Synthesis of a pseudo tetrasaccharide mimic of ganglioside GM1

Article abstract of DOI:10.1002/(SICI)1099-0690(199906)1999:6<1311::AID-EJOC1311>3.0.CO;2-W

The pseudo tetrasaccharide 2 was designed to mimic ganglioside GM1, the membrane receptor of both the cholera toxin and of the heat-labile toxin of E. coli. Compound 2 retains the Gal and Neu5Ac recognition determinant of GM1 and uses as the scaffold elem

Full text of DOI:10.1002/(SICI)1099-0690(199906)1999:6<1311::AID-EJOC1311>3.0.CO;2-W

FULL PAPER
Synthesis of a Pseudo Tetrasaccharide Mimic of Ganglioside GM1
Anna Bernardi,*[a] Giovanna Boschin,[a] Anna Checchia,[a] Maria Lattanzio,[a]
Leonardo Manzoni,[a] Donatella Potenza,[a] and Carlo Scolastico[a]
Keywords: Asymmetric synthesis / Carbohydrates / Pseudosugars / Carbohydrate mimics / Ganglioside GM1 / Cholera
toxin / Heat-labile toxin
The pseudo tetrasaccharide
2
was designed to mimic
diol 3 was enantioselectively synthesized by an asymmetric
Diels–Alder reaction, followed by dihydroxylation of the
resulting cyclohexene. Glycosylation of 3 with the sialyl
donor 17 and the Galβ(1-3)GalNAc donor 15, followed by
removal of the protecting groups, completed the synthesis
of 2.
ganglioside GM1, the membrane receptor of both the cholera
toxin and of the heat-labile toxin of E. coli. Compound 2
retains the Gal and Neu5Ac recognition determinant of GM1
and uses as the scaffold element a new, conformationally
restricted cyclohexanediol (DCCHD 3), with the same
relative and absolute configuration of natural galactose. The
nants Gal and Neu5Ac of 2 in the appropriate position for
protein recognition.[2b] In this paper we describe the syn-
thesis of 2.
Introduction
The cholera toxin (CT) and other related enterotoxins,
like the heat-labile toxin of E. Coli (LT), are recognized in
the epithelial cells of the host by the pentasaccharide por-
tion of the ganglioside GM1 1 (Figure 1).[1] In the course
of a program, directed toward the design and synthesis of
artificial cholera toxin binders,[2] the pseudo tetrasac-
charide 2 (Figure 1) was designed as a promising substitute
for the natural receptor. Saccharides 1 and 2 share the pres-
ence of a galactose (Gal) and a sialic acid (Neu5Ac) units
at the oligosaccharide non-reducing end. X-ray crystal-
lography of the GM1:CT complex has shown that these
residues interact directly with the toxin.[3] However, in 2 the
reducing end of the ganglioside has been substitued by the
conformationally
restricted
dicarboxycyclohexanediol
(DCCHD) 3 (Figure 1). The use of cyclohexanediols as core
sugar mimetics is an attractive strategy in order to simplify
the molecular structure of bioactive oligosaccharides, and
produce functional analogues which may be easier to syn-
thesize and of increased metabolic stability. This approach
has been used with success to design sialyl LewisX mimics
by replacing an N-acetylglucosamine unit with (R,R)-trans-
1,2-cyclohexanediol.[4] In order to replace a 3,4-disubsti-
tuted Gal residue, as it is found in the GM1 oligosaccharide
core region, a cis-cyclohexanediol is required, which must
be enantiomerically pure and conformationally stable in or-
der to distinguish unequivocally between the axial and the
equatorial substituents. The diol 3, which features the same
absolute configuration of natural galactose, and largely ex-
ists in a single chair conformation, appears to be a likely
candidate. Indeed, DCCHD 3 was calculated by molecular
mechanics to behave as an adequate replacement for the
core Gal unit of GM1 and to place the binding determi-
Figure 1. Ganglioside GM1 1, and its designed mimic 2
The retrosynthetic analysis is shown in Scheme 1. The
diol 3, which surprisingly has never been described in the
literature, can be synthesized by an enantioselective Diels
ϪAlder reaction between a chiral fumarate equivalent and
butadiene, followed by double-bond dihydroxylation. From
3, the pseudo tetrasaccharide could be assembled by reac-
tion of the axial hydroxy group with an appropriate donor
of Galβ(1-3)GalNAc, followed by sialylation of 4 (see
Scheme 1, Route a). Alternatively, sialylation of the equa-
[a]
Dipartimento di Chimica Organica e Industriale, and Centro
CNR Sostanze Organiche Naturali,
via Venezian 21, I-20133 Milano, Italy
E-mail: anna.bernardi@unimi.it
Eur. J. Org. Chem. 1999, 1311Ϫ1317
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/0606Ϫ1311 $ 17.50ϩ.50/0
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A. Bernardi et al.
FULL PAPER
torial hydroxy group followed by reaction of 5 with the catalytic amounts of OsCl3 and Me3NO in acetone/water[11]
Galβ(1-3)GalNAc donor could be planned (see Scheme 1, gave the diol 3 quantitatively. This was selectively protected
Route b). Both strategies have been successfully employed as a benzyl ether at the equatorial hydroxy group by
in the chemical synthesis of GM1,[5] although leaving the sequential reaction with Bu2SnO and benzyl bromide/
sialylation to the last step, as in Route a, was found to be Bu4NI in benzene,[12] to give 10 in a yield of 99%.
more problematic.[5b] Route a was initially followed, and
allowed the definition of optimal conditions for the selec-
tive formation of the β-glycosidic linkage between GalNAc
Synthesis of the Galβ(1-3)GalNAc Donor 15
and DCCHD. However, the sialylation of 4 was found to
be impractical, and the target molecule was eventually syn-
thesized following Route b.
2-Acetamido sugars are not usually regarded as valuable
glycosyl donors.[13] However, if the 2-acetamidotrichlo-
roacetimidate 15 (Scheme 3) could be used as the Galβ-
(1-3)GalNAc donor, it would straightforwardly introduce
the desired 2-acetamido functionality in the reaction prod-
uct, thus avoiding further manipulation of the oligosacchar-
ide, and possibly compensating for low yields in the glycos-
ylation reaction. Furthermore, an easy entry to 15 is en-
Results and Discussion
Enantioselective Synthesis of DCCHD 3
Although many methods for the enantioselective Diels sured by the elegant procedure reported by Russo and co-
ϪAlder reaction have been introduced in recent years,[6] few workers,[14] which allows a multigram-scale synthesis of the
of them were reported to be effective for the cycloaddition protected GalNAc derivative 11 in 5 steps and an overall
of butadiene to fumarates.[7] Among them, Helmchen’s yield of 30% from pentaacetylglucosamine. The above re-
methodology[7c] was chosen, which involves the low-tem- asons led to an investigation of the donor ability of 15
perature TiCl4-promoted reaction of butadiene with the (vide infra).
commercially available bis[ethyl (S)-lactate]fumaric ester 6
For the synthesis of 15, starting from 11, galactosylation
(Scheme 2). In the present study the desired (S,S)-dicar- with the trichloroacetimidate 12[15] was attempted. This re-
boxycyclohexene 7 was obtained as a 6:1 mixture with the action proved to be sluggish at room temp., with both
(R,R) isomer. The ratios were determined by 13C NMR of TMSOTf or BF3 · Et2O as promoters. Raising the reaction
the crude reaction mixture. The crude product was hy- temp. to 40°C in the presence of a catalytic amount of
drolyzed with LiOH in MeOH/H2O, and the acid 8 was TMSOTf resulted in the loss of β selectivity. Finally, stereo-
isolated in an overall yield of 69% by flash chromatogra- selective β-glycosylation of 11 with 12 was achieved with 1
phy.[8] At this stage, the (S,S) enantiomer of the acid was mol-equiv. of BF3 · Et2O at 40°C and afforded 13 in a yield
obtained in an enantiopure form by crystallization of its of 53%.[16] Deallylation of the anomeric oxygen atom[17]
quinine salt, following a published procedure.[9] Finally, the gave 14 in an overall yield of 60%. Finally, the trichloroacet-
di-tert-butyl ester 9 was synthesized in a yield of 73%, by imidate 15 was obtained from 14 by the standard pro-
reaction of (S,S)-8 with dimethylformamide di-tert-butyl cedure[15] in an almost quantitative yield, after silica gel
acetal in refluxing benzene.[10] Dihydroxylation of 9 with chromatography.
Scheme 1. Retrosynthesis of 2
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Eur. J. Org. Chem. 1999, 1311Ϫ1317

Synthesis of a Pseudo Tetrasaccharide Mimic of Ganglioside GM1
FULL PAPER
Scheme 2. Enantioselective synthesis of 3
The GalNAc β-anomeric configuration could be readily de-
rived from the J1,2 coupling constant of 9 ± 1 Hz.
Scheme 4. Route a
Hydrogenolysis of the benzyl ether in 16 gave the alcohol
4 which was subjected to sialylation with phosphite-acti-
vated sialic acid donors. However, probably due to the pres-
ence of the 2-acetamido group,[5a] 4 turned out not to be a
suitable sialyl acceptor in these reactions. Having shown
that 15 can perform as a reasonable Galβ(1-3)GalNAc do-
nor, it appeared that Route a could conveniently be aban-
doned in favor of Route b.
Scheme 3. Synthesis of the Galβ(1-3)GalNAc donor 15
Route b
If the α-sialylation reaction is to be carried out first, ad-
vantage can be taken of the markedly different reactivity of
equatorial and axial hydroxy groups in this type of reaction.
Neu5Ac donors are known to be very sensitive to the steric
hindrance of the acceptor, typically permitting regioselec-
Synthesis of 2
Route a
As mentioned above, 2-acetamido sugars tend to be poor tive sialylation of 3,4-unprotected galactose.[18] Indeed, sia-
glycosyl donors. Rapid intramolecular reaction in the pres- lylation of the unprotected diol 3 could be performed re-
ence of Lewis acids results in the formation of oxazolines, gioselectively at the equatorial position with the phosphite
which react very sluggishly with glycosyl acceptors. Con- 17[19][20] (Scheme 5). The reaction was carried out with
sidering the low reactivity of axial hydroxy groups, such as TMSOTf as a promoter in EtCN at Ϫ40°C.[19] The sialyl
the 4-OH of DCCHD (Figure 1), difficulties can be antici- derivative 5 was isolated in a yield of 20% as a 7:1 α/β mix-
pated in the formation of the Galβ(1-3)GalNAcϪDCCHD ture, and 60% of non-reacted 3 was recovered. The struc-
bond. Thus, the conditions for this bond formation were ture of 5 was assigned as follows. The sialylation site was
initially studied with the benzyl ether 10 as the glycosyl ac- determined by 13C NMR on the basis of the 4.3 ppm down-
ceptor (Scheme 4). After some experimentation, optimal field shift of the DCCHD 3-C signal. The diastereomeric
conditions were found with 2.5 mol-equiv. of acceptor, and α/β ratios were also measured by 13C NMR, exploiting the
adding a catalytic amount of TMSOTf to a refluxing relative intensity of the anomeric C-2 atoms of the sialic
CH2Cl2 solution of the glycosylation partners 10 and 15. acid moiety at δ ϭ 98.0 (α anomer) and δ ϭ 99.0 (β an-
The pseudotrisaccharide 16 (Scheme 4) was obtained in a omer). The α configuration was assigned to the major iso-
yield of 47%, and the excess acceptor was recovered by flash mer by measuring the JC-1,3-Hax coupling constant of Ne-
chromatography. The structure of compound 16 was as- u5Ac.[21] The α anomer gave a doublet at δ ϭ 168.5, with
signed with the help of H,H-COSY and HETCOR spectra. a JC-1,3-Hax coupling constant of 6.9 Hz, whereas a
Eur. J. Org. Chem. 1999, 1311Ϫ1317
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A. Bernardi et al.
FULL PAPER
solution cooled at Ϫ50°C and 1  TiCl4 in CH2Cl2 (13.3 mL,
13.3 mmol) was added under N2. Butadiene was added in small
portions over 6 h to the stirred mixture. After 24 h at Ϫ50°C, the
reaction was quenched with phosphate buffer and the solution fil-
tered through a Celite pad. The organic phase was washed with
phosphate buffer, then dried with Na2SO4, and concentrated in
vacuo to give 3.2 g of crude 7 contaminated by non-reacted 6. The
crude reaction product was dissolved in 3:1 MeOH/H2O (57 mL)
and LiOH (5.1 g) was added. After 24 h at room temp., the mixture
was concentrated at reduced pressure to about half the initial vol-
ume, 6  HCl was added to pH ϭ 1, and the resulting solution
extracted with AcOEt. The residue was purified by flash chroma-
tography (hexane/AcOEt/AcOH, 2:8:0.5) to give 1.1 g of 8 (69%).
JC-1,3-Hax coupling constant of 3.4 Hz was found for the β
isomer at δ ϭ 167.9.
20
Ϫ [α]D ϭ ϩ100 (c ϭ 2.7, EtOH). Ϫ (S,S)-8 was obtained in en-
antiomerically pure form by crystallization of its quinine salt,[9]
20
m.p. 145Ϫ147°C. Ϫ [α]D ϭ ϩ150 (c ϭ 2.7, EtOH) {ref.[9]
20
[α]D ϭ ϩ160 (c ϭ 2.7, EtOH)}. Ϫ 1H NMR (200 MHz, CDCl3):
δ ϭ 2.1Ϫ2.7 (m, 4 H), 2.85 (m, 2 H), 5.75 (d, 2 H, J ϭ 3 Hz), 9.6
(br. s, 2 H). Ϫ 13C NMR (50.3 MHz, CDCl3): δ ϭ 28.5, 42.1, 125.5,
181.5. Ϫ IR (CHCl3): ν˜ ϭ 3300Ϫ2900 cmϪ1 (OH), 1714 (CϭO).
Ϫ C8H10O4 (170.2): calcd. C 56.47, H 5.92; found C 56.75, H 6.03.
DCCHD 3: A solution of 8 (761 mg, 4.47 mmol) and (tBuO)2-
CHNMe2 (6.6 mL, 27.8 mmol) in dry benzene was refluxed under
N2 overnight. Water was added, the organic phase washed with
satd. NaHCO3 and brine, dried with Na2SO4, and concentrated in
vacuo. The crude di-tert-butyl ester 9 (822 mg, 2.9 mmol, 73% from
8) was dissolved in 14 mL of 4:1 acetone/water. OsCl3 (77 mg,
0.26 mmol) and Me3NO · H2O (648 mg, 5.8 mmol) were added,
and the solution was stirred at room temp. overnight, before
quenching with a satd. Na2SO3 solution. After stirring for 15 min,
the mixture was extracted with AcOEt. The organic phase was
dried with Na2SO4 and concentrated in vacuo. The crude product
was purified by flash chromatography (hexane/AcOEt, 1:1), to give
Scheme 5. Route b; synthesis of 2
The desired α anomer could be further purified by flash
chromatography (see Experimental Section) and submitted
to glycosylation by the Galβ(1-3)GalNAc donor 15
(Scheme 5). An excess of donor (2.3 mol-equiv.) was used,
otherwise the reaction conditions were the same as em-
ployed for the reaction between 15 and the acceptor 10
(catalytic TMSOTf, refluxing CH2Cl2). Thus, the pseudo-
tetrasaccharide 18, exhibiting the expected coupling con-
stants for the anomeric protons (J1a,2a ϭ 8.7 Hz, J1b,2b
ϭ
20
916 mg of 3 (97%), m.p. 98Ϫ100°C. Ϫ [α]D ϭ ϩ28.4 (c ϭ 1.2,
7.7 Hz), was isolated in a yield of 30%. Finally, MeONa/
MeOH hydrolysis gave the target pseudo GM1 2, which was
fully characterized by 500-MHz 1D- and 2D-NMR spec-
troscopy.
EtOH). Ϫ 1H NMR (200 MHz, CDCl3): δ ϭ 1.45 [s, 18 H,
COC(CH3)3], 1.5Ϫ1.68 (m, 1 H, 5ax-H), 1.7Ϫ1.9 (m, 1 H, 2ax-H),
2.0 (ddd, Jgem ϭ 13 Hz, J2eq,1 ϭ J
ϭ 4 Hz, 1 H, 2eq-H), 2.18
2eq,3
(ddd, Jgem ϭ 14 Hz, J5eq,6 ϭ J5eq,4 ϭ 4.5 Hz, 1 H, 5eq-H), 2.3 (br.
s, 2 H, OH), 2.55 (ddd, J1,6 ϭ 13 Hz, J1,2eq ϭ 4 Hz, J1,2ax ϭ 10 Hz,
1 H, 1-H), 2.86 (ddd, J6,1 ϭ 13 Hz, J6,5eq ϭ 4.5 Hz, J6,5ax ϭ 10 Hz,
1 H, 6-H), 3.63Ϫ3.75 (m, 1 H, 3-H), 3.95Ϫ4.2 (m, 1 H, 4-H). Ϫ
13C NMR (50.3 MHz, CDCl3): δ ϭ 27.8, 30.3, 33.0, 39.3, 44.0,
67.9, 70.3, 80.6, 80.7, 173.3, 174.3. Ϫ IR (CHCl3): ν˜ ϭ 3500Ϫ3200
cmϪ1 (OH), 1720 (CϭO). Ϫ C16H28O6 (316.4): calcd. C 60.74, H
8.92; found C 60.53, H 8.84.
ELISA and TLC overlay assays showed that 2 is as po-
tent as the GM1 oligosaccharide 1b in inhibiting the forma-
tion of the CT:GM1 complex.[2b]
Experimental Section
˚
General: Solvents: Pyridine and DMF were dried over 4-A molecu-
lar sieves. The other dry solvents were distilled under nitrogen
(from liquid nitrogen) shortly before use. THF and benzene were
distilled from Na; CH2Cl2, MeOH, TEA, DIPEA, and EtCN from
CaH2. Ϫ Flash chromatography: Silica gel (Kieselgel 60, 230Ϫ400
mesh). Ϫ M.p.s: Büchi 535 apparatus, uncorrected values. Ϫ MS:
VG 7070 EQ-HF. Ϫ NMR: Bruker AC-200, AC-300, and AMX-
500 (200 MHz, 300 MHz, and 500 MHz for 1H; 50.3 MHz,
75.4 MHz and 125.7 MHz for 13C), for 13C spectra only selected
signals are reported, for spectra in CDCl3 TMS as internal stand-
ard, for spectra in CD3OD δH ϭ 3.3, for spectra in [D6]DMSO
Benzyl Ether 10: A solution of the diol 3 (735 mg, 2.4 mmol) and
Bu2SnO (596 mg, 2.4 mmol) in dry benzene (13 mL) was refluxed
˚
under N2 and 4-A molecular sieves interposed between the flask
and the reflux condenser. After 6 h, the solution was concentrated
to half its volume, before adding Bu4NI (880 mg, 2.4 mmol) and
benzyl bromide (0.595 mL, 5 mmol). The resulting solution was
refluxed under N2 for 3 h, then the solvent evaporated, and the
crude product purified by flash chromatography (hexane/AcOEt,
20
8:2) to yield 10 (970 mg, 99%), m.p. 108Ϫ110°C. Ϫ [α]D ϭ ϩ15
1
(c ϭ 1, EtOH). Ϫ H NMR (200 MHz, CDCl3): δ ϭ 1.3Ϫ1.7 (m,
δH ϭ 2.5, for spectra in C6D6 δH ϭ 7.2. Ϫ IR: PerkinϪElmer FTIR
1 H, 5ax-H), 1.45 [s, 18 H, OC(CH3)3], 1.70Ϫ1.92 (m, 1 H, 2ax-H),
2.1 (ddd, Jgem ϭ 14 Hz, J2eq,1 ϭ J2eq,3 ϭ 4.6 Hz, 1 H, 2eq-H),
2.20Ϫ2.35 (m, 1 H, 5eq-H), 2.40Ϫ2.58 (m, 1 H, 1-H), 2.84Ϫ3.04
(m, 1 H, 6-H), 3.38Ϫ3.54 (m, 1 H, 3-H), 4.10Ϫ4.20 (m, 1 H, 4-H),
4.60 (m, 2 H, CH2Ph), 7.35 (s, 5 H, aromatic H). Ϫ 13C NMR
(75.4 MHz, CDCl3): δ ϭ 28.3, 28.6, 33.3, 39.9, 44.9, 66.5, 71.0,
78.0, 80.9, 81.1, 128.2, 128.5, 129.1, 173.4, 174.8. Ϫ IR (CHCl3):
1600 spectrometer. Ϫ Optical rotations: PerkinϪElmer 241 at
589 nm, using 1-mL cells. Ϫ Elemental analysis: PerkinϪElmer
240. Ϫ Starting materials: 11,[14] 12,[15] 17.[19] For spectral assign-
ments the DCCHD moiety was numbered as shown in Figure 1.
(1S,2S)-Cyclohex-4-ene-1,2-dicarboxylic Acid (8): The fumarate 6
(2.6 mL, 9.5 mmol) was dissolved in 60 mL of dry CH2Cl2, the
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Eur. J. Org. Chem. 1999, 1311Ϫ1317

Synthesis of a Pseudo Tetrasaccharide Mimic of Ganglioside GM1
ν˜ ϭ 3550 cmϪ1 (OH), 1710 (CϭO), 1600 (CϭC). Ϫ C23H34O6 2.2 (s, 3 H, COCH3), 3.9Ϫ4.4 (m, 7 H, 3a-H, 5a-H, 6a-H, 6Јa-H,
FULL PAPER
(406.5): calcd. C 67.96, H 8.43; found C 68.11, H 8.21.
5b-H, 6b-H, 6Јb-H), 4.6 (m, 1 H, 2a-H), 4.72 (d, J1,2 ϭ 7.5 Hz, 1
H, 1b-H), 5.0 (dd, J3,4 ϭ 3 Hz, J3,2 ϭ 10 Hz, 1 H, 3b-H), 5.2 (dd,
J2,1 ϭ 7.5 Hz, J2,3 ϭ 10 Hz, 1 H, 2b-H), 5.4 (dd, J4,3 ϭ 3 Hz, J4,5
< 1 Hz, 1 H, 4b-H), 5.47 (dd, J4,3 ϭ 2.5 Hz, J4,5 < 1 Hz, 1 H, 4a-
H), 5.72 (d, JNH,2 ϭ 7.4 Hz, 1 H, NHAc), 6.55 (d, J1,2 ϭ 3 Hz, 1
H, 1a-H), 8.74 (s, 1 H, NHϭC).
Allyl O-(2,3,4,6-Tetraacetyl-β- -galactopyranosyl)-(1؊3)-2-acetami-
D
do-2-deoxy-2,3-di-O-pivaloyl-β- -galactopyranoside (13): BF3 · Et2O
D
(0.07 mL, 0.58 mmol) was added to a refluxing solution of 11[14]
(226 mg, 0.52 mmol) under N2 in dry CH2Cl2 (2 mL), then a solu-
tion of 12[15] (285 mg, 0.58 mmol) in dry CH2Cl2 (2.3 mL) was
added dropwise. After 2 h, a few drops of Et3N were added (to
pH ϭ 6Ϫ7) and the mixture concentrated to dryness. Flash chro-
Synthesis of 16: TMSOTf (0.013 mL, 0.07 mmol) was added to a
refluxing solution of 15 (100 mg, 0.116 mmol) and 10 (118 mg,
0.29 mmol) in dry CH2Cl2 under N2 After 1 h, a few drops of Et3N
were added and the solvent evaporated. The product 16 (60 mg,
47%) was isolated by flash chromatography (AcOEt/hexane, 55:45),
which also allowed recovery of the excess of 10 (77 mg, 0.19 mmol).
matography (hexane/AcOEt, 2:8) gave 13 in a yield of 53%. Ϫ [α]
20
ϭ ϩ23 (c ϭ 1.07, CHCl3). Ϫ 1H NMR (200 MHz, CDCl3):
D
δ ϭ 1.22, 1.25 [2 s, 18 H, COC(CH3)3], 1.98, 2.0, 2.05, 2.08, 2.14
(5 s, 15 H, COCH3), 3.16Ϫ3.32 (m, 1 H, 2a-H), 3.81Ϫ3.96 (m, 2
H, 5a-H, 5b-H), 3.98Ϫ4.24 (m, 5 H, 6a-H, 6Јa-H, 6b-H, 6Јb-H,
ϪHCHϪCHϭCH2), 4.33 (dd, Jgem ϭ 13 Hz, Jvic ϭ 5.5 Hz, 1 H,
ϪHCHϪCHϭCH2), 4.61 (d, J1,2 ϭ 7.5 Hz, 1 H, 1b-H), 4.74 (dd,
J3,2 ϭ 10.5 Hz, J3,4 ϭ 4 Hz, 1 H, 3a-H), 4.96 (dd, J3,2 ϭ 10 Hz,
J3,4 ϭ 4 Hz, 1 H, 3b-H), 5.03Ϫ5.16 (m, 1 H, 2b-H), 5.08 (d, J1,2
8.5 Hz, 1 H, 1a-H), 5.18Ϫ5.38 (m, 2 H, ϪCHϭCH2), 5.32 (dd,
J4,3 ϭ 4 Hz, J4,5 < 1 Hz, 1 H, 4b-H), 5.41 (dd, J4,3 ϭ 4 Hz, J4,5
1 Hz, 1 H, 4a-H), 5.7 (d, JNH,2 ϭ 7 Hz, 1 H, NH), 5.77Ϫ5.98 (m,
1 H, ϪCHϭCH2). Ϫ 13C NMR (50.3 MHz, CDCl3): δ ϭ 20.6,
23.6, 27.0, 38.6, 55.6, 61.0, 62.5, 66.7, 68.5, 69.1, 70.2, 70.5, 70.8,
71.5, 97.9, 100.9, 118.0, 133.6. Ϫ C35H53NO17 (759.8): calcd. C
55.33, H 7.03, N 1.84; found C 55.05, H 7.11, N 1.71.
20
Ϫ [α]D ϭ ϩ11.9 (c ϭ 1.02, CHCl3). Ϫ 1H NMR (300 MHz,
C6D6): δ ϭ 1.25 (m, 1 H, 5cax-H), 1.27 [s, 9 H, COC(CH3)3], 1.3
[s, 9 H, COC(CH3)3], 1.4 [s, 9 H, OC(CH3)3], 1.45 [s, 9 H,
OC(CH3)3], 1.62, 1.65, 1.72, 1.74, 1.79 (5 s, 15 H, COCH3), 2.09
(m, 1 H, 2cax-H), 2.25 (ddd, Jgem ϭ 12 Hz, J2eq,1 ϭ J2eq,3 ϭ 4 Hz,
1 H, 2ceq-H), 2.43 (ddd, Jgem ϭ 14 Hz, J5eq,6 ϭ J5eq,4 ϭ 4 Hz, 1 H,
5ceq-H), 2.78 (ddd, J1,6 ϭ J1,2ax ϭ 12 Hz, J1,2eq ϭ 4 Hz, 1 H, 1c-
H), 3.05 (ddd, J3,2ax ϭ 12 Hz, J3,2eq ϭ 4 Hz, J3,4 ϭ 2 Hz, 1 H, 3c-
H), 3.25 (m, 2 H, 2a-H, 6c-H), 3.32 (ddd, J5,6 ϭ J5,6Ј ϭ 6.5 Hz, J5,4
< 1 Hz, 1 H, 5a-H), 3.71 (ddd, J5,6 ϭ J5,6Ј ϭ 6.5 Hz, J5,4 < 1 Hz,
1 H, 5b-H), 4.05Ϫ4.4 [m, 8 H, H-H COSY: 4.1 (1 H, 4c-H), 4.15
(2 H, 6a-H, 6Јa-H), 4.20 (1 H, 6b-H), 4.30 (1 H, 6Јb-H) 4.32 (2 H,
CH2Ph), 4.35 (d, J1,2 ϭ 8 Hz, 1 H, 1b-H)], 4.75 (dd, J3,2 ϭ 11 Hz,
J3,4 ϭ 4 Hz, 1 H, 3a-H), 4.8 (d, JNH,2 ϭ 7 Hz, 1 H, NH), 5.08 (dd,
J3,2 ϭ 11 Hz, J3,4 ϭ 3 Hz, 1 H, 3b-H), 5.35Ϫ5.5 [m, 4 H, H-H
COSY: 5.36 (1 H, 2b-H), 5.4 (d, J1,2 ϭ 9 ± 1 Hz, 1 H, 1a-H), 5.4
(1 H, 4a-H), 5.41 (1 H, 4b-H)], 7.2Ϫ7.4 (m, 5 H, aromatic H). Ϫ
13C NMR (75.4 MHz, C6D6): δ ϭ 20.9, 24.2, 28.0, 28.7, 29.7, 34.5,
41.0, 45.5, 56.6, 61.7, 63.4, 67.8, 69.9, 70.2, 71.3, 71.5, 72.1, 72.5,
73.5, 76.8, 79.9, 80.7, 80.9, 100.5, 102.4. Ϫ C55H81NO22 (1108.2),
calcd. C 59.61, H 7.37, N 1.27; found C 59.89, H 7.31, N 1.34.
ϭ
<
(2,3,4,6-Tetraacetyl-β-
deoxy-2,3-di-O-pivaloyl-Ͱ/β-
D
-galactopyranosyl)-(1؊3)-2-acetamido-2-
-galactopyranoside (14): DABCO
D
(9 mg, 0.08 mmol) and (Ph3P)3RhCl (22 mg, 0.024 mmol) were ad-
ded to a solution of 13 (300 mg, 0.39 mmol) in 9:1 EtOH/H2O (4
mL). The mixture was refluxed for 5 h, then concentrated to dry-
ness. The residue was filtered through a short silica gel column
(hexane/AcOEt, 3:7) and the crude product (225 mg) was dissolved
in 4:1 THF/H2O (3 mL). I2 (149 mg, 0.59 mmol) was added at
room temp., the solution stirred for 30 min, H2O added, and the
mixture extracted with CHCl3. The organic phase was washed with
5% Na2S2O5 (2 ϫ), dried with Na2SO4, and concentrated in vacuo.
The crude product was purified by flash chromatography (AcOEt/
CHCl3/MeOH, 9:1:0.2) to yield 14 (165 mg, 59%) as an α/β mix-
ture. Ϫ [α]D20 ϭ ϩ38.7 (c ϭ 1.14, CHCl3) (α anomer). Ϫ 1H NMR
(300 MHz, CDCl3): δ ϭ (α anomer) 1.2, 1.23 [2 s, 18 H,
COC(CH3)3], 1.75 (s, 3 H, COCH3), 2.0 (s, 3 H, COCH3), 2.05 (s,
6 H, COCH3), 2.16 (s, 3 H, COCH3), 3.1 (br. s, 1 H, OH), 3.87
(ddd, J5,6 ϭ J5,6Ј ϭ 6 Hz, J5,4 < 1 Hz, 1 H, 5b-H), 3.92Ϫ4.1 (m, 3
H, 3a-H, 6a-H, 6Јa-H), 4.13 (m, 2 H, 6b-H, 6Јb-H), 4.33 (ddd,
J5,6 ϭ J5,6Ј ϭ 6 Hz, J5,4 < 1 Hz, 1 H, 5a-H), 4.42 (m, 1 H, 2a-H),
Synthesis of 4: 10% Pd/C was added to a solution of 16 (61 mg,
0.055 mmol) in MeOH (3 mL) and the resulting mixture stirred for
1 h at room temp. under H2. The catalyst was filtered off, the sol-
vent evaporated, and the crude product purified by flash chroma-
tography (CHCl3/acetone/MeOH, 85:15:0.2) to yield 4 (40 mg,
20
1
73%). Ϫ [α]D ϭ ϩ33 (c ϭ 1.55, CHCl3). Ϫ H NMR (300 MHz,
CDCl3): δ ϭ 1.2 [s, 9 H, COC(CH3)3], 1.3 [s, 9 H, COC(CH3)3],
1.42 [s, 9 H, OC(CH3)3], 1.47 [s, 9 H, OC(CH3)3], 1.5 (m, 1 H, 2cax-
H), 1.63 (m, 1 H, 5cax-H), 1.86 (br. s, 1 H, OH), 1.96 (s, 3 H,
COCH3), 1.98 (s, 3 H, COCH3), 2.0 (m, 1 H, 5ceq-H), 2.06 (s, 6 H,
COCH3), 2.14 (s, 3 H, COCH3), 2.33 (ddd, Jgem ϭ 13 Hz, J2eq,1
ϭ
J2eq,3 ϭ 3.5 Hz, 1 H, 2ceq-H), 2.54 (ddd, J6,1 ϭ J6,5ax ϭ 11 Hz,
J6,5eq ϭ 3.3 Hz, 1 H, 6c-H), 2.83 (ddd, J1,6 ϭ J1,2ax ϭ 11 Hz,
4.61 (d, J1,2 ϭ 8 Hz, 1 H, 1b-H), 4.95 (dd, J3,2 ϭ 10 Hz, J3,4
4 Hz, 1 H, 3b-H), 5.1 (dd, J2,3 ϭ 10 Hz, J2,1 ϭ 8 Hz, 1 H, 2b-H),
5.35 (dd, J4,3 ϭ 4 Hz, J4,5 < 1 Hz, 1 H, 4b-H), 5.37 (br. s, 1 H, 1a-
ϭ
J
1,2eq ϭ 3.5 Hz, 1 H, 1c-H), 3.42 (ddd, J2,1 ϭ 8.7 Hz, J2,3 ϭ 11 Hz,
J2,NH ϭ 6.4 Hz, 1 H, 2a-H), 3.58 (m, 1 H, 4c-H), 3.7Ϫ4.1 [m, 7 H,
H-H COSY: 3.83 (2 H, 5a-H, 5b-H), 3.94 (2 H, 6a-H, 6b-H), 3.95
(1 H, 4c-H), 4.08 (2 H, 6Јa-H, 6Јb-H)], 4.38 (dd, J3,2 ϭ 11 Hz,
J3,4 ϭ 3.2 Hz, 1 H, 3a-H), 4.6 (d, J1,2 ϭ 7.4 Hz, 1 H, 1b-H), 4.98
(dd, J3,2 ϭ 10 Hz, J3,4 ϭ 3.5 Hz, 1 H, 3b-H), 5.1 (dd, J2,1 ϭ 7.4 Hz,
J2,3 ϭ 10 Hz, 1 H, 2b-H), 5.3 (d, J1,2 ϭ 8.7 Hz, 1 H, 1a-H), 5.35
(dd, J4,3 ϭ 3.5 Hz, J4,5 < 1 Hz, 1 H, 4b-H), 5.4 (dd, J4,3 ϭ 3.2 Hz,
J4,5 < 1 Hz, 1 H, 4a-H), 5.86 (d, JNH,2 ϭ 6.4 Hz, 1 H, NH). Ϫ 13C
NMR (50.3 MHz, CDCl3): δ ϭ 20.4, 20.5, 20.6, 20.7, 23.5, 26.9,
27.1, 27.8, 31.5, 33.0, 39.7, 44.1, 55.3, 60.9, 62.2, 66.8, 68.2, 69.1,
70.5, 70.6, 70.7, 71.7, 75.8, 76.7, 100.1, 100.8. Ϫ C48H75NO22
(1018.1): calcd. C 56.63, H 7.43, N 1.38; found C 56.48, H 7.65,
N 1.21.
H), 5.4 (dd, J4,3 ϭ 3.5 Hz, J4,5 < 1 Hz, 1 H, 4a-H), 5.7 (d, JNH,2
ϭ
8 Hz, 1 H, NH). Ϫ 13C NMR (50.3 MHz, CDCl3): δ (α anomer) ϭ
20.5, 23.1, 26.9, 39.6, 39.9, 49.4, 61.0, 62.0, 66.7, 67.0, 68.5, 68.7,
70.5, 70.9, 73.0, 91.9, 94.0, 169.5, 170.3, 176.7, 178.1.
C32H49NO17 (719.7): calcd. C 53.40, H 6.86, N 1.95; found C 52.62,
H 6.77, N 1.67.
Ϫ
Trichloroacetimidate 15: Cl3CCN (0.1 mL, 1 mmol) and DBU
(0.005 mL, 0.033 mmol) were added to a solution of 14 (132 mg,
0.18 mmol) in dry CH2Cl2 (1.8 mL). After 30 min at room temp.
under N2, the reaction mixture was concentrated in vacuo and the
residue filtered through a short silica gel column (hexane/AcOEt,
1
2:8) to give 15 (148 mg, 95%). Ϫ H NMR (200 MHz, CDCl3): δ
(α anomer) ϭ 1.15 [s, 9 H COC(CH3)3], 1.25 [s, 9 H, COC(CH3)3], 3-Sialyl-DCCHD 5: A solution of the sialyl phosphite 17[19]
1.98 (s, 6 H, COCH3), 2.07 (s, 3 H, COCH3), 2.1 (s, 3 H, COCH3),
Eur. J. Org. Chem. 1999, 1311Ϫ1317
(296 mg, 0.48 mmol) in dry EtCN (0.75 mL) was added dropwise
1315

A. Bernardi et al.
FULL PAPER
to a solution of 3 (151 mg, 0.48 mmol) and TMSOTf (0.02 mL, Ϫ C68H102N2O34 (1491.6): calcd. C 54.76, H 6.89, N 1.88; found C
0.11 mmol) in dry EtCN (0.8 mL) at Ϫ40°C under N2. After 3 h 54.57, H 6.81, N 2.24. Ϫ MS (FAB); m/z: 1512 [Mϩ ϩ Na].
the reaction was neutralized with Et3N, the solvent evaporated, and
the residue purified by flash chromatography (hexane/acetone/
MeOH, 6:4:0.1) to give 5 as a 7:1 α/β mixture contaminated by the
Neu5Ac glycal. A second flash chromatography (CHCl3/acetone,
Pseudo GM1 2: 1  MeONa in MeOH (0.2 mL) was added to a
solution of 18 (44 mg, 0.03 mmol) in dry MeOH (3 mL). After
stirring for 24 h at room temp. under N2, H2O (0.6 mL) was added
to the reaction mixture. The solution was stirred for additional
20
from 8:2 to 7:3) yielded the α anomer (76 mg, 20%). Ϫ [α]D
ϭ
12 h, then Amberlite IR 120 (Hϩ form) was added and the solvent
1
Ϫ7.6 (c ϭ 1.6, CHCl3). Ϫ H NMR (300 MHz, C6D6): δ ϭ 1.36,
1.42 [2 s, 18 H, OC(CH3)3], 1.55, 1.58 (2 s, 6 H, COCH3), 1.70 (m,
1 H, 5cax-H), 1.82 (s, 3 H, COCH3), 1.92 (s, 3 H, COCH3), 2.0 (m,
evaporated. The product 2 (24 mg, 82%) was isolated by flash chro-
20
matography (CHCl3/MeOH/H2O, 60:35:5). Ϫ [α]D ϭ ϩ8.6 (c ϭ
1
0.35, MeOH). Ϫ H NMR (500 MHz, D2O): δ ϭ 1.50, 1.53 [2 s,
1 H, 3dax-H), 2.02 (s, 3 H, COCH3), 2.22 (ddd, Jgem ϭ J2ax,3
2ax,1 ϭ 12.5 Hz, 1 H, 2cax-H), 2.35Ϫ2.5 [m, 2 H, H-H COSY: 2.38
(1 H, 2ceq-H), 2.42 (1 H, 5ceq-H)], 2.62 (dd, Jgem ϭ 12.5 Hz, J3eq,4 ϭ
4.5 Hz, 1 H, 3deq-H), 3.12 (ddd, J1,6 ϭ J1,2ax ϭ 12.5 Hz, J1,2eq
4 Hz, 1 H, 1c-H), 3.32 (s, 3 H, OCH3), 3.37 (m, 1 H, 6c-H), 3.95
(m, 1 H, 4c-H), 4.0Ϫ4.55 [m, 5 H, H-H COSY: 4.05 (dd, J6,5
ϭ
18 H, OC(CH3)], 1.65Ϫ4.97 [37 H, H-H COSY: 1.69 (m, 1 H, 5cax-
H), 1.72 (m, 1 H, 2cax-H), 1.85 (dd, Jgem ϭ J3ax,4 ϭ 12 Hz, 1 H,
3dax-H), 2.09 (m, 1 H, 2ceq-H), 2.07, 2.10 (2 s, 6 H, COCH3), 2.26
(ddd, Jgem ϭ 14 Hz, J5eq,6 ϭ J5eq,4 ϭ 4 Hz, 1 H, 5ceq-H), 2.66 (ddd,
J
ϭ
J1,6 ϭ J1,2ax ϭ 12 Hz, J1,2eq ϭ 4 Hz, 1 H, 1c-H), 2.76 (dd, Jgem
ϭ
ϭ
12 Hz, J3eq,4 ϭ 5 Hz, 1 H, 3deq-H), 2.79 (ddd, J6,1 ϭ J6,5ax ϭ 12 Hz,
J6,5eq ϭ 4 Hz, 1 H, 6c-H), 3.56Ϫ3.62 (m, 2 H, 2b-H, 6d-H), 3.69
(m, 1 H, 7d-H), 3.7 (m, 1 H, 3b-H), 3.75 (m, 2 H, 5b-H, 9d-H),
3.78 (m, 1 H, 5a-H), 3.80 (m, 1 H, 4d-H), 3.83 (m, 3 H, 6a-H, 6b-
H, 6Јb-H), 3.88 (m, 3 H, 6Јa-H, 5d-H, 8d-H), 3.92 (m, 1 H, 3a-H),
3.93 (m, 1 H, 9Јd-H), 3.98 (dd, J4,3 ϭ 3 Hz, J4,5 < 1 Hz, 1 H, 4b-
H), 4.11 (dd, J2,1 ϭ 8 Hz, J2,3 ϭ 10.5 Hz, 1 H, 2a-H), 4.12 (m, 1
H, 3c-H), 4.21 (m, 1 H, 4c-H), 4.24 (dd, J4,3 ϭ 3 Hz, J4,5 < 1 Hz,
1 H, 4a-H), 4.57 (d, J1,2 ϭ 7.5 Hz, 1 H, 1b-H), 4.96 (d, J1,2 ϭ 8 Hz,
1 H, 1a-H)]. Ϫ 13C NMR (125.7 MHz, D2O): δ ϭ 29.3, 32.5, 38.2,
39.4, 44.1, 51.0, 51.1, 60.5, 62.0, 67.6, 68.2, 68.4, 70.2, 71.6, 72.1,
72.4, 72.6, 74.3, 74.7, 74.8, 79.8, 82.3, 82.4, 101.7, 104.5. Ϫ
C41H68N2O24 (973.0): calcd. C 50.61, H 7.04, N 2.88; found C
50.74, H 6.85, N 2.71.
11 Hz, J6,7 ϭ 3 Hz, 1 H, 6d-H), 4.12 (d, JNH,5 ϭ 10 Hz, 1 H, NH),
4.3 (1 H, 3c-H), 4.4 (2 H, 5d-H, 9d-H)], 4.65 (dd, J9Ј,9 ϭ 13 Hz,
J
9Ј,8 ϭ 3 Hz, 1 H, 9Јd-H), 4.81 (ddd, J4,3ax ϭ J4,5 ϭ 11 Hz, J4,3eq ϭ
4.5 Hz, 1 H, 4d-H), 5.48 (dd, J7,8 ϭ 8 Hz, J7,6 ϭ 3 Hz, 1 H, 7d-H),
5.80 (ddd, J8,7 ϭ J8,9 ϭ 8 Hz, J8,9Ј ϭ 3 Hz, 1 H, 8d-H). Ϫ 13C
NMR (75.4 MHz, CDCl3) δ ϭ 20.4, 20.6, 21.1, 22.9, 28.0, 30.1,
33.6, 38.2, 39.6, 44.6, 49.0, 52.4, 62.7, 67.4, 67.6, 69.3, 70.0, 73.4,
74.6, 80.6, 98.0 (C-2d), 168.5 (1d-C, JC-1,3-Hax ϭ 6.9 Hz). Ϫ
C36H55NO18 (789.8): calcd. C 54.75, H 7.02, N 1.77; found C 54.96,
H 6.81, N 1.93.
Synthesis of 18: TMSOTf (0.007 mL, 0.043 mmol) was added to a
refluxing solution of 5 (57 mg, 0.072 mmol) and 15 (146 mg,
0.17 mmol) in dry CH2Cl2 (0.4 mL) under N2. After 5 h, the mix-
ture was neutralized with Et3N and the solvent evaporated. The
product 18 (32 mg, 30%) was isolated by flash chromatography
(CHCl3/acetone, 7:3) and further purified by a second chromatog-
Acknowledgments
20
This work was supported by CNR and MURST (COFIN
1998Ϫ99). We wish to thank Prof. Luigi Panza for helpful dis-
cussions and suggestions.
raphy (toluene/acetone, 6:4). Ϫ [α]D ϭ ϩ4.8 (c ϭ 0.8, CHCl3). Ϫ
1H NMR (500 MHz, C6D6): δ ϭ 1.25 [s, 9 H, COC(CH3)3], 1.32
[s, 9 H, COC(CH3)3], 1.38 [s, 9 H, OC(CH3)3], 1.47 [s, 9 H,
OC(CH3)3], 1.59 (s, 3 H, COCH3), 1.63 (s, 3 H, COCH3), 1.67 (s,
3 H, COCH3), 1.70 (s, 6 H, COCH3), 1.74 (s, 3 H, COCH3),
1.75Ϫ5.85 [48 H, H-H COSY: 1.80 (m, 1 H, 5cax-H), 1.83 (s, 3 H,
COCH3), 1.91 (s, 6 H, COCH3), 2.08 (s, 3 H, COCH3), 2.1 (m, 1
H, 3dax-H), 2.2 (m, 1 H, 2cax-H), 2.45 (ddd, Jgem ϭ 12 Hz, J2eq,1 ϭ
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J5eq,4 ϭ 4 Hz, 1 H, 5ceq-H), 3.08 (dd, Jgem ϭ 13.5 Hz, J3eq,4
ϭ
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1 Hz, 1 H, 5a-H), 4.02 (dd, J6,7 ϭ 2.2 Hz, J6,5 ϭ 10.7 Hz, 1 H, 6d-
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NHd), 4.30 (m, 2 H, 6a-H, 6Јa-H), 4.35 (m, 1 H, 3c-H), 4.40 (m,
1 H, 9d-H), 4.42 (m, 1 H, 5d-H), 4.55 (d, J1,2 ϭ 7.7 Hz, 1 H, lb-
H), 4.65 (dd, Jgem ϭ 12.5 Hz, J9Ј,8 ϭ 2.5 Hz, 1 H, 9Јd-H), 4.92
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U. Greilich, R. Brescello, K.-H. Jung, R. R.
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ϭ
[5c]
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J1,2 ϭ 8.7 Hz, 1 H, 1a-H), 5.42 (dd, J2,1 ϭ 7.7 Hz, J2,3 ϭ 11 Hz, 1
H, 2b-H), 5.45 (dd, J4,3 ϭ 3.5 Hz, J4,5 < 1 Hz, 1 H, 4b-H), 5.48
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3.8 Hz, J4,5 < 1 Hz, 1 H, 4a-H), 5.80 (ddd, J8,9 ϭ 2.5 Hz, J8,9Ј
ϭ
ϭ
6.6 Hz, J8,7 ϭ 7.7 Hz, 1 H, 8d-H)]. Ϫ 13C NMR (125.7 MHz,
C6D6): δ ϭ 27.3, 27.4, 28.0, 28.1, 30.0, 31.3, 38.6, 39.2, 40.4, 44.7,
49.4, 52.5, 55.8, 60.9, 62.4, 62.8, 67.1, 67.6, 69.3, 69.6, 69.8, 70.6,
71.5, 73.3, 74.3, 75.8, 80.0, 80.2, 99.3, 100.3, 101.8, 145.9, 149.4,
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Eur. J. Org. Chem. 1999, 1311Ϫ1317

Synthesis of a Pseudo Tetrasaccharide Mimic of Ganglioside GM1
FULL PAPER
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1317