Stereocontrolled and convergent entry to CF2-sialosides: Synthesis of CF2-linked ganglioside GM4

Article abstract of DOI:10.1021/ja075738w

Sialidase-resistant ganglioside analogues having biological activities similar to those of natural gangliosides are expected to be important probes for clarifying the biological functions of gangliosides. Focusing on difluoromethylene-linked (CF₂-linked) α(2,3)sialylgalactose as a core structure of sialidase-resistant ganglioside mimics, we have developed novel, stereocontrolled, and efficient methodologies to synthesize CF₂-sialosides based on Ireland-Claisen rearrangement. CF₂-linked α(2,3)sialylgalactose and CF₂-linked GM4 were synthesized. Copyright

Full text of DOI:10.1021/ja075738w

Published on Web 11/23/2007
Stereocontrolled and Convergent Entry to CF2-Sialosides: Synthesis of
CF2-Linked Ganglioside GM4
Go Hirai,† Toru Watanabe,† Kazunori Yamaguchi,‡ Taeko Miyagi,‡ and Mikiko Sodeoka*,†
Synthetic Organic Chemistry Laboratory, RIKEN, Hirosawa, Wako 351-0198, Japan, DiVision of Biochemistry,
Miyagi Cancer Center Research Institute, Natori 981-1293, Japan, and CREST, JST, Kawaguchi 332-1102, Japan
Received July 31, 2007; E-mail: sodeoka@riken.jp
Gangliosides (sialic acid-containing glycosphingolipids) are
known to be involved in intra- and intercellular cell signaling.1,2
For example, GM3 binds to epidermal growth factor receptor
(EGFR) and inhibits EGFR-dependent cell proliferation.3 Among
the four known human sialidases (neuraminidase),4 NEU2, NEU3,
and NEU4 but not NEU1 cleave the glycosidic linkage of sialic
acid in gangliosides.4a,5 Plasma membrane-associated sialidase
Figure 1. Structure of GM4 (1) and CF2-linked GM4 (2).
(NEU3) seems to play a critical role in cell survival because it is
upregulated in human cancer cells and tissues.6 However, the
physiological roles of gangliosides are still not fully clarified, partly
because rapid metabolism of gangliosides makes biological research
difficult. For example, sialidases hydrolyze GM3 and GM4 (1) to
lactosylceramide and galactosylceramide,7 which show different
biological activities.8 Therefore, chemically and biologically stable
ganglioside analogues would be very useful as probes for research
to elucidate the roles of these gangliosides in normal and cancer
cells. R-Difluorophosphonate derivatives are excellent nonhydro-
lyzable phosphate ester mimics, and the difluoromethylene group
is now recognized as bioisosteric to an oxygen atom.9 Therefore,
we envisioned the synthesis of difluoromethylene-linked (CF2-
linked) R(2,3)sialylgalactose (Figure 1) as a core structure of
sialidase-resistant ganglioside mimics,10 because the R(2,3)sialyl-
Figure 2. Strategy for the stereocontrolled synthesis of CF2-sialoside.
galactose structure is a key structure of not only GM3 and GM4,
but also most other gangliosides.
converted to the desired CF2-R(2,3)sialylgalactose unit 3 in a
stereocontrolled manner.
Various C-glycoside compounds, in which the oxygen atom of
the glycosidic linkage is replaced by a carbon atom, have already
been synthesized as hydrolytically stable glycoside mimics,11 but
efficient and stereoselective synthesis of C-sialoside is difficult,
because the anomeric position of the sialoside (C2′) is a tetra-
substituted carbon center.12 To our knowledge, only two method-
ologies for the stereoselective synthesis of the C-linked R(2,3)-
sialylgalactose have been developed,13 and synthesis of CF2-linked
R(2,3)sialylgalactose has not been reported. Linhardt and co-workers
have reported a convergent and stereoselective synthesis of CH-
(OH)-linked R(2,3)sialylgalactose derivatives based on the SmI2-
mediated coupling reaction13a,14 of the sialylsulfone derivative with
the aldehyde prepared from galactose. First, we examined trans-
formation of their CH(OH)-linked R(2,3)sialylgalactose to CF2-
linked R(2,3)sialylgalactose. However, this was unsuccessful,
probably due to the steric hindrance around the C-glycoside carbon
linkage.15 Therefore, for the synthesis of the CF2-linked R(2,3)-
sialylgalactose unit, a new strategy was required. Herein we report
a convergent and highly stereoselective method for the construction
of the CF2-linked R(2,3)sialylgalactose unit, and the conversion of
this unit to the CF2-linked analogue of ganglioside GM4 (2).16
As shown in Figure 2, we planned to construct the key C2′-
CF2 bond via Ireland-Claisen rearrangement of the ester III.17 We
anticipated that rearrangement would occur from the R-face of the
anomeric center (C2′) through the chairlike transition state II after
formation of the (Z)-silyl enolate. The resulting product I could be
The precursor for the Ireland-Claisen rearrangement 9 was
prepared by condensation of 418 with 8, which was prepared from
615 as shown in Scheme 1. Ireland-Claisen rearrangement of 9
proceeded smoothly even at ambient temperature on treatment with
LHMDS and TMSCl in THF. After treatment with TMS-
diazomethane, methyl ester 10 possessing a R-CF2-sialoside linkage
was obtained as a single isomer in 86% yield. The strong HMBC
correlation between R3′-H and 1′-C indicated that the C2′ tetra-
substituted carbon center has the desired R-stereochemistry.
To achieve stereocontrolled introduction of a C2 hydroxyl group
into the galactose unit, 10 was converted to the conformationally
fixed lactone 11. After removal of p-methoxybenzylidene acetal,
protection of the 6-OH group with TBS, and saponification of the
methyl ester, the desired lactone 11 was obtained in good yield by
treatment with carbodiimide (Scheme 2). Although attempts at
hydroboration or epoxidation of 11 were unsuccessful, we were
pleased to find that dihydroxylation of 11 using a stoichiometric
amount of OsO4 proceeded in a completely stereoselective manner
to afford the desired diol 12. Regio- and stereoselective reduction
of the C3-hydroxyl group was successfully achieved by radical
reduction of the cyclic thiocarbonate. Namely, treatment of 12 with
thiophosgene, followed by AIBN and Bu3SnH, gave 13 as a single
isomer. Hydrogenolysis of the four BOM groups, together with
removal of the TBS group, afforded the key 4-methoxyphenyl CF2-
linked R(2,3)sialylgalactose lactone unit 14. The stereochemical
assignment of the newly formed chiral carbon centers (C2′, C2,
and C3) was confirmed by X-ray crystallographic analysis of 14.
† Synthetic Organic Chemistry Laboratory, RIKEN.
‡ Miyagi Cancer Center Research Institute and CREST.
9
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J. AM. CHEM. SOC. 2007, 129, 15420-15421
10.1021/ja075738w CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1 a
sialylgalactose unit was confirmed to be suitable as a glycosyl donor
for the synthesis of a GM4 analogue. This new strategy based on
Ireland-Claisen rearrangement should be applicable not only for
the synthesis of CF2-sialylgalactose but also for the construction
of various other (un)substituted C-sialoside units, which are
expected to be useful for the synthesis of novel ganglioside-
mimicking molecules. Synthesis of larger gangliosides, such as a
GM3, and biological experiments using CF2-linked ganglioside
analogues are currently under way.
Acknowledgment. We thank Ms. Setsuko Moriya for evaluation
of sialidase inhibitory activity, Dr. Daisuke Hashizume for the X-ray
crystallographic analysis, and Dr. Hiroyuki Koshino for 1D and
2D-NMR measurements.
Supporting Information Available: Experimental procedure,
1
characterization of new compounds, biological evaluations, H NMR
a Reaction conditions: (a) CF2Br2, HMPT, THF, rt, 4 h, 76% (see ref
19); (b) conc. HCl, MeOH-CH2Cl2 (4:1), rt, 92%; (c) (4-OMe)Ph-
CH(OMe)2, TsOH, CH3CN, rt; (d) TBAF, THF, rt, 74% (two steps);
(e) EDC‚HCl, DMAP, 4, CH2Cl2, rt, 72%; (f) LHMDS, TMSCl, THF,
-78 °C then rt; (g) TMSCHN2, Et2O-MeOH (1:1), 86% (two steps).
and 13C NMR spectra, and CIF file for compound 14. This material is
available free of charge via the Internet at http://pubs.acs.org.
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The overall yield of this CF2-linked sialoside unit from 6 was 13%
(15 steps).
To demonstrate the potential of this novel CF2-linked sialylga-
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analogues, conversion of 14 to CF2-linked GM4 (2) was performed.
After acetylation of all hydroxyl groups, the MP group at the
anomeric position was converted to trichloroacetoimidate to give
a glycosyl donor 15. Glycosylation of 15 with the ceramide
derivative 1620 was conducted in the presence of TfOH in CH2Cl2.
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in a completely stereoselective manner, and this nonhydrolyzable
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