 1H; NHd), 5.63 (s, 1H;
PhCH), 5.32 (d, J3,4 =3.4 Hz, 1H; H-4c), 5.10 (d, J1,2 =8.9 Hz, 1H; H-1c),
5.04 (dd, J2,3 =11.7 Hz, J3,4 =3.4 Hz, 1H; H-3c), 5.01 (d, Jgem =10.3 Hz,
1H; CH2), 4.78 (t, J1,2 =J2,3 =8.9 Hz, 1H; H-2a), 4.77 (d, Jgem =12.4 Hz,
1H; CH2), 4.69 (d, Jgem =11.0 Hz, 1H; CH2), 4.65 (d, J1,2 =8.9 Hz, 1H; H-
1d), 4.61 (d, Jgem =12.4 Hz, 1H; CH2), 4.56 (d, Jgem =11.0 Hz, 1H; CH2),
4.53 (d, J1,2 =8.9 Hz, 1H; H-1a), 4.47 (d, Jgem =10.3 Hz, 1H; CH2), 4.42
(d, Jgem =11.7 Hz, 1H; CH2), 4.37 (m, 1H; H-2c), 4.35 (d, J1,2 =8.2 Hz,
J
J
gem =11.7 Hz, 1H; CH2), 4.28 (d, Jgem =12.3 Hz, 1H; CH2), 4.10 (dd,
5,6 =5.5 Hz, Jgem =11.0 Hz, 1H; H-6c), 4.05 (dd, J5,6’ =6.2 Hz, Jgem =11.0
Hz, 1H; H-6’c), 4.00 (brs, 1H; H-5c), 3.96 (d, Jgem =12.3 1H; CH2), 3.90
(s, 1H; H-4b), 3.84 (m, 1H; H-2c), 3.83 (t, J3,4 =J4,5 =9.6 Hz, 1H; H-4a),
3.73 (dd, J5,6 =4.2 Hz, Jgem =11.0 Hz, 1H; H-6a), 3.70–3.61 (m, 4H; H-3a,
H-5a, H-6’a, H-3b), 3.53 (dd, J5,6 =4.2 Hz, Jgem =10.5 Hz, 1H; H-6b),
3.50–3.46 (m, 2H; H-2b, H-5b), 3.25 (dd, J5,6’ =6.6 Hz, Jgem =10.5 Hz, 1H;
H-6’b), 2.16, 1.97 and 1.87 (3ꢄs, 9H; 3 Ac), 1.14 ppm (s, 9H; tBu);
13C NMR (150 MHz, [D6]DMSO): d=176.2, 170.1, 170.1, 169.6, 153.9,
139.3, 139.0, 138.8, 138.4, 137.6, 128.8, 128.4, 128.2, 128.2, 127.8, 127.7,
127.5, 127.5, 127.4, 127.3, 127.2, 102.3, 101.5, 99.7, 96.5, 80.7, 80.3, 76.4,
76.3, 74.8, 74.4, 73.6, 73.5, 73.0, 72.5, 72.3, 70.9, 70.2, 69.9, 69.6, 67.9, 66.9,
61.6, 52.6, 38.4, 27.0, 20.6, 20.6 ppm; HRMS: m/z calcd for
C67H78Cl3NO21Na+: 1360.4024 [M+Na]+; found: 1360.4026.
1H; H-1b), 4.30–4.26 (m, 3H; H-6d, 2 CH2), 4.17 (dd, J5,6 =5.5 Hz, Jgem
9.6 Hz, 1H; H-6c), 4.08 (d, J3,4 =2.8 Hz, 1H; H-4b), 4.07–3.98 (m, 4H; H-
5c, H-6’c, H-2d, CH2), 3.86 (t, J3,4 =J4,5 =9.3 Hz, 1H; H-4a), 3.81 (t, J5,6’
gem =10.0 Hz, 1H; H-6’d), 3.77 (t, J2,3 =J3,4 =9.7 Hz, 1H; H-3d), 3.72–
3.66 (m, 3H; H-6a, H-4d, OH), 3.62–3.59 (m, 2H; H-3a, H-3b), 3.55 (d,
gem =9.6 Hz, 1H; H-6’a), 3.51–3.43 (m, 3H; H-5a, H-6b, H-5d), 3.40–3.37
=
=
J
J
(m, 2H; H-2b, H-5b), 3.18 (dd, J5,6’ =6.2 Hz, Jgem =10.3 Hz, 1H; H-6’b),
2.18, 1.96, 1.95, 1.92 and 1.75 (5ꢄs, 15H; 5ꢄAc), 1.16 ppm (s, 9H; tBu);
594
www.chemeurj.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 588 – 597

Synthesis of a Hybrid Ganglioside X1
FULL PAPER
13C NMR (150 MHz, CD3CN): d=176.2, 170.3, 170.1, 169.9, 169.1, 139.0,
139.0, 138.8, 138.4, 137.9, 137.6, 129.1, 128.8, 128.3, 128.2, 128.1, 127.8,
127.6, 127.5, 127.5, 127.2, 127.2, 127.0, 126.6, 103.5, 101.5, 101.0, 101.0,
99.7, 81.5, 81.2, 80.2, 79.3, 76.3, 74.9, 74.8, 74.4, 74.2, 72.9, 72.4, 72.4, 72.2,
71.6, 70.2, 70.1, 69.7, 69.4, 68.1, 67.6, 67.1, 66.0, 61.9, 56.4, 49.1, 38.4, 27.0,
23.1, 23.1, 20.7, 20.6 ppm; HRMS: m/z calcd for C81H96N2O25Na+:
1519.6194 [M+Na]+; found: 1519.6194.
0.8H; H-1ab); HRMS: m/z calcd for 1/2 (C107H140N4O59)+Na+:
1235.3931 [1/2M+Na]+; found: 1235.3931.
Compound 31: Hydrazine acetate (10 mg, 111 mmol) was added to a solu-
tion of 30 (97 mg, 40.0 mmol) in DMF (800 mL) and the mixture was
stirred for 10 min at 508C (completion of the reaction was confirmed by
TLC analysis; CHCl3/MeOH, 15:1; developed twice). The reaction mix-
ture was diluted with EtOAc and the solution was washed with water
and brine, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH, 20:1!15:1) to give
the 1-OH derivative, which was exposed to high vacuum for 7 h. The resi-
due was then treated with a solution of trichloroacetonitrile (80.0 mL,
79.8 mmol) and DBU (7.5 mL, 50.1 mmol) in CH2Cl2 for 50 min at RT
(completion of the reaction was confirmed by TLC analysis; CHCl3/
MeOH, 10:1). The mixture was concentrated and the residue was puri-
fied by column chromatography on silica gel (CHCl3/MeOH, 25:1) to
give 31 (83 mg, 82%). [a]D =ꢀ2.0 (c=1.0, CHCl3); 1H NMR (600 MHz,
CDCl3): d=8.65 (s, 1H; NH), 8.14 (d, J=6.9 Hz, 2H; Ph), 8.05 (d, J=
7.6 Hz, 2H; Ph), 7.59 (t, J=7.2 Hz, 1H; Ph), 7.55 (t, J=7.2 Hz, 1H; Ph),
7.48–7.43 (m, 4H; Ph), 6.49 (d, J1,2 =3.5 Hz, 1H; H-1a), 6.43–6.35 (m,
2H; NHc, NHf), 5.85 (d, J2,NH =11.0 Hz, 1H; NHd), 5.53 (m, 1H; H-8g),
5.52 (t, J2,3 =J3,4 =10.0 Hz, 1H; H-3a), 5.37 (d, J3,4 =2.7 Hz, 1H; H-4c),
5.36 (d, J3,4 =3.4 Hz, 1H; H-4f), 5.29 (dd, J1,2 =7.5 Hz, J2,3 =9.6 Hz, 1H;
Compound 29: AW-300 molecular sieves (4 ꢃ, 500 mg) were added to a
solution of 5 (153 mg, 115 mmol) and 28 (113 mg, 75.5 mmol) in CH2Cl2
(1.90 mL). The suspension was stirred for 30 min at 08C, whereupon
TMSOTf (1.0 mL, 5.54 mmol) was added. Stirring was continued for
30 min at 08C (completion of the reaction was indicated by TLC analysis;
CHCl3/MeOH, 12:1; developed twice). The reaction mixture was filtered
through Celite and the removed molecular sieves were washed with
CHCl3. The combined filtrate and washings were extracted with CHCl3,
and organic layer was washed with saturated aqueous NaHCO3 and
brine, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH, 40:1!35:1!30:1!
25:1) to give 29 (174 mg, 86%). [a]D =ꢀ16.5 (c=1.0, CHCl3); 1H NMR
(600 MHz, CD3CN): d=8.30 (d, J=7.5 Hz, 2H; Ph), 7.83 (d, J=7.6 Hz,
2H; Ph), 7.65 (t, J=7.3 Hz, 1H; Ph), 7.58 (t, J=7.2 Hz, 1H; Ph), 7.50 (t,
J=7.3 Hz, 2H; Ph), 7.46–7.11 (m, 32H; 7 Ph), 7.03 (d, J2,NH =9.6 Hz, 1H;
NHc), 6.41 (d, J2,NH =9.7 Hz, 1H; NHf), 6.30 (d, J2,NH =9.6 Hz, 1H;
NHd), 6.12 (d, J5,NH =10.3 Hz, 1H; NHg), 5.62 (s, 1H; PhCH), 5.34 (d,
H-2e), 5.23–5.20 (m, 3H; H-3c, H-3f, H-7g), 5.04 (dd,J1,2 =3.5 Hz, J2,3
10.0 Hz, 1H; H-2a), 5.02–4.98 (m, 3H; H-1c, H-1f, NHg), 4.95 (dd, J1,2
=
=
8.3 Hz, J2,3 =10.3 Hz, 1H; H-2b), 4.88 (d, J1,2 =7.5 Hz, 1H; H-1e), 4.83
(td, J3ax,4 =J4,5 =11.2 Hz, J3eq,4 =4.2 Hz, 1H; H-4g), 4.76 (brs, 1H; H-4d),
4.62 (dd, J5,6 =7.2 Hz, Jgem =11.0 Hz, 1H; H-6e), 4.40–4.38 (m, 2H; H-6c,
H-3e), 4.32 (dd, J5,6’ =4.8 Hz, Jgem =11.0 Hz, 1H; H-6’e), 4.26 (d, J1,2 =8.3
J
3,4 =3.5 Hz, 1H; H-4c), 5.22 (d, J3,4 =2.8 Hz, 1H; H-4f), 5.18–5.12 (m,
3H; H-3c, H-7g, H-8g), 5.08–4.99 (m, 5H; H-1c, H-1e, H-2e, H-3f, H-4g),
4.98 (d, Jgem =10.3 Hz, 1H; CH2), 4.86 (d, J1,2 =8.3 Hz, 1H; H-1f), 4.76 (t,
J
1,2 =J2,3 =9.0 Hz, 1H; H-2a), 4.75 (d, Jgem =12.4 Hz, 1H; CH2), 4.62–4.48
Hz, 1H; H-1b), 4.26–4.23 (m, 3H; H-6b, H-1d, H-6f), 4.20 (dd, J8,9
=
(m, 7H; H-1a, H-1d, H-3e, H-9g, 3 CH2), 4.46 (d, Jgem =10.3 Hz, 1H;
CH2), 4.40 (d, Jgem =12.4 Hz, 1H; CH2), 4.37 (q, J1,2 =J2,3 =J2,NH =9.6 Hz,
1H; H-2c), 4.29 (d, J1,2 =8.2 Hz, 1H; H-1b), 4.26–4.22 (m, 3H; H-6d, 2
CH2), 4.18–4.02 (m, 12H; H-4b, H-5c, H-6c, H-6’c, H-2d, H-3d, H-6e, H-
2f, H-5f, H-5g, H-9g, CH2), 3.99 (d, J3,4 =2.1 Hz, 1H; H-4e), 3.95–3.92
(m, 3H; H-5e, H-6’e, H-6f), 3.84–3.81 (m, 3H; H-4a, H-6’d, H-6g), 3.75
(s, 3H; COOCH3), 3.71 (dd, J5,6 =6.2 Hz, Jgem =11.0 Hz, 1H; H-6’f), 3.63–
3.56 (m, 2H; H-6a, H-4d), 3.54 (dd, J2,3 =9.6 Hz, J3,4 =2.7 Hz, 1H; H-3b),
3.50 (d, Jgem =10.3 Hz, 1H; H-6’a), 3.45–3.40 (m, 3H; H-5a, H-6b, H-5d),
3.36 (t, J5,6 =J5,6’ =6.2 Hz, 1H; H-5b), 3.33 (dd, J1,2 =8.2 Hz, J2,3 =9.6 Hz,
1H; H-2b), 3.16 (dd, J5,6ꢀ =6.2 Hz, Jgem =11.0 Hz, 1H; H-6’b), 2.22–2.15
(m, 7H; H-3geq, 2 Ac), 2.11, 2.05 and 1.98 (3ꢄs, 9H; 3 Ac), 1.96–1.89 (m,
19H; H-3gax, 6 Ac), 1.82, 1.78 and 1.75 (3ꢄs, 9H; 3 Ac), 1.16 ppm (s,
9H; tBu); 13C NMR (150 MHz, CD3CN): d=177.6, 171.5, 171.3, 171.2,
171.1, 171.0, 171.0, 170.8, 170.6, 170.5, 170.4, 169.2, 166.4, 165.3, 140.0,
139.9, 139.7, 139.3, 138.4, 138.4, 134.3, 134.1, 130.9, 130.7, 130.7, 130.1,
129.8, 129.6, 129.4, 129.4, 129.2, 129.2, 129.1, 129.0, 128.9, 128.9, 128.7,
128.6, 128.4, 128.3, 128.2, 128.1, 127.9, 127.0, 103.9, 103.1, 102.9, 102.2,
101.9, 100.9, 100.8, 100.2, 82.2, 81.4, 81.3, 80.3, 79.0, 78.7, 77.0, 75.9, 75.8,
75.5, 75.3, 74.3, 74.0, 73.7, 73.5, 73.2, 72.6, 72.4, 72.2, 72.1, 71.7, 71.3, 71.2,
70.8, 70.6, 70.3, 69.1, 68.6, 68.5, 68.3, 67.8, 67.6, 67.0, 63.8, 63.0, 62.4, 62.2,
55.4, 53.9, 50.8, 50.4, 48.7, 39.3, 35.7, 27.4, 23.4, 23.2, 23.1, 23.0, 21.4, 21.0,
21.0, 20.8, 20.8, 20.7, 20.5 ppm; HRMS: m/z calcd for 1/2
(C135H160N4O52)+Na+: 1357.4892 [1/2M+Na]+; found: 1357.4893.
1.4 Hz, Jgem =12.3 Hz, 1H; H-9g), 4.16–4.11 (m, 2H; H-6’b, H-6’c), 4.08–
4.05 (m, 3H; H-5a, H-6a, H-6d), 4.03 (d, J3,4 =2.0 Hz, 1H; H-4b), 3.99
(dd, J8,9’ =5.5 Hz, Jgem =12.3 Hz, 1H; H-9g), 3.96–3.78 (m, 10H; H-4a, H-
6’a, H-3d, H-5d, H-6’d, H-4e, H-5e, H-5f, H-6’f, H-5g), 3.76 (dd, J5,6
11.0 Hz, J6,7 =2.0 Hz, 1H; H-6g), 3.73 (s, 3H; COOCH3), 3.62–3.57 (m,
5H; H-3b, H-5b, H-2c, H-2d, H-2f), 2.67 (dd, J3eq,4 =4.2 Hz, Jgem
=
=
12.3 Hz, 1H; H-3geq), 2.15, 2.14, 2.14, 2.09, 2.08, 2.06, 2.05, 2.05, 2.05,
2.04, 2.02, 2.01, 2.01 and 1.96 (14ꢄs, 42H; 14 Ac), 1.84–1.79 (m, 10H; H-
3gax, 3 Ac), 1.77 (s, 3H; Ac), 1.13 ppm (s, 9H; tBu); 13C NMR (150 MHz,
CDCl3): d=178.0, 171.5, 171.3, 171.3, 171.2, 171.1, 171.0, 171.0, 170.7,
170.4, 170.3, 170.0, 169.1, 166.9, 165.3, 160.7, 134.4, 134.3, 131.0, 130.6,
130.6, 130.3, 129.6, 129.4, 103.6, 103.0, 102.0, 101.6, 100.8, 100.3, 93.4,
91.3, 80.0, 79.0, 78.7, 76.6, 75.9, 75.2, 74.4, 72.9, 72.8, 72.6, 72.3, 72.2, 72.1,
71.6, 71.3, 70.9, 70.5, 70.4, 70.3, 70.0, 68.5, 68.0, 67.9, 67.4, 64.4, 64.1, 63.2,
62.9, 62.5, 62.4, 62.1, 53.9, 50.9, 50.0, 48.7, 39.3, 35.8, 27.0, 23.4, 23.2, 23.0,
22.9, 21.4, 21.2, 21.0, 21.0, 20.9, 20.8, 20.8, 20.7, 20.7 ppm; HRMS: m/z
calcd for C107H138Cl3N5O58Na+: 2548.6961 [M+Na]+; found: 2548.6960.
Compound 32: Molecular sieves (4 ꢃ, 250 mg) were added to a solution
of 31 (88 mg, 34.8 mmol) and 4 (35 mg, 52.2 mmol) in CHCl3 (880 mL).
The suspension was stirred for 30 min at RT, whereupon BF3·OEt2
(5.2 mL, 41.0 mmol) was added. Stirring was continued for 6.5 h at RT
(completion of the reaction was indicated by TLC analysis; CHCl3/
MeOH, 20:1; developed twice). The reaction mixture was filtered
through Celite and the removed molecular sieves were washed with
CHCl3. The combined filtrate and washings were extracted with CHCl3,
and the organic layer was washed with saturated aqueous NaHCO3 and
brine, dried (Na2SO4), and concentrated. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH, 40:1!30:1!20:1,
then acetone/hexane, 3:2) to give 32 (27 mg, 26%). [a]D =ꢀ14.6 (c=2.3,
CHCl3); 1H NMR (600 MHz, CDCl3): d=8.12 (d, J=6.9 Hz, 2H; Ph),
8.05 (d, J=6.8 Hz, 2H; Ph), 8.00 (d, J=6.9 Hz, 2H; Ph), 7.60–7.53 (m,
Compound 30: Pd(OH)2/C (20%, 234 mg) was added to a solution of 29
(153 mg, 115 mmol) in EtOH (1.90 mL) and the suspension was stirred
under a hydrogen stream for 62 h at RT (completion of the reaction was
confirmed by TLC analysis; CHCl3/MeOH/H2O, 14:3:0.1). The reaction
mixture was filtered through Celite and washed thoroughly with CHCl3.
The combined filtrate and washings were concentrated and exposed to
high vacuum for 4 h. The residue was then treated with a solution of
Ac2O (500 mL, 5.29 mmol) and DMAP (1 mg, 8.15 mmol) in pyridine
(500 mL) for 21 h (completion of the reaction was confirmed by TLC
analysis; CHCl3/MeOH, 18:1; developed twice), then the reaction mix-
ture was coevaporated with toluene. The residue was extracted with
EtOAc and the solution was washed with 2m aqueous HCl, saturated
aqueous NaHCO3, and brine, dried (Na2SO4), and concentrated. The resi-
due was purified by column chromatography on silica gel (CHCl3/MeOH,
30:1!25:1) to give 30 (97 mg, 93%, a/b=5/4). 1H NMR (600 MHz,
CD3CN): d=6.16 (d, J1,2 =3.4 Hz, 1H; H-1aa), 5.75 ppm (d, J1,2 =8.3 Hz,
3H; 3 Ph), 6.44–6.31 (m, 2H; NHc, NHf), 5.86 (dt, J4,5 =15.2 Hz, J5,6
=
J
J
J
5,6’ =7.2 Hz, 1H, H-5Cer), 5.82 (d, J2,NH =10.3 Hz, 1H; NHd), 5.72 (d,
2,NH =9.6 Hz, 1H; NHCer), 5.54–5.51 (m, 2H; H-8g, H-3Cer), 5.45 (dd,
3,4 =7.9 Hz, J4,5 =15.2 Hz,1H, H-4Cer), 5.37 (d, J3,4 =2.7 Hz, 1H; H-4c),
5.36 (d, J3,4 =3.4 Hz, 1H; H-4f), 5.28 (t, J1,2 =J2,3 =9.0 Hz, 1H; H-2e),
5.22–5.20 (m, 3H; H-3c, H-3f, H-7g), 5.15 (t, J2,3 =J3,4 =8.6 Hz, 1H; H-
3a), 5.02–4.97 (m, 2H; H-1c, NHg), 4.91–4.87 (m, 4H; H-2a, H-2b, H-1e,
H-1f), 4.83 (td, J3ax,4 =J4,5 =11.0 Hz, J3eq,4 =4.4 Hz, 1H; H-4g), 4.72 (brs,
1H; H-4d), 4.62 (dd, J5,6 =7.6 Hz, Jgem =10.3 Hz, 1H; H-6e), 4.46 (m, 1H;
Chem. Eur. J. 2011, 17, 588 – 597
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
595

H. Ando, M. Kiso et al.
H-2Cer), 4.38 (d, J1,2 =7.6 Hz, 1H; H-1a), 4.36 (m, 1H; H-3e), 4.32 (dd,
5,6’ =4.5 Hz, Jgem =10.3 Hz, 1H; H-6’e), 4.25–4.17 (m, 6H; H-1b, H-6c,
Acknowledgements
J
H-1d, H-6d, H-6f, H-9g), 4.12 (dd, J5,6’ =7.2 Hz, Jgem =12.0 Hz, 1H; H-
6’c), 4.08–3.79 (m, 14H; H-6a, H-6’a, H-4b, H-6b, H-6’b, H-3d, H-5d, H-
This work was financially supported by MEXT of Japan (WPI program
and a grant-in-aid for Scientific Research to M.K., nos. 1701007 and
22380067). We thank Kiyoko Ito for technical assistance.
6’d, H-4e, H-5e, H-6’f, H-5g, H-9g, H-1Cer), 3.75 (dd, J5,6 =11.0 Hz, J6,7
2.1 Hz, 1H; H-6 g), 3.73 (s, 3H; COOCH3), 3.69 (dd, J3,4 =8.6 Hz, J4,5
=
=
9.6 Hz, 1H; H-4a), 3.66–3.55 (m, 8H; H-3b, H-5b, H-2c, H-5c, H-2d, H-
2f, H-5f, H-1’Cer), 3.49 (m, 1H; H-5a), 2.64 (dd, J3eq,4 =4.4 Hz, Jgem
=
[1] a) L. Freddo, R. K. Yu, N. Latov, P. D. Donofrio, A. P. Hays, H. S.
Greenberg, J. W. Albers, A. G. Allessi, D. Keren, Neurology 1986,
36, 454–458; b) A. Pestronk, D. R. Cornblath, A. A. Ilyas, H. Baba,
R. H. Quarles, J. W. Griffin, K. Alderson, R. N. Adams, Ann.
Neurol. 1988, 24, 73–78.
10.3 Hz, 1H; H3geq), 2.15, 2.14, 2.13, and 2.10 (4ꢄs, 12H; 4 Ac), 2.06–
2.00 (m, 30H; H-3gax, H-6Cer, H-6’Cer, 9 Ac), 1.96, 1.92, 1.88, 1.83, 1.83,
1.81, and 1.75 (7ꢄs, 21H; 7 Ac), 1.33–1.22 (m, 54H; 27 CH2), 1.14 (s,
9H; tBu), 0.88 ppm (2ꢄt, J=6.6 Hz, 6H;
2
CH2CH3); 13C NMR
(150 MHz, CDCl3): d=177.0, 172.6, 171.8, 171.1, 170.8, 170.8, 170.7,
170.7, 170.5, 170.3, 170.3, 170.2, 170.1, 170.1, 170.0, 169.9, 169.9, 169.6,
168.9, 168.3, 165.7, 165.0, 164.8, 164.7, 137.7, 133.3, 133.2, 132.9, 130.3,
130.3, 129.8, 129.7, 129.6, 128.5, 128.5, 128.3, 124.7, 100.4, 100.2, 100.1,
99.6, 99.6, 97.9, 74.7, 73.8, 73.6, 73.5, 73.4, 73.0, 72.7, 72.1, 71.9, 71.9, 71.4,
71.2, 70.7, 70.5, 70.1, 69.9, 68.7, 68.6, 67.4, 67.1, 66.9, 66.9, 66.5, 63.2, 62.6,
62.4, 62.3, 62.1, 61.4, 61.2, 52.9, 52.8, 50.5, 49.2, 38.8, 36.9, 36.5, 32.3, 31.9,
30.0, 29.9, 29.8, 29.8, 29.7, 29.6, 29.5, 29.5, 29.4, 29.3, 29.2, 28.9, 27.1, 26.9,
25.7, 23.6, 23.3, 23.2, 23.1, 22.7, 21.2, 20.9, 20.8, 20.7, 20.7, 20.6, 20.6, 20.5,
20.4, 20.3, 14.1 ppm; HRMS: m/z calcd for 1/2 (C148H211N5O61)+Na+:
[2] N, Yuki, S. Sato, T. Miyatake, K. Sugiyama, T. Katagiri, H. Sasaki,
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1540.1674ACHTUNGTRENNUNG
[1/2M+Na]+; found: 1540.1673.
Ganglioside X1 (1): A solution of sodium methoxide (28% in MeOH,
0.4 mg) was added to a solution of 32 (22 mg, 7.25 mmol) in MeOH
(500 mL) and THF (500 mL). The mixture was stirred for 45 h at RT,
whilst monitoring the reaction by TLC (CHCl3/MeOH/12 mm MgCl2 aq,
5:4:1). The mixture was then heated at 408C and stirring was continued
for 32 h at 408C. Water (200 mL) was added and stirring was continued
for 35 h at 408C. After neutralization with Dowex-50 (H+), the mixture
was filtered through cotton wool, and the removed resin was washed
with mixed solvent (CHCl3/MeOH, 1:1). The combined filtrate and wash-
ings were concentrated and the residue was purified by column chroma-
tography on Sephadex LH-20 (CHCl3/MeOH, 1:1) and column chroma-
tography on silica gel (CHCl3/MeOH/H2O, 5:4:0.6!5:4:0.7) to give 1
(13.5 mg, 95%). [a]D =+1.4 (c=0.7, MeOH); 1H NMR (600 MHz,
CD3OD): d=5.67 (dt, J4,5 =15.1 Hz, J5,6 =J5,6’ =7.2 Hz, 1H; H-5Cer), 5.44
(dd,J4,5 =15.1 Hz, J3,4 =7.8 Hz, 1H; H-4Cer), 4.88–4.85 (m, 2H; 2 anomeric
H), 4.60 (d, J1,2 =8.9 Hz, 1H; anomeric H), 4.40 (d, J1,2 =8.3 Hz, 1H;
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anomeric H), 4.31 (d, J1,2 =7.6 Hz, 1H; anomeric H), 4.28 (d, J1,2
7.5 Hz, 1H; anomeric H), 4.26 (d, J3,4 =2.0 Hz, 1H; H-4), 4.19 (dd, J=
9.9 Hz, J=4.5 Hz, 1H), 4.12 (d, J3,4 =2.8 Hz, 1H; H-4), 2.75 (dd, Jgem
=
=
12.8 Hz, J3eq,4 =5.2 Hz, 1H; H-3geq), 2.03–2.00 (m, 14H; H-6Cer, H-6’Cer, 4
Ac), 2.16 (t, J=7.6 Hz, 2H; NHCOCH2), 1.89 (t, J3ax,4 =12.0 Hz, 1H; H-
3gax), 1.57 (m, 2H; NHCOCH2CH2),1.39–1.28 (m, 50H; 25 CH2),
0.90 ppm (2ꢄt, J=7.3 Hz, 6H; 2 CH2CH3); 13C NMR (150 MHz, CDCl3):
d=175.9, 175.6, 175.0, 174.7, 174.7, 174.5, 135.1, 131.4, 105.2, 105.0, 104.6,
104.4, 104.3, 103.4, 103.4, 101.3, 84.7, 83.5, 78.9, 77.7, 76.9, 76.5, 76.5, 76.3,
76.1, 75.8, 75.7, 75.1, 74.9, 74.2, 73.8, 73.4, 73.0, 71.3, 70.7, 70.4, 70.3, 70.0,
69.9, 69.7, 65.4, 63.0, 62.9, 61.8, 61.8, 56.2, 54.7, 54.3, 54.1, 53.8, 38.8, 37.4,
33.5, 33.1, 30.9, 30.8, 30.8, 30.8, 30.7, 30.6, 30.5, 30.5, 30.4, 27.2, 23.7, 23.6,
23.6, 23.5, 22.6, 14.5 ppm; HRMS: m/z calcd for C89H157N5O41ꢀH:
1951.0281 [MꢀH]ꢀ; found: 1951.0280.
TLC with immunostaining: The novel GM2-epitope-containing ganglio-
sides, X1 and X2, and authentic gangliosides GM2, GM1, GD1a, GD1b,
and GT1b were prepared from bovine brain gangliosides as described
elsewhere.[3,22] These gangliosides and the synthesized X1 were layered
on precoated Silica Gel 60 plates (Merck, Darmstadt, Germany). The
plates were developed with a solvent system of chloroform/methanol/
12 mm magnesium chloride in water (5:4:1, by volume), dipped in n-
hexane containing 0.4%
> polyisobutylmethacrylate for 1 min, then
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dried under an air stream. The TLC plate was overlaid with serum from
the patient with ALS-like disorder (1:50 dilution with phosphate-buffered
saline/0.5% casein) and kept at 48C overnight. The plates were washed
and overlaid with peroxidase-conjugated anti-human m-chain-specific an-
tibodies (Dako, Glostrup, Denmark; 1:250 dilution with phosphate-buf-
fered saline/0.5% casein), kept at 208C for 2 h, then washed. Binding ac-
tivities were made visible with 4-chloro-1-naphtol.
596
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Chem. Eur. J. 2011, 17, 588 – 597

Synthesis of a Hybrid Ganglioside X1
FULL PAPER
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Received: July 30, 2010
Published online: November 5, 2010
Chem. Eur. J. 2011, 17, 588 – 597
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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