First Total Synthesis of Ganglioside GAA-7 from Starfish Asterias amurensis versicolor

Article abstract of DOI:10.1002/ejoc.201500606

The first total synthesis of neuritogenic ganglioside GAA-7 was achieved using the glucosyl ceramide (Glc-Cer) cassette approach. The stereocenter triad within the ceramide moiety of the target molecule was efficiently established from D-lyxose. The assem

Full text of DOI:10.1002/ejoc.201500606

FULL PAPER
DOI: 10.1002/ejoc.201500606
First Total Synthesis of Ganglioside GAA-7 from Starfish Asterias amurensis
versicolor[‡]
Hideki Tamai,[a,b] Akihiro Imamura,[a] Junya Ogawa,[a] Hiromune Ando,*[a,b]
Hideharu Ishida,[a] and Makoto Kiso*[a,b]
Keywords: Total synthesis / Natural products / Glycosylation / Glycolipides / Gangliosides / Echinoderms
The first total synthesis of neuritogenic ganglioside GAA-7
was achieved using the glucosyl ceramide (Glc-Cer) cassette
approach. The stereocenter triad within the ceramide moiety
ride moiety, followed by global deprotection. By using the
most suitable Glc-Cer cassette, the target molecule was suc-
cessfully synthesized. In vitro evaluation indicated that the
synthesized GAA-7 and its glycan moiety both showed
strong neuritogenic activity towards neuron-like rat adrenal
pheochromocytoma (PC12) cells in the presence of neurite
of the target molecule was efficiently established from
D-
lyxose. The assembly of the ceramide moiety was followed
by glycosylation with glucosyl donors to give Glc-Cer cas-
settes, which underwent conjugation with the oligosaccha- growth factor.
Thus, it is very important to supply homogeneous EGs for
Introduction
molecular-level mechanism studies. Therefore, the synthesis
of EGs and their glycan parts have been investigated inten-
sively by many research groups, including our group.[4–8]
Recently, we have been tackling the synthesis of LLG-3 and
GAA-7, which are highly potent neuritogenic EGs that
have 8-O-methyl sialic acids as the common monosaccha-
ride residues. In 2011, we demonstrated that synthetic LLG-
3 potentiates the neuritogenesis of PC-12 cells in the pres-
ence of NGF.[9]
Ganglioside GAA-7 (1) was found in the starfish Asterias
amurensis versicolor Sladen in 1993.[10] GAA-7 has a unique
glycan structure linked to a cerebroside ceramide moiety,
which is composed of an α-hydroxy fatty acid and an unsat-
urated phytosphingosine base (Figure 1). Glycan moiety 2,
which features the terminal branch of the 8-O-Me-N-Gc-
sialic acid (Gc = glycolyl) residue stemming from N-acetyl-
In mammals, sialic-acid-containing glycosphingolipids
called gangliosides are crucial in many biological processes,
including cell-type-specific adhesion, the binding of bacte-
rial toxins, and the entry of pathogens into host cells.[1] Di-
verse gangliosides have also been found in echinoderms
(e.g., starfish and sea cucumbers), and their unique struc-
tures have been identified. It was found that echinoderma-
tous gangliosides (EGs) show neuritogenic activity towards
neuron-like rat adrenal pheocromocytoma (PC12) cells in
the presence of the nerve growth factor (NGF).[2] Because
the activities of EGs are comparable or superior to that of
mammalian ganglioside GM1, which has been used in the
development of a drug for Alzheimer’s disease,[3] EGs are
thought to have great potential as a source for neurothera-
peutics. Most EGs contain partially modified sialic acid res-
idues and/or repeated sequences of sialic acid, both of
which are unique to EGs. In addition, the structures of the
ceramide lipid moieties in EGs are composed of various
pairs of fatty acids and sphingoid bases. Due to the struc-
tural diversity of EGs, it is not understood which structures
are responsible for the neuritogenic activity, and neither is
the mechanism by which EGs potentiate neurite outgrowth.
[‡] Synthetic Studies on Sialoglycoconjugates, 160. Part 159:
Ref.[12f]
[a] Department of Applied Bioorganic Chemistry, Gifu University,
1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan
E-mail: hando@gifu-u.ac.jp
kiso@gifu-u.ac.jp
http://www1.gifu-u.ac.jp/~kassei1/top%20eng%20frame.html
[b] Institute for Integrated Cell-Material Sciences (WPI-iCeMS),
Kyoto University,
Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500606.
Figure 1. Structures of ganglioside GAA-7 (1) and its glycan moi-
ety (2).
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galactosamine residue, has recently been synthesized as a the C-3 and C-6 hydroxy groups was expected to enhance
glycoside by our research group through the double sialyl- the reactivity of Glc donor 5 and also the 4-OH in Glc-Cer
ation of a GalN-Gal acceptor with an 8-O-Me-N-Troc cassette 4. For ceramide construction, the ceramide moiety
(Troc = 2,2,2-trichloroethoxycarbonyl) sialyl donor, and was retrosynthetically disconnected at the amide bond at C-
coupling of the tetrasaccharyl donor with a Glc acceptor.[11] 2, the C-5–C-6 bond, and the homoallylic carbon–carbon
Based on the successful synthesis of the glycan part of bond at C-11, giving α-hydroxy fatty acid 7, lactol 8, and
GAA-7, in this paper we report the synthesis of the entire alkyl bromide 9 for the Wittig reaction, and alkenyl brom-
structure of GAA-7, and examine the neuritogenic activity ide 10 for Grignard coupling.
of the synthetic GAA-7 and its glycan part.
The synthesis of the ceramide moiety started with the
construction of the inner segment of the phytosphingosine
moiety (Scheme 2). To establish the stereocenter triad at C-
2, C-3, and C-4 (2S,3S,4R), d-lyxose was converted into a
Results and Discussion
Taking advantage of the efficacy of the glucosyl ceramide suitably protected lactol (8) by following a reported pro-
(Glc-Cer) cassette approach,[12] target molecule 1 was dis- cedure.[13] Next, lactol 8 underwent Wittig olefination with
connected at the Galβ(1,4)Glc linkage to give tetrasaccharyl the phosphonium ylid obtained by treatment of 11 (pre-
donor 3 and Glc-Cer cassette 4 (Scheme 1). Because the pared from 9) with LHMDS in THF. Subsequent hydrogen-
glycosylation at the C-4 hydroxy group of a glucosyl ac- ation gave enantiomeric alcohol 12 in 82% yield. The
ceptor with tetrasaccharyl donor 3 gave a high yield in the hydroxy group at C-2 was then substituted with an azido
synthesis of the glycan moiety (i.e., 2),[11] donor 3 was also group in a Mitsunobu reaction[14] using DPPA, Ph3P, and
used for this total synthesis. Glc-Cer cassette 4 was designed DEAD. This gave 13, which has the desired stereocenters.
as a coupling partner for 3 based on our previous work on Finally, removal of the pivaloyl group with DIBAL-H at
the total syntheses of complex gangliosides.[9,12] Then, 4 –80 °C,[15] and oxidation with Dess–Martin periodinane,[16]
was disconnected at the glycoside bond to give glucosyl do- provided aldehyde 14 (72% over two steps). Although we
nor 5 and ceramide acceptor 6. In keeping with previously examined Zemplén conditions (NaOMe in MeOH) for the
reported ganglioside syntheses, protecting groups for the removal of the pivaloyl group, the reaction was sluggish,
hydroxy groups of the Glc donor were chosen so as to and the TBDPS group at the C-1 position was also partially
achieve a high-yielding, stereoselective glycosylation of cer- cleaved.
amide 6, and to obtain a high yield in the final coupling
To construct the counterpart of aldehyde 14, alkynyl tos-
between Glc-Cer cassette 4 and oligosaccharyl donor 3. ylate 16 was prepared from known alkynyl alcohol 15[17] in
Thus, a pivaloyl group was used for 2-OH protection to 95% yield, and was transformed quantitatively into Z-ole-
impart β-selectivity, and a TBS group was used at O-4 to fin 17 by hydrogenation with Lindlar catalyst (0.1 equiv.)
temporarily protect the 4-OH group, and achieve a high- and quinoline (1.0 equiv.) (Scheme 3). Subsequently, alkenyl
yielding glycosylation. The use of PMB groups to protect tosylate 17 was treated with LiBr (3.0 equiv.) in acetone un-
Scheme 1. Retrosynthetic analysis of ganglioside GAA-7; LG = leaving group, TBS = tert-butyldimethylsilyl, PMB = p-methoxybenzyl,
TBDPS = tert-butyldiphenylsilyl, Piv = pivaloyl.
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Total Synthesis of Ganglioside GAA-7
Scheme 2. Preparation of phytosphingosine derivative 14. Reagents and conditions: a) (i) 11, LHMDS, THF, 0 °C to room temp., (ii) H2,
Pd/C, EtOAc, room temp., 82% (two steps); b) DPPA, DEAD, PPh3, THF, 0 °C to room temp., 79%; c) DIBAL-H, toluene, CH2Cl2,
–80 °C; d) Dess–Martin periodinane, NaHCO3, CH2Cl2, room temp., 72% (two steps). LHMDS = lithium bis(trimethylsilyl)amide, DPPA
= diphenylphosphoryl azide, DEAD = diethyl azodicarboxylate, DIBAL-H = diisobutylaluminum hydride.
Scheme 3. Synthesis of GAA-7 ceramide acceptor 24. Reagents and conditions: a) 14, Et2O, 0 °C to r.t.; b) PPh3, THF, H2O, room temp.;
c) 7, EDC·HCl, CH2Cl2, room temp.; d) phenyl chlorothionocarbonate, DMAP, pyridine, 40 °C; e) nBu3SnH, AIBN, toluene, 100 °C,
30% (five steps); Ts = p-tolylsulfonyl, AIBN = 2,2Ј-azobis(isobutyronitrile), EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,
TBAF = tetra-n-butylammonium fluoride, DMAP = 4-(dimethylamino)pyridine.
der reflux to give alkenyl bromide 10[18] quantitatively. C-1 hydroxy group was selectively deprotected with TBAF
Upon treatment with Mg and a catalytic amount of iodine, (1.5 equiv.) in the presence of AcOH (2.0 equiv.) to give cer-
this bromide was then converted into Grignard reagent 18, amide acceptor 24 in 94% yield.
the coupling partner for aldehyde 14. We then carried out
With phytoceramide 24 in hand, we synthesized the
the coupling reaction with aldehyde 14 (1.0 equiv.). This glucosyl ceramide cassette for the final conjugation with the
was followed by amide formation with α-hydroxy fatty acid oligosaccharyl donor (Scheme 4). Glucosyl imidate donor
derivative 7,[6] thiocarbonylation, and radical reduction 26 was prepared from known phenylthioglycoside 25.[9]
with nBu3SnH (10.0 equiv.) and AIBN (0.05 equiv.) to give Treatment of 25 with NBS in acetone/H2O[19] at room tem-
phytoceramide 23 in 30% yield over five steps. Finally, the perature, and subsequent imidoylation of the resulting 1-
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Scheme 4. Preparation of Glc-Cer acceptor 28 and examination of the cassette approach. PMB = p-methoxybenzyl, NBS = N-bromosuc-
cinimide, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, MS = molecular sieves, TMSOTf = trimethylsilyl trifluoromethanesulfonate.
OH with CCl3CN and DBU[20] in CH2Cl2 at 0 °C gave 26 the PMB group was retained at C-6 in glucosyl donor 33,
in 90% yield over two steps. Then, the glycosylation of ac- in which the C-2 and C-3 hydroxy groups were protected
ceptor 24 with donor 26 was carried out under mild condi- with acetyl groups (Scheme 5).
tions to give glucosyl ceramide 27 in high yield. Subsequent
An analogous triacetylated glucosyl donor 36 was also
removal of the TBS group at O-4 yielded glucosyl ceramide used for comparison with 33. Donors 33 and 36 were de-
cassette 28 in high yield, and this was then subjected to the rived from 31[23] in a straightforward manner. For donor 33,
final glycosylation. Similarly to previously reported gangli- the PMB ether at C-6 was established by a reductive ring-
oside syntheses based on the Glc-Cer cassette approach, tet- opening of the 4,6-anisylidene group,[24] which was followed
rasaccharyl donor 3 and cassette 28 were coupled with by silylation to give 32 in high yield. Then, 32 was converted
TMSOTf (0.1 equiv.) at –10 °C, and the reaction was con- into glucosyl imidate 33. For donor 36, compound 31 was
tinued at 0 °C for 1 h. This gave the protected GAA-7 (i.e., transformed into known intermediate 34,[25] which was then
29) in 54% yield in a stereoselective manner, with none of silylated to give triacetylated thioglycoside 35. Unlike 32,
the orthoester analog formed. Considering that the cou- subsequent hydroxylation of 35 generated a mixture of 1-
pling yield with the glucosyl acceptor [2-(trimethylsilyl)ethyl OH and 2-OH derivatives. Therefore, in the next step, the
glycoside analog of 28] in the synthesis of the GAA-7 gly- mixture was acetylated, and the product was treated with
can moiety (i.e., 2) was 73%,[11] ganglioside framework 29 hydrazine acetate to give the hemiacetal, which was then
was generated in an acceptable yield, which suggested that converted into trichloroacetimidate 36. Next, phytoceramide
this glucosyl cassette approach would be a viable synthetic acceptor 24 was treated with glucosyl donors 33 and 36, and
route. However, cleavage of the PMB groups failed unex- this was followed by deprotection of the C-4 hydroxy group
pectedly in the next step. We examined various reaction to give Glc-Cer cassettes 38 and 40, respectively.
conditions for the removal of the PMB groups [(i) TFA (tri-
Consistent with our initial speculation, reducing the
fluoroacetic acid), CH2Cl2; (ii) TFA, anisole, CH2Cl2; number of PMB groups in the Glc-Cer cassette decreased
(iii) DDQ
(2,3-dichloro-5,6-dicyano-1,4-benzoquinone), the yield of the glycosylation with tetrasaccharyl donor 3
CH3CN, H2O;[21] (iv) SnCl4, PhSH, CH2Cl2[22]]. Under all (Scheme 6). Taking the results for 28 (glycosylation yield:
the reaction conditions tested, an unknown by-product was 50%) together with the results for 38 and 40, a decrease by
generated along with the desired product (i.e., 30), giving one in the number of PMB groups caused a 50% decrease
an inseparable mixture of reaction products.
in the glycosylation yields.
To solve the deprotection problem, we reconsidered the
We used the protected GAA-7 (i.e., 41) to examine global
design of the glucosyl donor. Because the electron-donating deprotection. However, we again failed to obtain ganglio-
properties of the PMB group are crucial for increasing the side GAA-7 because of the generation of an unknown by-
reactivity of the C-4 hydroxy group in the Glc-Cer cassette, product during the cleavage of the PMB groups that could
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Total Synthesis of Ganglioside GAA-7
Scheme 5. Synthesis of Glc-Cer acceptors 38 and 40. Reagents and conditions: a) NaBH3CN, HCl (2 m in Et2O), THF, MS (3 Å), 0 °C;
b) TBSOTf, 2,6-lutidine, CH2Cl2, 0 °C to room temp.; c) NBS, acetone, H2O, room temp.; d) CCl3CN, DBU, CH2Cl2, 0 °C; e) TsOH·H2O,
MeOH, 0 °C, 89%; f) AcCl, pyridine, CH2Cl2, –40 °C to room temp., 88%; g) TBSCl, pyridine, 40 °C, 91%; h) Ac2O, pyridine, room
temp.; i) hydrazine acetate, THF, room temp.; j) TBAF, AcOH, THF, room temp., PMP = p-methoxyphenyl.
Scheme 6. Final coupling and global deprotection to give the target molecule.
not be separated from the desired product. As a result, tri-
acetyl Glc-Cer 40 was used for the synthesis of the target
compound. To minimize the loss of valuable oligosaccharyl
donor 3, 3.0 equiv. of 40 was used in the reaction to give
42, and this resulted in an increase in the glycosylation yield
to 25%. Finally, 42 was treated with TFA at 0 °C, followed
by basic hydrolysis of ester groups, to give ganglioside
GAA-7 (1) in 77% yield over two steps (Scheme 6).
The synthesized GAA-7 (i.e., 1) and its glycan moiety (2)
were evaluated in terms of neuritogenic activity towards
PC-12 cells. The results are summarized in Figure 2. Con-
sistent with the results obtained with natural GAA-7,[2] the
Figure 2. Neurite outgrowth evaluation. PC-12 cells were incubated
with or without the synthesized GAA-7 (1) or the glycan moiety
(2) (10 nm each) in the presence of NGF (5 ng/mL) for 5 d. (a) Neu-
rites were counted, and the total length per cell was calculated.
(b) The number of neurites that were longer than twice the cell-
body diameter was counted per cell. Results are shown as average
synthesized GAA-7 (1) potentiated the neurite outgrowth
in the presence of NGF (5.0 ng/mL). GAA-7 (1) at 10 nm
increased the total length of neurites by around 1.5 times,
compared to the length without added GAA-7 (Figure 2,
a). In particular, the number of neurites with a length of total length or number of neurites per cell Ϯ the standard error.
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2 h at 0 °C, and then for a further 6 h at ambient temperature. The
progress of the reaction was monitored by TLC (n-hexane/EtOAc,
2:1). After the consumption of starting material was confirmed, the
reaction was quenched with satd. aq. NH4Cl, and the mixture was
extracted with EtOAc. The organic layer was successively washed
with H2O, satd. aq. NaHCO3, and brine, dried with Na2SO4, and
concentrated. The resulting residue was purified by silica gel col-
umn chromatography (n-hexane/EtOAc, 8:1) to give an E/Z mix-
ture as a colorless syrup.
more than twice the cell-body diameter was significantly
increased by exogenous GAA-7 (1) (Figure 2, b). More
interestingly, the glycan moiety (2) showed activity similar
to that of GAA-7, which indicates that the glycan moiety is
crucial for neurite outgrowth activation.
Conclusions
Next, Pd/C (5% on activated carbon; 1.4 g) was added to a solution
of the E/Z mixture in EtOAc (190 mL). The mixture was stirred
for 12 h under a hydrogen atmosphere, and the progress of the reac-
tion was monitored by TLC (n-hexane/EtOAc, 3:1). Next, the reac-
tion mixture was diluted with EtOAc, and filtered through a pad
of Celite. The filtrate was concentrated, and the residue was puri-
fied by silica gel column chromatography (n-hexane/EtOAc, 8:1) to
give compound 12 (7.70 g, 82%) as a colorless syrup. [α]D = –6.8
The first total synthesis of ganglioside GAA-7 (1) was
achieved, using tetrasaccharyl donor 3 and Glc-Cer cassette
38 as the key building blocks to construct the glycolipid
framework of the target molecule. Furthermore, evaluation
of the neuritogenic activity of the synthesized molecules 1
and 2 revealed that the glycan moiety of GAA-7 is essential
for the potentiation of neurite outgrowth in the presence of
NGF.
1
(c = 1.0, CHCl3). H NMR (500 MHz, CDCl3): δ = 7.69–7.36 (m,
10 H, Ar), 4.17 (dd, J2,3 = 2.7, J3,4 = 6.4 Hz, 1 H, 3-H), 4.12 (m,
1 H, 4-H), 4.04 (t, J9,10 = 6.6 Hz, 2 H, 10-H), 3.72–3.65 (m, 3 H,
1a-H, 1b-H, 2-H), 2.35 (d, 1 H, OH), 1.74–1.21 (m, 16 H, 5 CH2,
2 Me), 1.19 (s, 9 H, tBu), 1.06 (s, 9 H, tBu) ppm. 13C NMR
(125 MHz, CDCl3): δ = 178.6, 135.6, 135.6, 133.3, 129.8, 129.7,
127.7, 127.7, 107.7, 77.4, 76.4, 69.8, 65.2, 64.4, 38.7, 29.8, 29.7,
29.5, 29.4, 29.2, 28.6, 27.3, 27.2, 26.8, 26.7, 25.9, 25.1, 19.2 ppm.
HRMS (ESI): calcd. for C34H52O6Si [M + Na]+ 607.3425; found
607.3426.
Experimental Section
General Remarks: All reactions were carried out under a positive
pressure of argon, unless otherwise noted. All chemicals were pur-
chased from commercial suppliers, and were used without further
purification, unless otherwise noted. Molecular sieves were pur-
chased from Wako Chemicals Inc. (Miyazaki, Japan) and dried at
300 °C for 2 h in a muffle furnace before use. Reaction solvents
were dried with molecular sieves, and used without purification.
TLC analysis was carried out on Merck TLC plates (silica gel
60F254 on glass plate). Compounds were detected either by expo-
sure to UV light (253.6 nm) or by soaking in H2SO4 (10% solution
in ethanol) followed by heating. Silica gel (80 mesh and 300 mesh;
Fuji Silysia Co., Aichi, Japan) was used for flash column
chromatography. The quantity of silica gel was usually 100 to 200
times the weight of the crude sample. Solvent systems for
chromatography are specified as v/v ratios. Evaporation and con-
centration were carried out in vacuo. 1H and 13C NMR spectra
were recorded with 500 and 800 MHz spectrometers (Biospin
(2S,3S,4R)-2-Azido-1-O-tert-butyldiphenylsilyl-3,4-O-isopropyl-
idene-10-O-pivaloyl-1,3,4,10-decanetetrol (13): DEAD (40% solu-
tion in toluene; 421 μL, 1.07 mmol) and PPh3 (281 mg, 1.07 mmol)
were added to a solution of compound 12 (569 mg, 0.972 mmol) in
THF. The mixture was stirred for 10 min at 0 °C, then DPPA
(231 μL, 1.07 mmol) was added. The mixture was then stirred for
1 h at 0 °C, and the reaction was monitored by TLC (n-hexane/
EtOAc, 3:1). The reaction mixture was concentrated, and the resi-
due was purified by silica gel column chromatography (n-hexane/
EtOAc, 8:1) to give compound 13 (471 mg, 79%) as a colorless
1
syrup. [α]D = +8.3 (c = 1.0, CHCl3). H NMR (500 MHz, CDCl3):
δ = 7.73–7.34 (m, 10 H, Ar), 4.12 (m, 1 H, 4-H), 4.06 (t, J9,10
=
1
AVANCE III, Bruker, Billerica, MA, USA). Chemical shifts in H
6.6 Hz, 2 H, 10-H), 4.03 (dd, Jgem = 10.9, J1a,2 = 2.6 Hz, 1 H, 1a-
H), 3.95 (dd, J2,3 = 9.8, J3,4 = 5.5 Hz, 1 H, 3-H), 3.42 (td, 1 H, 2-
H), 1.66–1.39 (m, 10 H, 5 CH2), 1.27 (2 s, 6 H, 2 Me), 1.20 and
1.08 (2 s, 18 H, 2 tBu) ppm. 13C NMR (125 MHz, CDCl3): δ =
178.6, 135.7, 135.6, 133.0, 132.9, 129.8, 129.7, 129.4, 128.9, 128.6,
127.7, 127.7 127.5, 108.2, 77.7, 75.2, 65.2, 64.4, 61.7, 38.7, 29.3,
29.2, 28.6, 28.1, 27.2, 26.9, 26.7, 26.3, 25.9, 25.7, 25.1, 19.1 ppm.
HRMS (ESI): calcd. for C34H51N3O5Si [M + Na]+ 632.3490; found
632.3491.
NMR spectra are expressed in ppm (δ) relative to the Me4Si signal,
adjusted to δ = 0.00 ppm. Data are presented as follows: chemical
shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet,
dd = doublet of doublets, td = triplet of doublets, m = multiplet
and/or multiple resonances), integration, coupling constant in
Hertz (Hz), and position of the corresponding proton. COSY
methods were used to confirm the NMR peak assignments. High-
resolution mass spectra (ESI-TOF) were obtained with a mass
spectrometer (micrOTOF, Bruker). Optical rotations were mea-
(4R,5S)-4-{2,2-Dimethyl-5-[(1S)-1-azido-2-tert-butyldiphenylsilyl-
oxyethyl]-1,3-dioxolan-4-yl}hexanal (14): DIBAL-H (1 m solution in
toluene; 5.9 mL, 5.84 mmol) was added to a solution of compound
13 (1.62 g, 2.66 mmol) in CH2Cl2 (27 mL) at –80 °C. The progress
of the reaction was monitored by TLC (n-hexane/EtOAc, 3:1). The
mixture was stirred for 30 min at –80 °C, then the reaction was
quenched with satd. aq. NH4Cl, and satd. aq. sodium potassium
tartrate was added. The mixture was stirred vigorously for 30 min,
then it was extracted with CHCl3. The organic layer was success-
ively washed with H2O and brine, dried with Na2SO4, and concen-
trated. The resulting residue was purified by silica gel column
chromatography (n-hexane/EtOAc, 6:1) to give a hydroxy deriva-
tive.
sured with
(Kyoto, Japan)].
a high-sensitivity polarimeter [SEPA-300, Horiba
(2R,3S,4R)-1-O-tert-Butyldiphenylsilyl-3,4-O-isopropylidene-10-
O-pivaloyl-1,2,3,4,10-decanepentol (12): Triphenylphosphine (45 g,
171 mmol) was added to a solution of 5-bromopentyl pivaloylate
(9; 14.2 g, 56.7 mmol) in toluene (200 mL). The mixture was stirred
for 2 d under reflux, then it was diluted with toluene, and the super-
natant was decanted. The resulting residue was dried in vacuo for
6 h.
The residue was then suspended in THF (60 mL), the mixture was
cooled to 0 °C, and LHMDS (1.0 m solution in THF; 57 mL,
57 mmol) was added. The mixture was stirred for 90 min, then a
solution of lactol 8 (8.10 g, 18.9 mmol) in THF (60 mL) was added
to the stirred mixture at 0 °C. The reaction mixture was stirred for
This hydroxy derivative was exposed to high vacuum for 10 h, then
it was dissolved in CH2Cl2 (27 mL). Dess–Martin periodinane
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Total Synthesis of Ganglioside GAA-7
(1.74 g, 4.11 mmol) and NaHCO3 (345 mg, 4.11 mmol) were added
to the solution at ambient temperature, and the reaction mixture
was stirred for 25 min at ambient temperature. The progress of the
reaction was monitored by TLC (n-hexane/EtOAc, 4:1). Then, the
reaction mixture was extracted with Et2O. The organic layer was
successively washed with H2O and brine, dried with Na2SO4, and
concentrated. The resulting residue was purified by silica gel col-
umn chromatography (n-hexane/EtOAc, 6:1) to give compound 14
EtOAc. The organic phase was successively washed with H2O and
brine, and dried with Na2SO4. The solution was concentrated to
1
give compound 10[18] (1.17 g, quant.) as a colorless oil. H NMR
(500 MHz, CDCl3): δ = 5.53 (m, 1 H, 4-H), 5.35 (m, 1 H, 3-H),
3.36 (t, J1,2 = 7.2 Hz, 2 H, 1-H), 2.63 (near q, J2,3 = 7.4 Hz, 2 H,
2-H), 2.03 (q, J3,4 = 7.2 Hz, 2 H, 5-H), 1.37–1.27 (m, 12 H, 6 CH2),
0.88 (t, 3 H, Me) ppm. 13C NMR (125 MHz, CDCl3): δ = 133.2,
125.7, 32.5, 31.9, 30.8, 29.5, 29.4, 29.3, 27.4, 22.7, 14.1 ppm.
(1.04 g, 72% over two steps) as a colorless syrup. [α]D = +31.6 (c
(2S,3S,4R,13Z)-2-[(R)-2-(Benzoyloxy)tetracosanoylamino]-1-O-
tert-butyldiphenylsilyl-3,4-O-isopropylidenedocos-13-ene-1,3,4-triol
(23): Magnesium foil (78 mg, 3.21 mmol) and I2 (10 mg,
0.040 mmol) were suspended in Et2O (10 mL). The mixture was
stirred for 30 min under reflux, and then a solution of bromoalkene
10 (594 mg, 2.41 mmol) in Et2O (10 mL) was added to the suspen-
sion dropwise at 35 °C. The reaction mixture was stirred for a fur-
ther 60 min under reflux. The mixture was then added dropwise to
a solution of aldehyde 14 (267 mg, 0.510 mmol) in Et2O (10 mL)
at 0 °C. The progress of the reaction was monitored by TLC (n-
hexane/EtOAc, 5:1). The mixture was stirred for 2 h at ambient
temperature, then the reaction was quenched with satd. aq. NH4Cl,
and the mixture was filtered through a pad of Celite. The mixture
was extracted with Et2O, and the organic phase was successively
washed with H2O and brine, dried with Na2SO4, and concentrated.
1
= 0.6, CHCl3). H NMR (500 MHz, CDCl3): δ = 9.77 (t, J9,CHO
=
1.8 Hz, 1 H, CHO), 7.73–7.37 (m, 10 H, Ar), 4.11 (m, 1 H, 4-H),
4.03 (dd, Jgem = 10.8, J1a,2 = 2.6 Hz, 1 H, 1a-H), 3.95 (dd, J2,3
=
9.8, J3,4 = 5.5 Hz, 1 H, 3-H), 3.85 (dd, J1b,2 = 6.9 Hz, 1 H, 1b-H),
3.40 (td, 1 H, 2-H), 2.45 (td, J8,9 = 7.3 Hz, 2 H, 9-H), 1.70–1.37
(m, 8 H, 4 CH2), 1.27 (2 s, 6 H, 2 Me), 1.08 (s, 9 H, tBu) ppm. 13
C
NMR (125 MHz, CDCl3): δ = 202.6, 135.7, 135.6, 133.1, 132.9,
129.8, 129.7, 127.8, 127.7, 108.2, 77.6, 75.2, 65.2, 61.6, 43.8, 29.3,
29.1, 28.1, 26.7, 26.2, 25.7, 22.0, 19.1 ppm. HRMS (ESI): calcd. for
C29H41N3O4Si [M + Na]+ 546.2759; found 546.2757.
1-O-(p-Tolylsulfonyl)dodec-3-yn-1-ol (16): p-Toluenesulfonyl chlor-
ide (2.40 g, 11.8 mmol) was added to a solution of dodec-3yn-1-ol
15 (1.80 g, 9.87 mmol) in pyridine/CH2Cl2 (1:4; 33 mL) at ambient
temperature. The progress of the reaction was monitored by TLC
(n-hexane/EtOAc, 7:1). The mixture was stirred for 4 h at ambient
temperature, then it was coevaporated with toluene, and extracted
with EtOAc. The organic layer was successively washed with HCl
(2 m), H2O, and brine, dried with Na2SO4, and concentrated. The
residue was purified by silica gel column chromatography (n-hex-
ane/Et2O, 10:1) to give compound 16 (3.15 g, 95%) as a colorless
oil. 1H NMR (500 MHz, CDCl3): δ = 7.80 and 7.35 (2 d, 4 H, Ar),
4.06 (t, J = 7.3 Hz, 2 H, 1-H), 2.52 (m, 2 H, 2-H), 2.45 (s, 3 H,
Me), 2.07 (m, 2 H, 5-H), 1.43–1.26 (m, 12 H, 6 CH2), 0.88 (s, 3 H,
Me) ppm. 13C NMR (125 MHz, CDCl3): δ = 144.8, 133.2, 129.8,
127.9, 83.0, 73.8, 68.3, 31.8, 29.2, 29.1, 28.8, 28.7, 22.6, 21.6, 19.8,
18.6, 14.1 ppm. HRMS (ESI): calcd. for C19H28O3S [M + Na]+
359.1651; found 359.1651.
The resulting crude material (compound 19) was dried in vacuo for
6 h, and then it was dissolved in a mixture of THF (16 mL) and
H2O (0.8 mL). Next, PPh3 (820 mg, 3.20 mmol) was added, and
the mixture was stirred for 18 h at ambient temperature. The pro-
gress of the reaction was monitored by TLC (n-hexane/EtOAc, 5:1,
developed twice). Then, the reaction mixture was concentrated, and
the residue was purified by silica gel column chromatography (n-
hexane/EtOAc, 7:1) to give amino derivative 20.
This compound was dissolved in CH2Cl2 (16 mL), and EDC·HCl
(154 mg, 0.802 mmol) and compound 7 (392 mg, 0.802 mmol) were
added to the solution at ambient temperature. The progress of the
reaction was monitored by TLC (n-hexane/EtOAc, 8:1). The mix-
ture was stirred for 2 h, and then it was diluted and extracted with
Et2O. The organic layer was successively washed with H2O and
brine, dried with Na2SO4, and concentrated. The residue was puri-
fied by silica gel column chromatography (n-hexane/EtOAc,
15:1Ǟ8:1Ǟ5:1) to give amide derivative 21 (326 mg, 0.287 mmol),
which was dried under high vacuum.
(3Z)-1-O-(p-Tolylsulfonyl)dodec-3-en-1-ol (17): Lindlar catalyst
(0.502 g, 0.237 mmol) and quinoline (0.56 mL, 4.75 mmol) were
added to a solution of compound 16 (1.60 g, 4.75 mmol) in n-hex-
ane (47 mL). The flask containing the mixture was evacuated and
filled with H2 gas, and this process was repeated three times. The
mixture was then stirred at ambient temperature under a hydrogen
atmosphere, and the progress of the reaction was monitored by
TLC (n-hexane/EtOAc, 12:1). The mixture was stirred for 2 h, then
it was filtered through a pad of Celite, and the filter residue was
washed with EtOAc. The filtrate was successively washed with HCl
(2 m), H2O, and brine. The organic layer was dried with Na2SO4,
and concentrated to give compound 17 (1.61 g, quant.) as a color-
The dried compound was dissolved in pyridine (6 mL). PhOC(=S)
Cl (115 μL, 0.860 mmol) and DMAP (7.0 mg, 57.4 μmol) were
added to the solution, and the mixture was stirred for 2 h at 40 °C.
The reaction was monitored by TLC (n-hexane/EtOAc, 8:1). The
reaction mixture was coevaporated with toluene, and the residue
was roughly purified by silica gel chromatography (n-hexane/
EtOAc, 100:1Ǟ20:1). The resulting material (compound 22) was
dried in vacuo for 6 h.
1
less oil. H NMR (500 MHz, CDCl3): δ = 7.79 and 7.34 (2 d, 4 H,
Ar), 5.48 (m, J3,4 = 10.8, J4,5 = 6.9 Hz, 1 H, 4-H), 5.21 (m, J2,3
=
This compound was then dissolved in toluene (29 mL), and
nBu3SnH (0.386 mL, 1.43 mmol) and AIBN (4.7 mg, 0.028 mmol)
were added to the resulting solution at ambient temperature. The
mixture was stirred for 2 h at 100 °C, then it was concentrated, and
the residue was purified by silica gel column chromatography (n-
hexane/EtOAc, 100:1Ǟ40:1Ǟ25:1Ǟ15:1) to give compound 23
(269 mg, 30% over five steps) as a colorless syrup. [α]D = +25.8 (c
6.9 Hz, 1 H, 3-H), 4.01 (t, J1,2 = 7.0 Hz, 2 H, 1-H), 2.45 (s, 3 H,
Me), 2.39 (near q, 2 H, 2-H), 1.95 (q, J5,6 = 6.9 Hz, 2 H, 5-H),
1.30–1.19 (m, 12 H, 6 CH2), 0.88 (s, 3 H, Me) ppm. 13C NMR
(125 MHz, CDCl3): δ = 144.6, 134.0, 129.8, 127.9, 122.5, 69.8, 31.9,
29.4, 29.2, 27.3, 27.1, 22.6, 21.6, 14.1 ppm. HRMS (ESI): calcd. for
C19H30O3S [M + Na]+ 361.1808; found 361.1807.
1
(3Z)-1-Bromododec-3-ene (10): Lithium bromide (0.604 g,
14.3 mmol) was added to a solution of compound 17 (1.61 g,
4.75 mmol) in acetone (47 mL). The mixture was then stirred for
3 h under reflux, and the progress of the reaction was monitored
by TLC (n-hexane/EtOAc, 12:1). The reaction mixture was filtered
through paper. The filtrate was then concentrated, and diluted with
= 0.3, CHCl3). H NMR (500 MHz, CDCl3): δ = 8.04–7.29 (m, 15
H, Ar), 6.52 (d, J2,NH = 9.8 Hz, 1 H, NH), 5.48 (t, J2,3 = 5.9 Hz,
1 H, 2Ј-H), 5.36 (m, 2 H, 13-H, 14-H), 4.35 (dd, J2,3 = 9.5, J3,4
=
5.4 Hz, 1 H, 3-H), 4.21 (near t, 1 H, 2-H), 4.13 (m, 1 H, 4-H), 3.95
(dd, Jgem = 10.1, J1a,2 = 2.3 Hz, 1 H, 1a-H), 3.62 (dd, J1b,2 = 2.4 Hz,
1 H, 1b-H), 2.07–1.96 (m, 6 H, 12-H, 15-H, 3Ј-H), 1.58–1.23 (m,
Eur. J. Org. Chem. 2015, 5199–5211
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5205

H. Tamai, A. Imamura, J. Ogawa, H. Ando, H. Ishida, M. Kiso
FULL PAPER
72 H, 33 CH2, 2 Me), 0.88 (2 t, 6 H, 2 Me), 0.81 (s, 9 H, tBu) ppm. oxy)tetracosanoylamino]-3,4-O-isopropylidenedocos-13-ene-1,3,4-
13C NMR (125 MHz, CDCl3): δ = 169.0, 165.2, 135.6, 135.4, 133.5, triol (27): Molecular sieves (4 Å, MS AW300; 0.28 g), compound
132.8, 132.8, 129.9, 129.9, 129.8, 129.7, 129.6, 129.1, 128.6, 127.7,
26 (83 mg, 109 μmol), and compound 24 (96 mg, 109 μmol) were
127.5, 108.1, 77.9, 75.5, 74.5, 63.7, 49.0, 32.1, 31.9, 31.9, 29.8, 29.8, suspended in CH2Cl2 (17.5 mL) at ambient temperature. The mix-
29.7, 29.6, 29.6, 29.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.3, 29.3, 29.3,
28.3, 27.2, 27.2, 27.1, 26.5, 26.3, 26.0, 24.9, 22.7, 19.0, 14.1 ppm.
HRMS (ESI): calcd. for C72H117NO6Si [M + Na]+ 1142.8542;
found 1142.8544.
ture was stirred for 30 min, then it was cooled to –10 °C. Then,
TMSOTf (1.0 μL, 5.5 μmol) was added to the suspension, which
was then stirred for 1 h at 0 °C. The progress of the reaction was
monitored by TLC (toluene/EtOAc, 6:1). Next, the reaction was
quenched with satd. aq. NaHCO3, the mixture was filtered through
a pad of Celite, and the pad was washed with CHCl3. The com-
bined filtrate and washings were washed with satd. aq. NaHCO3
and brine, dried with Na2SO4, and concentrated. The resulting resi-
due was purified by silica gel column chromatography (toluene/
(2S,3S,4R,13Z)-2-[(R)-2-(Benzoyloxy)tetracosanoylamino]-3,4-O-
isopropylidenedocos-13-ene-1,3,4-triol (24): Tetrabutylammonium
fluoride (1.0 m solution in THF; 233 μL, 0.233 mmol) was added
to a solution of compound 23 (131 mg, 0.117 mmol) and AcOH
(10 μL, 0.175 mmol) in THF (1.2 mL) at ambient temperature. The
mixture was stirred for 6 h, and the progress of the reaction was
monitored by TLC (n-hexane/Et2O, 5:2). The mixture was evapo-
rated. The residue was purified by silica gel column chromatog-
raphy (n-hexane/Et2O, 4:1Ǟ5:2) to give 24 (96 mg, 94%) as a color-
less syrup. [α]D = +2.2 (c = 1.1, CHCl3). 1H NMR (500 MHz,
CDCl3): δ = 8.08–7.47 (m, 5 H, Ar), 6.60 (d, J2,NH = 8.4 Hz, 1 H,
NH), 5.39–5.32 (m, 3 H, 13-H, 14-H, 2Ј-H), 4.19–4.15 (m, 2 H, 3-
H, 4-H), 4.09 (m, 1 H, 2-H), 3.87 (br. dt, 1 H, 1a-H), 3.65 (m, 1
H, 1b-H), 2.45 (br. s, 1 H, OH), 2.03–1.94 (m, 6 H, 12-H, 15-H,
3Ј-H), 1.61–1.13 (m, 72 H, 33 CH2, 2 Me), 0.88 (2 t, 6 H, 2 Me)
ppm. 13C NMR (125 MHz, CDCl3): δ = 169.9, 165.5, 133.6, 129.9,
129.8, 129.8, 129.2, 128.6, 108.2, 77.9, 77.7, 77.6, 74.8, 63.1, 50.2,
32.6, 31.9, 31.9, 31.9, 29.8, 29.8, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5,
29.4, 29.3, 29.3, 29.3, 29.2, 27.5, 27.2, 26.8, 25.2, 25.0, 22.7,
14.1 ppm. HRMS (ESI): calcd. for C56H99NO6 [M + Na]+
904.7365; found 904.7367.
EtOAc, 15:1) to give 27 (124 mg, 77%) as a colorless syrup. [α]D
=
–16.5 (c = 0.5, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 8.10–
6.81 (m, 13 H, Ar), 6.60 (d, J2,NH = 8.6 Hz, 1 H, NH), 5.37–5.32
(m, 2 H, 13-H, 14-H), 5.22 (t, J2,3 = 6.3 Hz, 1 H, 2Ј-H), 4.84 (t,
J1,2 = J2,3 = 8.2 Hz, 1 H, 2a-H), 4.56–4.34 (5 d, 5 H, 4 ArCH2O,
1a-H), 4.33 (dd, J2,3 = 8.9, J3,4 = 5.6 Hz, 1 H, 3-H), 4.06–3.97 (m,
3 H, 1a-H, 2-H, 4-H), 3.79–3.76 (m, 7 H, 2 OMe, 1b-H), 3.62 (dd,
Jgem = 10.3, J5,6a = 1.7 Hz, 1 H, 6aa-H), 3.44–3.32 (m, 4 H, 3a-H,
4a-H, 5a-H, 6ba-H), 2.03–1.90 (m, 6 H, 12-H, 15-H, 3Ј-H), 1.62–
1.15 (m, 72 H, 33 CH2, 2 Me), 1.11 (s, 9 H, tBu), 0.88 (2 t, 6 H, 2
Me), 0.79 (s, 9 H, tBu), –0.09 and –0.12 (2 s, 6 H, SiMe2tBu) ppm.
13C NMR (125 MHz, CDCl3): δ = 176.6, 169.6, 165.6, 159.3, 158.8,
133.3, 130.3, 130.3, 130.0, 129.9, 129.9, 129.9, 129.7, 129.3, 128.5,
128.4, 113.9, 113.5, 107.9, 101.2, 82.8, 77.9, 76.1, 75.4, 75.0, 73.9,
73.6, 73.0, 71.0, 69.3, 69.0, 55.2, 55.2, 49.2, 38.7, 32.6, 31.9, 31.9,
31.9, 29.8, 29.8, 29.7, 29.7, 29.7, 29.6, 29.5, 29.5, 29.4, 29.4, 29.3,
29.3, 29.2, 28.1, 27.3, 27.2, 27.0, 26.7, 25.9, 25.8, 25.2, 22.7, 17.9,
14.1, –3.8, –4.8 ppm. HRMS (ESI): calcd. for C89H147NO14Si [M
+ Na]+ 1505.0483; found 1505.0483.
4-O-tert-Butyldimethylsilyl-3,6-di-O-p-methoxybenzyl-2-O-pivalo-
yl-α-D-glucopyranosyl Trichloroacetimidate (26): NBS (98 mg,
546 μmol) was added to a solution of compound 25 (259 mg,
364 μmol) in acetone/H2O (95:5; 3.8 mL) at ambient temperature.
The progress of the reaction was monitored by TLC (n-hexane/
EtOAc, 5:1). After the consumption of starting material was con-
firmed, the reaction mixture was extracted with EtOAc. The or-
ganic layer was successively washed with satd. aq. Na2S2O3 and
brine, dried with Na2SO4, and concentrated. The residue was puri-
fied by silica gel column chromatography (CHCl3/acetone,
3:2Ǟ2:3) to give a hemiacetal intermediate, which was then dried
in vacuo.
3,6-Di-O-p-methoxybenzyl-2-O-pivaloyl-β-D-glucopyranosyl-
(1Ǟ1)-(2S,3S,4R,13Z)-2-[(R)-2-(benzoyloxy)tetracosanoylamino]-
3,4-O-isopropylidenedocos-13-ene-1,3,4-triol (28): Tetrabutylammo-
nium fluoride (1.0 m solution in THF; 269 μL, 0.269 mmol) was
added to a solution of compound 27 (133 mg, 0.0896 mmol) and
AcOH (7.7 μL, 134 μmol) in THF (2.0 mL) at ambient tempera-
ture. The mixture was stirred for 6 h, and the reaction was moni-
tored by TLC (n-hexane/EtOAc, 3:1). The mixture was evaporated.
The residue was purified by silica gel column chromatography (n-
hexane/EtOAc, 3:1) to give 28 (111 mg, 91%) as a colorless syrup.
[α]D = +4.3 (c = 0.5, CHCl3). 1H NMR (500 MHz, CDCl3): δ =
8.08–6.84 (m, 13 H, Ar), 6.50 (d, J2,NH = 8.6 Hz, 1 H, NH), 5.34
(m, 2 H, 13-H, 14-H), 5.25 (t, J2Ј,3Ј = 5.7 Hz, 1 H, 2Ј-H), 4.83 (dd,
J1,2 = 7.9, J2,3 = 9.1 Hz, 1 H, 2a-H), 4.61–4.44 (4 d, 4 H, 4 Ar-
CH2O), 4.40 (d, 1 H, 1a-H), 4.27 (dd, J2,3 = 8.7, J3,4 = 5.6 Hz, 1
H, 3-H), 4.07–3.98 (m, 3 H, 1a-H, 2-H, 4-H), 3.79 and 3.78 (2 s, 6
H, 2 OMe), 3.73 (dd, Jgem = 10.7, J1b,2 = 3.4 Hz, 1 H, 1b-H), 3.64
This compound was dissolved in CH2Cl2 (4.2 mL), and trichloro-
acetonitrile (549 μL, 5.47 mmol) and 1,8-diazabicyclo[5.4.0]undec-
7-ene (55 μL, 365 μmol) were added to the solution at 0 °C. The
progress of the reaction was monitored by TLC (n-hexane/EtOAc,
3:1). The reaction mixture was concentrated, and the residue was
purified by silica gel column chromatography (n-hexane/EtOAc,
5:1) to give 26 (44 mg, 94%) as an amorphous powder. [α]D = +64.6
1
(c = 1.1, CHCl3). H NMR (500 MHz, CDCl3): δ = 8.57 (s, 1 H,
(dd, Jgem = 10.1, J5,6a = 4.1 Hz, 1 H, 6aa-H), 3.57 (dd, J5,6b
=
C=NH), 7.25–6.80 (4 d, 8 H, 2 MeOC6H4CH2), 6.52 (d, J1,2
3.6 Hz, 1 H, 1-H), 5.12 (dd, J2,3 = 9.7 Hz, 1 H, 2-H), 4.77–4.41 (4
d, 4 H, 4 ArCH2O), 3.95 (td, J4,5 = 9.5, J5,6a = 2.9 Hz, J5,6b
=
5.6 Hz, 1 H, 6ba-H), 3.52–3.40 (m, 3 H, 3a-H, 4a-H, 5a-H), 2.55 (s,
1 H, OH), 2.04–1.89 (m, 6 H, 12-H, 15-H, 3Ј-H), 1.69–1.11 (m, 72
H, 33 CH2, 2 Me), 0.88 (2 t, 6 H, 2 Me) ppm. 13C NMR (125 MHz,
CDCl3): δ = 176.6, 169.5, 165.5, 159.5, 159.3, 133.4, 129.9, 129.9,
129.8, 129.6, 129.5, 129.3, 129.2, 128.5, 113.9, 113.9, 107.9, 101.2,
82.2, 77.7, 75.5, 74.8, 73.9, 73.8, 73.4, 72.7, 71.6, 70.1, 68.8, 55.2,
55.2, 49.2, 38.7, 31.9, 31.9, 31.8, 29.8, 29.8, 29.7, 29.6, 29.5, 29.5,
29.5, 29.4, 29.4, 29.3, 29.3, 29.3, 29.2, 28.0, 27.2, 27.2, 27.1, 26.6,
25.8, 25.1, 22.7, 14.1 ppm. HRMS (ESI): calcd. for C83H133NO14
[M + Na]+ 1390.9624; found 1390.9624.
=
2.5 Hz, 1 H, 5-H), 3.89 (t, J3,4 = 9.2 Hz, 1 H, 3-H), 3.83 (t, 1 H,
4-H), 3.78 and 3.77 (2 s, 6 H, OMe), 3.67–3.62 (m, 2 H, 6a-H, 6b-
H), 1.08 (s, 9 H, tBu), 0.83 (s, 9 H, tBu), 0.0 and –0.1 (2 s, 6 H,
SiMe2tBu) ppm. 13C NMR (125 Hz, CDCl3): δ = 177.5, 160.8,
160.1, 158.8, 130.4, 130.2, 129.1, 129.0, 128.1, 128.1, 125.3, 113.8,
113.7, 113.5, 93.7, 91.2, 79.5, 74.9, 74.3, 72.8, 72.8, 70.2, 68.1, 60.3,
55.2, 55.1, 38.7, 27.0, 25.9, 25.8, 21.4, 21.0, 18.0, 14.2, 14.1, –3.7,
–4.9 ppm. HRMS (ESI): calcd. for C35H50Cl3NO9Si [M + Na]+
784.2213; found 784.2213.
[Methyl 5-Acetoxyacetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-
4-O-tert-Butyldimethylsilyl-3,6-di-O-p-methoxybenzyl-2-O-pivalo- methyl-
D
-glycero-α-
D-galacto-2-nonulopyranosylonate]-(2Ǟ6)-
yl-β-
D-glucopyranosyl-(1Ǟ1)-(2S,3S,4R,13Z)-2-[(R)-2-(benzoyl- {[methyl 5-Acetoxyacetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-
5206
www.eurjoc.org
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 5199–5211

Total Synthesis of Ganglioside GAA-7
methyl-
acetamido-4-O-acetyl-2-deoxy-β-
O-acetyl-2-O-benzoyl-β-
methoxybenzyl-2-O-pivaloyl-β-
(2S,3S,4R,13Z)-2-[(R)-2-(benzoyloxy)tetracosanoylamino]-3,4-O-iso- syrup. [α]D = –32.5 (c = 0.6, CHCl3). 1H NMR (500 MHz, CDCl3):
D
-glycero-α-
D
-galacto-2-nonulopyranosylonate]-(2Ǟ3)}-(2-
tracted with CHCl3, and washed with H2O and brine. The organic
D-galactopyranosyl)-(1Ǟ3)-(4,6-di-
layer was dried with Na2SO4, and concentrated. The resulting resi-
D
-galactopyranosyl)-(1Ǟ4)-(3,6-di-O-p- due was purified by silica gel column chromatography (n-hexane/
-glucopyranosyl)-(1Ǟ1)- EtOAc, 7:1) to give 32 (427 mg, 71%, over two steps) as a colorless
D
propylidenedocos-13-ene-1,3,4-triol (29): Molecular sieves (4 Å, MS
AW300; 0.20 g), compound 3 (54.6 mg, 30.9 μmol), and compound
28 (55.9 mg, 40.8 μmol) were suspended in CH2Cl2 (1 mL) at ambi-
ent temperature. The mixture was stirred for 30 min, then the mix-
ture was cooled to –10 °C. TMSOTf (0.3 μL, 1.55 μmol) was added
to the suspension, which was then stirred for 1 h at 0 °C. The reac-
tion was monitored by TLC (CHCl3/acetone, 1:1). Next, the reac-
δ = 7.51–6.86 (m, 9 H, Ar), 5.09 (t, J2,3 = J3,4 = 9.0 Hz, 1 H, 3-H),
4.84 (near t, J1,2 = 10.1 Hz, 1 H, 2-H), 4.72 (d, 1 H, 1-H), 4.54 and
4.46 (2 d, Jgem = 11.5 Hz, 2 H, ArCH2O), 3.81 (s, 3 H, OMe), 3.78
(t, J4,5 = 9.2 Hz, 1 H, 4-H), 3.73 (dd, Jgem = 10.9, J5,6a = 1.8 Hz,
1 H, 6a-H), 3.63 (dd, J5,6b = 5.4 Hz, 1 H, 6b-H), 3.50 (m, 1 H, 5-
H), 2.05 and 2.01 (2 s, 6 H, 2 Ac), 0.80 (s, 9 H, tBu), 0.03 and 0.00
(2 s, 6 H, 2 Me) ppm. 13C NMR (125 MHz, CDCl3): δ = 170.0,
tion was quenched with satd. aq. NaHCO3, the mixture was filtered 169.6, 159.1, 132.6, 132.2, 131.6, 130.3, 129.4, 129.1, 128.8, 113.7,
through a pad of Celite, and the pad was washed with CHCl3. The
combined filtrate and washings were washed with satd. aq.
NaHCO3 and brine, dried with Na2SO4, and concentrated. The
resulting residue was purified by gel filtration using Sephadex LH-
20 (CHCl3/MeOH, 1:1), and silica gel column chromatography
(CHCl3/acetone, 5:1Ǟ5:2Ǟ1:1) to give 29 (49.9 mg, 54%) as a
white solid. [α]D = –7.2 (c = 1.0, CHCl3) 1H NMR (500 MHz,
CDCl3): δ = 8.00–6.80 (m, 18 H, Ar), 6.35 (d, J2,NH = 8.8 Hz, 1 H,
85.2, 80.4, 77.1, 72.9, 70.9, 68.8, 68.4, 55.2, 31.5, 25.4, 22.5, 21.1,
20.7, 17.8, –4.3, –4.8 ppm. HRMS (ESI): calcd. for C30H42O8SSi
[M + Na]+ 613.2262; found 613.2262.
2,3-Di-O-acetyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-
α-D-glucopyranosyl Trichloroacetimidate (33): NBS (192 mg,
1.08 mmol) was added to a solution of compound 32 (426 mg,
721 μmol) in acetone/H2O (10:1; 7.2 mL) at ambient temperature.
The progress of the reaction was monitored by TLC (n-hexane/
EtOAc, 5:1). After the starting material had been consumed, the
reaction mixture was extracted with EtOAc. The organic layer was
successively washed with satd. aq. Na2S2O3 and brine, dried with
Na2SO4, and concentrated. The residue was purified by silica gel
column chromatography (CHCl3/acetone, 3:1Ǟ3:2).
NHCer), 5.84 and 5.82 (2 d, 2 H, NHa, NHb), 5.66 (d, J2,NH
=
8.0 Hz, 1 H, NHc), 5.38 (dd, J1,2 = 8.2, J2,3 = 9.8 Hz, 1 H, 2d-H),
5.37–5.32 (m, 3 H, 4d-H, 13Cer-H, 14Cer-H), 5.18 (t, J2Ј,3Ј = 6.2 Hz,
1 H, 2ЈCer-H), 5.11 (dd, J6,7 = 1.6, J7,8 = 9.3 Hz, 1 H, 7Neu-H),
5.04–4.97 (m, 4 H, 4a-H, 4b-H, 7Neu-H, 4c-H), 4.82–4.78 (m, 3 H,
3c-H, 2e-H, ArCH2O), 4.65 (d, 1 H, 1d-H), 4.59–4.52 (m, 4 H, 1c-
H, ArCH2O, 2 AcOCH2CO), 4.35 (d, 1 H, ArCH2O), 4.31–4.23
(m, 4 H, 3Cer-H, 2 AcOCH2CO, ArCH2O), 4.18 (d, 1 H, 1e-H),
4.15–3.77 (m, 27 H, 1aCer-H, 2Cer-H, 4Cer-H, 6a-H, 6b-H, 9aa-H,
9ba-H, 4e-H, 6ad-H, 5a-H, 5b-H, 8a-H, 8b-H, 3d-H, 6ac-H, 2 OMe,
2 COOMe), 3.72–3.62 (m, 5 H, 9ba-H, 9bb-H, 1bCer-H, 6bc-H, 5d-
H), 3.58–3.47 (m, 6 H, 6ae-H, 2c-H, 6bd-H, OMe), 3.43–3.26 (m, 6
H, 3e-H, 6be-H, 5c-H, OMe), 3.07 (near td, 1 H, 5e-H), 2.57 and
2.52 (2 dd, 2 H, 3eqa-H, 3eqb-H), 2.17–1.95 (12 s, 36 H, 12 Ac),
1.88–1.78 (m, 7 H, 3axNeu-H, 12Cer-H, 15Cer-H), 1.66 (t, 1 H, 3ax-
Neu-H), 1.49–1.12 (m, 72 H, 33 CH2, 2 Me), 1.09 (s, 9 H, tBu), 0.88
(2 t, 6 H, 2 Me) ppm. 13C NMR (125 MHz, CDCl3): δ = 176.4,
171.0, 170.9, 170.4, 169.9, 169.7, 169.6, 169.5, 169.5, 169.3, 167.9,
167.7, 167.6, 167.5, 165.5, 164.5, 159.4, 158.9, 133.6, 133.5, 130.8,
130.1, 129.9, 129.8, 129.7, 129.5, 129.3, 128.6, 113.9, 113.4, 107.9,
101.1, 99.8, 98.7, 97.6, 79.9, 77.6, 77.5, 75.7, 75.5, 75.2, 75.0, 74.8,
73.3, 73.1, 72.6, 72.2, 71.8, 71.5, 71.1, 70.4, 69.4, 68.5, 68.4, 68.0,
67.4, 66.7, 62.7, 62.7, 62.6, 61.4, 61.2, 60.9, 58.5, 58.1, 55.3, 55.2,
53.1, 52.7, 49.4, 49.3, 48.8, 38.6, 37.5, 37.2, 32.6, 31.9, 31.9, 31.7,
29.9, 29.8, 29.7, 29.7 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 29.3,
29.2, 28.1, 27.2, 27.2, 27.0, 26.5, 25.7, 25.0, 22.7, 22.6, 20.9, 20.8,
20.8, 20.7, 20.7, 20.7, 20.6, 20.5, 14.1 ppm. HRMS (ESI): calcd. for
Then, the resulting compound was dissolved in CH2Cl2 (7.2 mL).
Trichloroacetonitrile (549 μL, 5.47 mmol) and 1,8-diazabicy-
clo[5.4.0]undec-7-ene (55 μL, 365 μmol) were added to the solution
at 0 °C. The progress of the reaction was monitored by TLC (n-
hexane/EtOAc, 3:1). When the reaction was complete, the reaction
mixture was concentrated. The residue was purified by silica gel
column chromatography (n-hexane/EtOAc, 5:1) to give 33 (218 mg,
47%) as an amorphous powder. [α]D = +57.9 (c = 1.1, CHCl3). 1H
NMR (500 MHz, CDCl3): δ = 8.59 (s, 1 H, C=NH), 7.26–6.86 (2
d, 4 H, Ar), 6.49 (d, J1,2 = 3.7 Hz, 1 H, 1-H), 5.45 (dd, J2,3 = 9.9,
J3,4 = 8.5 Hz, 1 H, 3-H), 4.99 (dd, 1 H, 2-H), 4.51 and 4.44 (2 d,
Jgem = 11.6 Hz, 2 H, ArCH2O), 3.99–3.93 (m, 2 H, 4-H, 5-H), 3.79
(s, 3 H, OMe), 3.70 (dd, Jgem = 10.8, J5,6a = 3.4 Hz, 1 H, 6a-H),
3.63 (dd, J5,6b = 1.4 Hz, 1 H, 6b-H), 2.04 and 1.97 (2 s, 6 H, 2 Ac),
0.82 (s, 9 H, tBu), 0.05 and 0.03 (2 s, 6 H, 2 Me) ppm. 13C NMR
(125 MHz, CDCl3): δ = 170.1, 169.6, 161.1, 159.1, 130.0, 129.2,
129.1, 113.8, 113.7, 93.5, 90.9, 74.5, 72.9, 70.6, 68.3, 67.6, 55.2,
25.7, 25.6, 21.2, 20.4, 17.9, –4.3, –4.9 ppm. HRMS (ESI): calcd. for
C26H38Cl3NO9Si [M + Na]+ 664.1274; found 664.1272.
Phenyl 2,3,6-Tri-O-acetyl-4-O-tert-butyldimethylsilyl-1-thio-β-D-
151H222N4O54 [M + 2Na]2+ 1507.7345; found 1507.7346.
Phenyl 2,3-Di-O-acetyl-4-O-tert-butyldimethylsilyl-6-O-p-meth-
oxybenzyl-1-thio-β- -glucopyranoside (32): HCl (2 m in Et2O;
glucopyranoside (35): TBSCl (2.30 g, 15.2 mmol) was added to a
solution of compound 34 (604 mg, 1.52 mmol) in pyridine (15 mL)
under an argon atmosphere. The reaction was monitored by TLC
(n-hexane/EtOAc, 2:1). The mixture was stirred for 12 h at 40 °C,
then the reaction mixture was coevaporated with toluene. The resi-
due was extracted with EtOAc, and the organic layer was washed
with HCl (2 m aq.), NaHCO3, and brine, then it was dried with
Na2SO4, and concentrated. The resulting residue was purified by
silica gel column chromatography (n-hexane/EtOAc, 6:1) to give 35
(708 mg, 91%) as a colorless syrup. [α]D = –25.8 (c = 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 7.48–7.28 (m, 5 H, Ph), 5.11 (t,
J2,3 = J3,4 = 9.1 Hz, 1 H, 3-H), 4.85 (near t, J1,2 = 10.1 Hz, 1 H,
2-H), 4.72 (d, 1 H, 1-H), 4.48 (dd, Jgem = 12.0, J5,6a = 2.1 Hz, 1 H,
6a-H), 4.09 (dd, J5,6b = 5.4 Hz, 1 H, 6b-H), 3.77 (t, J4,5 = 9.3 Hz,
1 H, 4-H), 3.56 (m, 1 H, 5-H), 2.09–2.04 (3 s, 9 H, 3 Ac), 0.83 (s,
9 H, tBu), 0.05 and 0.03 (2 s, 6 H, 2 Me) ppm. 13C NMR
C
D
3.2 mL, 6.40 mmol) was added to a suspension of compound 31
(498 mg, 0.842 mmol), NaBH3CN (529 mg, 8.42 mmol), and mo-
lecular sieves (3 Å; 850 mg) in THF (8.5 mL) under an argon atmo-
sphere. The mixture was stirred for 30 min, then it was diluted with
EtOAc, and filtered through a pad of Celite. The filtrate was ex-
tracted with EtOAc, and the organic phase was washed with satd.
aq. NaHCO3 and brine, dried with Na2SO4, and concentrated.
The resulting residue was dissolved in CH2Cl2 (8.5 mL), then 2,6-
lutidine (390 mL, 3.37 mmol) and TBSOTf (290 mL, 1.26 mmol)
were added at 0 °C under an argon atmosphere. The reaction was
monitored by TLC (n-hexane/EtOAc, 4:1), and when the reaction
was complete, it was quenched with MeOH. The mixture was ex-
Eur. J. Org. Chem. 2015, 5199–5211
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5207

H. Tamai, A. Imamura, J. Ogawa, H. Ando, H. Ishida, M. Kiso
FULL PAPER
(125 MHz, CDCl3): δ = 170.5, 170.0, 169.7, 132.8, 132.0, 128.8, NMR (500 MHz, CDCl3): δ = 8.08–6.85 (m, 9 H, Ar), 6.38 (d,
128.1, 85.4, 78.3, 70.7, 68.9, 62.8, 25.6, 21.2, 20.8, 20.7, 17.8, –4.1, J2,NH = 8.0 Hz, 1 H, NH), 5.35–5.32 (m, 2 H, 13-H, 14-H), 5.25
–4.9 ppm. HRMS (ESI): calcd. for C24H36O8SSi [M + Na]+
535.1792; found 535.1792.
(t, J2Ј,3Ј = 6.2 Hz, 1 H, 2Ј-H), 4.96 (t, J2,3 = J3,4 = 9.1 Hz, 1 H, 3a-
H), 4.53–4.42 (3 d, Jgem = 11.7, J1,2 = 8.0 Hz, 3 H, ArCH2O-, 1a-
H), 4.19 (dd, J = 5.3, J = 9.0 Hz, 1 H, 3-H), 4.13 (m, 1 H, 2-H),
4.05–4.00 (m, Jgem = 10.8, J1a,2 = 3.9 Hz, 2 H, 1a-H, 4-H), 3.80 (s,
2,3,6-Tri-O-acetyl-4-O-tert-butyldimethylsilyl-α-D-glucopyranosyl
Trichloroacetimidate (36): NBS (708 mg, 4.00 mmol) was added to
a solution of compound 35 (680 mg, 1.33 mmol) in acetone/H2O
(10:1; 14 mL) at ambient temperature. The progress of the reaction
was monitored by TLC (n-hexane/EtOAc, 5:1). After the starting
material had been consumed, the reaction mixture was extracted
with EtOAc. Then, the organic layer was successively washed with
satd. aq. Na2S2O3 and brine, and dried with Na2SO4. The solution
was concentrated to give a crude mixture including a hemiacetal
and 2-OH derivatives.
3 H, OMe), 3.72 (dd, J1b,2 = 3.5 Hz, 1 H, 1b-H), 3.61 (dd, Jgem
=
11.6, J5,6a = 1.7 Hz, 1 H, 6aa-H), 3.57 (t, J4,5 = 9.1 Hz, 1 H, 4a-H),
3.45 (dd, J5,6b = 5.9 Hz, 1 H, 6ba-H), 3.38 (m, 1 H, 5a-H), 2.03–
1.91 (m, 12 H, 3 Ac, 12-H, 15-H, 3Ј-H), 1.50–1.13 (m, 74 H, 34
CH2, 2 Me), 0.88 (2 t, 6 H, 2 Me), 0.78 (s, 9 H, tBu) ppm. 13C
NMR (125 MHz, CDCl3): δ = 170.1, 169.6, 169.4, 159.3, 133.4,
130.0, 129.9, 129.8, 129.5, 129.2, 128.6, 113.8, 108.0, 100.4, 75.9,
74.8, 73.1, 72.3, 69.2, 68.6, 68.3, 68.2, 55.2, 48.5, 31.92, 31.90, 31.7,
29.8, 29.78, 29.71, 29.70, 29.6, 29.56, 29.53, 29.51, 29.41, 29.38,
29.35, 29.33, 29.31, 29.2, 29.1, 28.9, 28.1, 27.24, 27.22, 26.7, 25.8,
25.6, 25.1, 22.7, 21.2, 29.6, 17.9, 14.1, 14.0, –4.3, –4.7 ppm. HRMS
(ESI): calcd. for C80H135NO14Si [M + Na]+ 1384.9544; found
1384.9544.
This residue was dried in vacuo, then it was dissolved in pyridine
(14 mL), and Ac2O (7 mL) was added to the solution at 0 °C under
an argon atmosphere. The mixture was stirred for 10 h, then it was
coevaporated with toluene. The residue was purified by silica gel
column chromatography (n-hexane/EtOAc, 4:1) to give the tetra-
acetylated derivative.
2,3-Di-O-aceyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1Ǟ1)-
(2S,3S,4R,13Z)-2-[(R)-2-(benzoyloxy)tetracosanoylamino]-3,4-O-iso-
propylidenedocos-13-ene-1,3,4-triol (38): Tetrabutylammonium
fluoride (1.0 m solution in THF; 116 μL, 0.116 mmol) was added
to a solution of compound 37 (106 mg, 0.077 mmol) and AcOH
(9.0 μL, 154 μmol) in THF (1.5 mL) at ambient temperature. The
mixture was stirred for 6 h, and the reaction was monitored by TLC
(n-hexane/EtOAc, 3:1). The mixture was evaporated. The resulting
residue was purified by silica gel column chromatography (n-hex-
ane/EtOAc, 3:1) to give 38 (96 mg, 98%) as a colorless syrup. [α]D
This compound was dissolved in DMF (14 mL), and hydrazine
acetate (180 mg, 2.00 mmol) was added to the resulting solution at
ambient temperature. After TLC confirmed that the reaction was
complete, the reaction mixture was extracted with EtOAc, and the
organic phase was washed with H2O and brine. The organic layer
was dried with Na2SO4 and concentrated. The resulting residue was
purified by silica gel column chromatography (n-hexane/EtOAc,
2:1) to give a hemiacetal, which was dried under high vacuum.
1
= +24.4 (c = 0.2, CHCl3). H NMR (500 MHz, CDCl3): δ = 8.07–
6.86 (m, 9 H, Ar), 6.28 (m, 1 H, NH), 5.38–5.30 (m, 2 H, 13-H,
The hemiacetal was dissolved in in CH2Cl2 (20 mL), and the solu-
tion was cooled to 0 °C. Trichloroacetonitrile (2.6 mL, 26 mmol)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (219 μL, 1.47 mmol) were
added, and the mixture was stirred for 30 min. The progress of the
reaction was monitored by TLC (n-hexane/EtOAc, 3:1). Then, the
reaction mixture was concentrated, and the residue was purified by
silica gel column chromatography (n-hexane/EtOAc, 5:1) to give 36
14-H), 5.28 (t, J2Ј,3Ј = 6.1 Hz, 1 H, 2Ј-H), 4.96 (t, J2,3 =, J3,4
=
9.4 Hz, 1 H, 3a-H), 4.79 (dd, J1,2 = 8.0 Hz, 1 H, 2a-H), 4.51–4.43
(m, 3 H, 1a-H, ArCH2O), 4.15–4.12 (m, 2 H, 2-H, 3-H), 4.03–4.02
(m, 2 H, 1a-H, 4-H), 3.79 (s, 3 H, OMe), 3.70–3.63 (m, 3 H, 1b-H,
6aa-H, 6ba-H), 3.59 (t, J4,5 = 9.1 Hz, 1 H, 4a-H), 3.47 (m, 1 H, 5a-
H), 2.99 (br. s, 1 H, OH), 2.16–1.89 (m, 12 H, 2 Ac, 12-H, 15-H,
3Ј-H), 1.52–1.23 (m, 74 H, 34 CH2, 2 Me), 0.88 (2 t, 6 H, 2 Me)
ppm. 13C NMR (125 MHz, CDCl3): δ = 171.2, 169.34, 169.31,
165.5, 159.4, 133.5, 129.9, 129.83, 129.80, 129.42, 129.39, 129.34,
128.5, 113.9, 108.0, 100.5, 77.7, 75.6, 74.7, 73.9, 73.4, 71.1, 70.9,
69.8, 68.5, 55.2, 48.6, 31.89, 31.87, 29.8, 29.74, 29.67, 29.64, 29.62,
29.60, 29.52. 29.50, 29.45, 29.34, 29.32, 29.29, 29.28, 29.1, 28.0,
27.2, 27.19, 26.6, 25.7, 25.0, 22.6, 20.8, 20.5, 14.1 ppm. HRMS
(ESI): calcd. for C74H121NO14 [M + Na]+ 1270.8679; found
1270.8679.
(576 mg, 77%, over four steps) as an amorphous powder. [α]D
=
1
+75.0 (c = 2.3, CHCl3). H NMR (500 MHz, CDCl3): δ = 8.62 (s,
1 H, C=NH), 6.42 (d, J1,2 = 3.7 Hz, 1 H, 1-H), 5.43 (t, J2,3 = J3,4
= 9.6 Hz, 1 H, 3-H), 4.98 (dd, 1 H, 2-H), 4.41 (dd, Jgem = 12.1,
J5,6a = 1.7 Hz, 1 H, 6a-H), 4.08 (dd, J5,6b = 4.2 Hz, 1 H, 6b-H),
3.99 (m, 1 H, 5-H), 3.89 (t, J4,5 = 9.4 Hz, 1 H, 4-H), 2.05–1.96 (3
s, 9 H, 3 Ac), 0.83 (s, 9 H, tBu), 0.04 (2 s, 6 H, 2 Me) ppm. 13C
NMR (125 MHz, CDCl3): δ = 170.4, 170.1, 169.5, 163.7, 161.0,
93.2, 72.6, 72.3, 70.4, 68.6, 62.1, 25.6, 25.5, 21.1, 20.7, 20.4, 17.9,
14.0, –4.2, –5.1 ppm. HRMS (ESI): calcd. for C20H32Cl3NO9Si [M
+ Na]+ 586.0804; found 586.0805.
2,3,6-Tri-O-acetyl-4-O-tert-butyldimethylsilyl-β-
D-glucopyranosyl-
2,3-Di-O-acetyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-
(1Ǟ1)-(2S,3S,4R,13Z)-2-[(R)-2-(Benzoyloxy)tetracosanoylamino]-
β-
racosanoylamino]-3,4-O-isopropylidenedocos-13-ene-1,3,4-triol (37):
Molecular sieves (4 Å, MS AW300, 0.22 g), compound 33 (83 mg, compound 24 (184 mg, 206 μmol) were suspended in CH2Cl2
D-glucopyranosyl-(1Ǟ1)-(2S,3S,4R,13Z)-2-[(R)-2-(benzoyloxy)tet- 3,4-O-isopropylidenedocos-13-ene-1,3,4-triol (39): Molecular sieves
(4 Å, MS AW300; 0.410 g), compound 36 (232 mg, 411 μmol), and
129 μmol), and compound 24 (95 mg, 109 μmol) were suspended
in CH2Cl2 (2.2 mL) at ambient temperature. The mixture was
stirred for 30 min, then it was cooled to –10 °C. TMSOTf (0.23 μL,
1.3 μmol) was added to the suspension, and the mixture was stirred
for 1 h at 0 °C. The reaction was monitored by TLC (toluene/
EtOAc, 6:1). The reaction mixture was quenched with satd. aq.
NaHCO3. The mixture was filtered through a pad of Celite, and
the pad was washed with CHCl3. The combined filtrate and wash-
ings were washed with satd. aq. NaHCO3 and brine, dried with
Na2SO4, and concentrated. The resulting residue was purified by
silica gel column chromatography (toluene/EtOAc, 15:1) to give 37
(4.1 mL) at ambient temperature. The mixture was stirred for
30 min, then the mixture was cooled to –10 °C. TMSOTf (3.6 μL,
20.6 μmol) was added to the suspension. The mixture was stirred
for 1 h at 0 °C, and the reaction was monitored by TLC (toluene/
EtOAc, 6:1). The reaction mixture was quenched with satd. aq.
NaHCO3. The mixture was filtered through a pad of Celite, and
the pad was washed with CHCl3. The combined filtrate and wash-
ings were washed with satd. aq. NaHCO3 and brine, dried with
Na2SO4, and concentrated. The resulting residue was purified by
silica gel column chromatography (toluene/EtOAc, 15:1) to give 39
(202 mg, 75%) as a colorless syrup. [α]D = –40 9 (c = 0.1, CHCl3).
(93 mg, 64%) as a white solid. [α]D = +8.4 (c = 1.0, CHCl3). 1H 1H NMR (500 MHz, CDCl3): δ = 8.05–7.44 (m, 5 H, Ph), 6.20
5208
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Eur. J. Org. Chem. 2015, 5199–5211

Total Synthesis of Ganglioside GAA-7
(J2,NH = 8.8 Hz, 1 H, NH), 5.35–5.30 (m, 2 H, 13-H, 14-H), 5.25
resulting residue was purified by gel filtration using Sephadex LH-
(t, J2Ј,3Ј = 6.2 Hz, 1 H, 2Ј-H), 4.96 (t, J2,3 = J3,4 = 9.3 Hz, 1 H, 3a- 20 (CHCl3/MeOH, 1:1), followed by silica gel column chromatog-
H), 4.67 (dd, J1,2 = 7.9 Hz, 1 H, 2a-H), 4.44 (d, 1 H, 1a-H), 4.36 raphy (CHCl3/MeOH, 40:1Ǟ35:1Ǟ30:1) to give 41 (19.7 mg, 26%)
(dd, Jgem = 11.9, J5,6a = 2.1 Hz, 1 H, 6aa-H), 4.14–4.01 (m, 3 H, 2-
as a white solid. [α]D = +5.8 (c = 0.4, CHCl3) 1H NMR (500 MHz,
H, 3-H, 4-H), 3.98 (dd, J5,6b = 5.3 Hz, 1 H, 6ba-H), 3.95 (dd, Jgem CDCl3): δ = 8.02–6.92 (m, 14 H, Ar), 6.22 (d, J2,NH = 9.0 Hz, 1 H,
= 10.7, J1a,2 = 4.1 Hz, 1 H, 1a-H), 3.67 (dd, J1b,2 = 3.3 Hz, 1 H, NHCer), 5.81 and 5.77 (2 d, J5,NH = 10.0 Hz, 2 H, NHa, NHb), 5.59
1b-H), 3.64 (t, J4,5 = 9.1 Hz, 1 H, 4a-H), 3.41 (m, 1 H, 5a-H), 2.05– (d, J2,NH = 7.8 Hz, 1 H, NHc), 5.38–5.33 (m, 3 H, 13f-H, 14f-H,
1.86 (m, 15 H, 3 Ac, 12-H, 15-H, 3Ј-H), 1.47–1.18 (m, 74 H, 34 4d-H), 5.27–5.23 (m, 2 H, 2d-H, 1Јf-H), 5.11 (dd, J6,7 = 1.7, J7,8
=
CH2, 2 Me), 0.86–0.78 (m, 15 H, 2 Me, tBu), 0.02 and 0.00 (2 s, 6 9.3 Hz, 1 H, 7Neu-H), 5.05–4.98 (m, 5 H, 7Neu-H, 4a-H, 4b-H, 3e-
H, 2 Me) ppm. 13C NMR (125 MHz, CDCl3): δ = 170.5, 169.9, H), 4.91 (d, J1,2 = 8.0 Hz, 1 H, 1c-H), 4.78 (dd, J1,2 = 8.0, J2,3
169.6, 165.6, 133.5, 130.3, 130.2, 129.9, 129.8, 129.4, 128.6, 108.3,
9.7 Hz, 1 H, 2e-H), 4.64 (br. d, J2,3 = 10.8 Hz, 1 H, 3c-H), 4.59–
=
108.1, 100.4, 77.8, 75.9, 75.6, 74.8, 74.4, 72.2, 69.1, 68.7, 62.7, 48.4, 44.52 (m, 3 H, 2 AcOCH2CO, 1d-H), 4.31–4.23 (m, 5 H, ArCH2O-,
32.6, 31.9, 31.8, 31.6, 29.8, 29.7, 29.67, 29.62, 29.59, 29.57, 29.50,
29.46, 29.35, 29.32, 29.30, 29.28, 29.18, 29.17, 29.10, 28.0, 27.5,
2 AcOCH2CO-, 1e-H, 3d-H), 4.16 (dd, Jgem = 11.7, J8.9a = 4.3 Hz,
1 H, 9aNeu-H), 4.13–3.80 (m, 23 H, ArCH2O-, 2Cer-H, 4Cer-H, 9ab-
27.21, 27.20, 26.6, 25.7, 25.6, 25.1, 22.6, 21.1, 20.9, 20.7, 20.5, 17.9, H, 9ba-H, 6a-H, 6b-H, 1aCer-H, 5a-H, 5b-H, 4e-H, 8a-H, 8b-H, 2
17.8, 14.1, –4.2, –4.9 ppm. HRMS (ESI): calcd. for C74H129NO14Si COOMe, OMe), 3.78–3.66 (m, 2 H, 6ac-H, 1bCer-H), 3.65–3.63 (m,
[M + Na]+ 1306.9075; found 1306.9077.
2 H, 6bc-H, 3Cer-H), 3.57–3.35 (m, 10 H, 9bb-H, 6ad-H, 6bd-H, 5e-
H, 2 OMe), 3.26 (t, J5,6 = 8.6 Hz, 1 H, 5c-H), 3.15 (near d, Jgem
9.6 Hz, 1 H, 6be-H), 2.58 and 2.50 (2 dd, Jgem = 12.6, J3eq,4
=
=
2,3,6-Tri-O-acetyl-β- -glucopyranosyl-(1Ǟ1)-(2S,3S,4R,13Z)-2-
D
[(R)-2-(benzoyloxy)tetracosanoylamino]-3,4-O-isopropylidenedocos-
13-ene-1,3,4-triol (40): Tetrabutylammonium fluoride (1.0 m solu-
tion in THF; 233 μL, 0.233 mmol) was added to a solution of com-
pound 39 (202 mg, 0.156 mmol) and AcOH (13.7 μL, 233 μmol) in
THF (3.1 mL) at ambient temperature. The mixture was stirred for
6 h, and the reaction was monitored by TLC (n-hexane/EtOAc,
3:1). The mixture was evaporated. The resulting residue was puri-
fied by silica gel column chromatography (n-hexane/EtOAc, 3:1) to
give 40 (155 mg, 85%) as a colorless syrup. [α]D = –3.7 (c = 0.5,
CHCl3). 1H NMR (500 MHz, CDCl3): δ = 8.08–7.47 (m, 5 H, Ph),
6.23 (d, J2,NH = 9.0 Hz, 1 H, NH), 5.39–5.33 (m, 2 H, 13-H, 14-
H), 5.31 (t, J2Ј,3Ј = 6.1 Hz, 1 H, 2Ј-H), 4.95 (t, J2,3 = J3,4 = 9.2 Hz,
4.7 Hz, 2 H, 3eqa-H, 3eqb-H), 2.19–1.82 (m, 36 H, 3axNeu-H, 11
Ac, 2 CH2), 1.67–1.49 (m, 12 H, 3axNeu-H, 3 Ac, CH2), 1.47–1.19
(m, 72 H, 33 CH2, 2 Me), 0.88 and 0.87 (2 t, 6 H, 2 Me) ppm. 13C
NMR (125 MHz, CDCl3): δ = 171.0, 170.9, 170.8, 170.6, 170.5,
169.9, 169.8, 169.7, 169.6, 169.5, 169.4, 169.2, 167.9, 167.7, 167.6,
167.5, 165.5, 164.3, 159.4, 133.6, 133.5, 130.3, 130.1, 129.9, 129.9,
129.83, 129.80, 129.7, 129.5, 129.2, 128.6, 113.9, 108.0, 100.7,
100.4, 100.1, 98.7, 97.7, 77.7, 77.6, 75.6, 75.5, 74.7, 74.3, 73.1, 72.7,
72.6, 72.3, 71.5, 71.4, 71.2, 70.2, 69.4, 68.6, 68.2, 68.0, 67.9, 66.9,
62.8, 62.7, 61.2, 61.1, 58.3, 58.1, 55.3, 53.1, 52.18, 49.4, 48.3, 37.5,
37.2, 32.6, 31.92, 31.91, 31.7, 29.8, 29.78, 29.71, 29.66, 29.61, 29.54,
29.46, 29.36, 29.33, 29.32, 29.2, 29.1, 28.1, 27.24, 27.23, 26.6, 25.8,
25.0, 22.72, 22.68. 20.98, 20.87, 20.86, 20.84, 20.79, 20.77, 20.71,
20.69, 20.58, 20.47, 14.1 ppm. HRMS (ESI): calcd. for
1 H, 3a-H), 4.81 (dd, J1,2 = 7.8 Hz, 1 H, 2a-H), 4.39 (dd, Jgem
=
11.9, J5,6a = 4.0 Hz, 1 H, 6aa-H), 4.27 (near d, 1 H, 6ba-H), 4.17
(m, 1 H, 2-H), 4.11–4.04 (m, 2 H, 3-H, 4-H), 4.01 (dd, Jgem = 10.5,
J1a,2 = 4.1 Hz, 1 H, 1a-H), 3.70 (dd, J1b,2 = 3.2 Hz, 1 H, 1b-H),
3.50–3.44 (m, 2 H, 4a-H, 5a-H), 3.03 (d, J4,OH = 3.2 Hz, 1 H, OH),
2.10–1.89 (m, 15 H, 3 Ac, 12-H, 15-H, 3Ј-H), 1.52–1.14 (m, 74 H,
34 CH2, 2 Me), 0.88 (2 t, 6 H, 2 Me) ppm. 13C NMR (125 MHz,
CDCl3): δ = 171.6, 171.3, 169.3, 169.2, 165.6, 133.6, 130.3, 130.2,
129.9, 129.8, 128.7, 128.6, 108.1, 100.6, 77.7, 75.7, 75.4, 74.7, 74.2,
71.0, 68.9, 68.5, 62.8, 48.4, 32.6, 31.89, 31.88, 31.7, 29.80, 29.75,
29.68, 29.63, 29.59, 29.51, 29.45, 29.33, 29.29, 29.20, 29.17, 29.12,
28.0, 27.21, 27.20, 26.6, 25.7, 25.0, 22.7, 20.8, 20.4, 14.1 ppm.
HRMS (ESI): calcd. for C68H115NO14 [M + Na]+ 1192.8210; found
1192.8210.
C
143H212N4O54 [M + Na]+ 2872.3858; found 2872.3858.
[Methyl 5-Acetoxyacetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-
methyl- -glycero-α- -galacto-2-nonulopyranosylonate]-(2Ǟ6)-
{[methyl 5-Acetoxyacetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-
methyl- -glycero-α- -galacto-2-nonulopyranosylonate]-(2Ǟ3)}-(2-
acetamido-4-O-acetyl-2-deoxy-β- -galactopyranosyl)-(1Ǟ3)-(4,6-O-
acetyl-2-O-benzoyl-β- -galactopyranosyl)-(1Ǟ4)-(2,3,6-tri-O-
acetyl-β- -glucopyranosyl)-(1Ǟ1)-(2S,3S,4R,13Z)-2-[(R)-2-
D
D
D
D
D
D
D
(benzoyloxy)tetracosanoylamino]-3,4-O-isopropylidenedocos-13-
ene-1,3,4-triol (42): Molecular sieves (4 Å, MS AW300; 0.060 g),
compound 3 (100 mg, 56.6 μmol), and compound 40 (22.0 mg,
18.9 μmol) were suspended in CH2Cl2 (0.6 mL) at ambient tem-
perature. The mixture was stirred for 30 min, and then it was cooled
to –10 °C. TMSOTf (0.34 μL, 1.89 μmol) was added, and the mix-
ture was stirred for 1 h at 0 °C. The reaction was monitored by
TLC (CHCl3/acetone, 1:1). Next, the reaction mixture was
quenched with satd. aq. NaHCO3. The mixture was filtered
through a pad of Celite, and the pad was washed with CHCl3. The
[Methyl 5-Acetoxyacetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-
methyl-
{[methyl 5-Acetoxyacetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-
methyl- -glycero-α- -galacto-2-nonulopyranosylonate]-(2Ǟ3)}-(2-
acetamido-4-O-acetyl-2-deoxy-β- -galactopyranosyl)-(1Ǟ3)-(4,6-di-
O-acetyl-2-O-benzoyl-β- -galactopyranosyl)-(1Ǟ4)-(2,3-di-O-
acetyl-6-O-p-methoxybenzyl-β- -glucopyranosyl)-(1Ǟ1)-
D-glycero-α-D-galacto-2-nonulopyranosylonate]-(2Ǟ6)-
D
D
D
D
D
(2S,3S,4R,13Z)-2-[(R)-2-(benzoyloxy)tetracosanoylamino]-3,4-O-iso- combined filtrate and washings were washed with satd. aq.
propylidenedocos-13-ene-1,3,4-triol (41): Molecular sieves (4 Å, MS
AW300, 0.075 g), compound 3 (45 mg, 25.5 μmol), and compound
38 (32 mg, 25.5 μmol) were suspended in CH2Cl2 (0.75 mL) at am-
NaHCO3 and brine, dried with Na2SO4, and concentrated. The
resulting residue was purified by gel filtration using Sephadex LH-
20 (CHCl3/MeOH, 1:1), followed by silica gel column chromatog-
bient temperature. The mixture was stirred for 30 min, then it was raphy (CHCl3/MeOH, 40:1Ǟ35:1Ǟ30:1) to give 42 (13.2 mg, 25%)
cooled to –10 °C. TMSOTf (0.46 μL, 2.55 μmol) was added, and
the mixture was stirred for 1 h at 0 °C. The reaction was monitored
by TLC (CHCl3/acetone, 1:1). Next, the reaction mixture was
quenched with satd. aq. NaHCO3. The mixture was filtered
through a pad of Celite, and the pad was washed with CHCl3. The
combined filtrate and washings were washed with satd. aq.
NaHCO3 and brine, dried with Na2SO4, and concentrated. The
as a white solid. [α]D = –7.7 (c = 1.5, CHCl3) 1H NMR (500 MHz,
CDCl3): δ = 8.07–7.43 (m, 10 H, 2 Ph), 6.15 (d, J2,NH = 9.1 Hz, 1
H, NHCer), 5.78 (d, J5,NH = 9.4 Hz, 1 H, NHNeu), 5.74 (d, J5,NH
=
10.0 Hz, 1 H, NHNeu), 5.66 (d, J2,NH = 8.0 Hz, 1 H, NHc), 5.37–
5.31 (m, 4 H, 13Cer-H, 14Cer-H, 2d-H, 4d-H), 5.26 (t, J1,2 = 6.2 Hz,
1 H, 1ЈCer-H), 5.11 (dd, J6,7 = 1.6, J7,8 = 9.3 Hz, 1 H, 7Neu-H), 5.05
(t, J2,3 =, J3,4 = 9.4 Hz, 1 H, 3e-H), 5.03–4.97 (m, 4 H, 4a-H, 4b-H,
Eur. J. Org. Chem. 2015, 5199–5211
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5209

H. Tamai, A. Imamura, J. Ogawa, H. Ando, H. Ishida, M. Kiso
FULL PAPER
7Neu-H, 4c-H), 4.87 (d, J1,2 = 7.9 Hz, 1 H, 1c-H), 4.76 (dd, J1,2
=
(Life Technologies Japan Ltd., Tokyo, Japan) supplemented with
5% heat-inactivated fetal bovine serum (FBS; PAA Laboratories
GmbH, Pasching, Austria) and 10% heat-inactivated horse serum
(HS; PAA Laboratories GmbH) in 5% CO2 at 37 °C. Cells were
7.9 Hz, J2,3 = 9.6 Hz, 1 H, 2e-H), 4.60–4.53 (m, 3 H, 3c-H, 2 Ac-
OCH2), 4.48 (d, J1,2 = 7.9 Hz, 1 H, 1d-H), 4.32 (d, 1 H, 1e-H),
4.31–4.23 (m, 4 H, 1af-H, 3ЈCer-H, 2 AcOCH2), 4.17 (dd, Jgem
=
11.7, J8,9a = 5.6 Hz, 1 H, 9aNeu-H), 4.13–3.91 (m, 13 H, 5a-H, 5b- plated onto transparent 96-well microplates precoated with poly-l-
H, 2Cer-H, 3Cer-H, 4Cer-H, 1bCer-H, 6a-H, 6b-H, 9aNeu-H, 3d-H, 4d- lysine (Nacalai Tesque, Kyoto, Japan).
H, 6ae-H, 6be-H), 3.85–3.80 (m, 9 H, 8a-H, 8b-H, 5c-H, 2 COOMe),
PC-12 Differentiation with NGF: The transparent 96-well micro-
3.67–3.52 (m, 9 H, 6ac-H, 6bc-H, 6ad-H, 6bd-H, 9ba-H, 9bb-H,
plates were seeded with 1ϫ104 cells per well, and then cells were
cultured with the RPMI medium containing both sera. After 24 h,
the medium was replaced by RPMI medium with low sera (0.05%
heat-inactivated FBS and 0.1% heat-inactivated HS), which was
supplemented with NGF (Alomone Labs Ltd., Jerusalem, Israel)
at 5 ng/mL to induce differentiation. After 3 d, the culture medium
was replaced by new low-serum medium with 5 ng/mL of NGF,
and then the cells were cultured for a further 2 d.
OMe), 3.38–3.33 (m, 4 H, 5d-H, OMe), 3.21 (t, J4,5 = J5,6a =, J5,6b
= 8.5 Hz, 1 H, 5e-H), 2.54 and 2.52 (2 dd, Jgem = 12.5, J3eq,4
=
4.6 Hz, 3eqa-H, 3eqb-H), 2.17–1.87 (m, 47 H, 13 Ac, 4 CH2), 1.81
(t, J3ax,4 = 12.5 Hz, 1 H, 3axNeu-H), 1.79–1.64 (m, 4 H, 3axNeu-H,
Ac), 1.50–1.19 (m, 75 H, Ac, 33 CH2, 2 Me), 0.88 and 0.87 (2 t, 6
H, 2 Me) ppm. 13C NMR (125 MHz, CDCl3): δ = 170.93, 170.91,
170.87, 170.84, 170.6, 170.5, 170.4, 169.91, 169.89, 169.74, 169.67,
169.54, 169.50, 169.48, 169.40, 169.2, 167.8, 167.7, 167.6, 167.5,
165.6, 164.3, 133.6, 133.5, 130.4, 130.3, 130.0, 129.9, 129.8, 129.5,
129.3, 128.6, 108.1, 100.9, 100.7, 100.3, 98.7, 97.6, 77.7, 77.6, 75.6,
75.4, 74.7, 72.7, 72.5, 72.3, 71.7, 71.4, 71.3, 70.6, 69.1, 68.6, 68.5,
68.0, 66.7, 62.8, 62.7, 62.6, 62.1, 61.4, 61.0, 60.9, 58.6, 58.0, 53.1,
52.7, 49.4, 49.3, 48.3, 37.6, 37.1, 32.6, 31.9, 31.8, 31.6, 30.0, 29.81,
29.76, 29.69, 29.64, 29.59, 29.52, 29.48, 29.44, 29.34, 29.32, 29.30,
29.2, 29.1, 28.0, 27.23, 27.21, 27.1, 26.6, 25.7, 25.0, 22.8, 22.7, 21.0,
20.9, 20.85, 20.82, 20.80, 20.75, 20.68, 20.65, 20.63, 20.59, 20.42,
14.1 ppm. HRMS (ESI): calcd. for C137H206N4O54 [M + Na]+
2794.3389; found 2794.3387.
Neurite Outgrowth Evaluation: Compound 1 or 2 (10 nm) was
added to the low-serum culture medium with 5 ng/mL of NGF
for neurite outgrowth evaluation. Cells were fixed for 30 min using
paraformaldehyde (4% in PBS; phosphate-buffered saline) at room
temperature, and were then stained using toluidine blue O (Sigma–
Aldrich). Morphological changes were observed with an inverted
microscope (IX70, Olympus, Tokyo, Japan) with a CCD camera
(DP71, Olympus). For the analysis of morphological changes, four
random areas per well were selected and photographed. The
lengths of the neurites and cell bodies in the image were quantified
by ImageJ software (National Institutes of Health, Bethesda,
USA). Measurements were carried out in triplicate. Sufficient mea-
surements were acquired for the analysis of at least 120 cells. Cells
containing neurite(s) were counted. In addition, the number of neu-
rites per well with lengths longer than two cell-body diameters was
counted, and then an average number of neurites per cell was calcu-
lated.
Ganglioside GAA-7 (1): Trifluoroacetic acid (90% aq.; 125 μL) was
added to a solution of compound 42 (13.2 mg, 4.76 μmol) in
CH2Cl2 (0.5 mL) at 0 °C. The progress of the reaction was moni-
tored by TLC (CHCl3/MeOH, 12:1). The mixture was stirred for
30 min at 0 °C, then it was diluted and extracted with CHCl3. The
organic layer was successively washed with satd. aq. NaHCO3,
H2O, and brine, dried with Na2SO4, and concentrated. The re-
sulting residue was purified by gel filtration (Sephadex LH-20,
CHCl3/MeOH, 1:1) to give a diol compound, which was dried in
vacuo.
Acknowledgments
The iCeMS is supported by the Japanese World Premier Inter-
national Research Center Initiative (WPI), MEXT. This work was
financially supported in part by the Japanese Ministry of Educa-
tion, Culture, Sports, Science and Technology (MEXT) [Grant-in-
Aid for Scientific Research (B), grant number 22380067 to M. K.,
and a Grant-in-Aid for Young Scientists (A), grant number
23688014 and a Grant-in-Aid on Innovative Areas, grant number
24110505, to H. A.]. The authors deeply thank Dr. Tomio Yabe
and Ritsuko Hosoda-Yabe for their technical guidance related to
the examination of neurite outgrowth. Kiyoko Ito is thanked for
technical assistance.
The dried residue was dissolved in THF/MeOH (2:1; 300 μL), and
a solution of NaOH (0.5 m aq.; 257 μL) was then added to this
solution at ambient temperature. The mixture was stirred for 36 h
at ambient temperature [monitored by TLC; CHCl3/MeOH/CaCl2
(30 mm aq.), 5:4:1], and the mixture was then concentrated. The
resulting residue was purified by column chromatography on Se-
phadex LH-20 (CHCl3/MeOH/H2O, 5:4:1) and subsequent column
chromatography on Iatrobeads 6RS-8060 (CHCl3/MeOH/H2O,
5:4:0Ǟ5:4:0.4Ǟ5:4:1) to give compound 1 (7.0 mg, 77%). [α]D
=
+3.3 (c = 0.5, CHCl3/MeOH/H2O, 5:4:1) 1H NMR (500 MHz,
CD3OD): δ = 5.24 (td, 2 H, 13Cer-H, 14Cer-H), 4.51 (d, 1 H, anom-
eric H), 4.29 (d, 1 H, anomeric H), 4.22 (d, 1 H, anomeric H), 3.42
(s, 3 H, OMe), 3.41 (s, 3 H, OMe), 2.56 (m, 2 H, 3eqa-H, 3eqb-H),
2.00 (s, 3 H, Ac), 1.32–1.04 (m, 66 H, 33 CH2), 0.80 (t, 6 H, 2 Me)
ppm. 13C NMR (200 MHz, [D6]DMSO): δ = 177.2, 173.9, 173.8,
171.1, 159.6, 130.1, 130.0, 129.6, 129.5, 103.3, 103.2, 103.1, 100.4,
98.9, 82.5, 81.8, 81.0, 80.6, 75.2, 74.9, 74.7, 73.8, 73.4, 73.1, 72.9,
72.7, 72.3, 71.5, 70.9, 70.6, 69.8, 69.5, 69.2, 68.7, 68.2, 67.2, 67.0,
66.6, 62.9, 61.5, 61.3, 61.2, 60.9, 60.2, 59.8, 58.0, 57.8, 52.8, 50.6,
49.9, 34.4, 32.0, 31.9, 31.6, 31.3, 29.4, 29.3, 29.2, 29.1, 29.08, 29.05,
29.02, 28.9, 28.8, 26.7, 26.6, 25.5, 24.4, 23.3, 22.1, 14.0 ppm.
HRMS (ESI): calcd. for C90H160N4O38 [M – 2H]2– 952.5355; found
952.5350.
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