Synthesis of immunostimulatory α- C -galactosylceramide glycolipids via sonogashira coupling, asymmetric epoxidation, and trichloroacetimidate- mediated epoxide opening

Article abstract of DOI:10.1021/ol1009976

Stereocontrolled syntheses of α-C-GalCer (2) and its α-C-acetylenic analogue 6 were accomplished in high efficiency by a convergent construction strategy from 1-hexadecene and d-galactose. The key transformations include Sonogashira coupling, Sharpless asymmetric epoxidation, and Et₂AlCl-catalyzed cyclization of an epoxytrichloroacetimidate to generate protected dihydrooxazine 21.

Full text of DOI:10.1021/ol1009976

ORGANIC
LETTERS
2010
Vol. 12, No. 13
2974-2977
Synthesis of Immunostimulatory
r-C-Galactosylceramide Glycolipids via
Sonogashira Coupling, Asymmetric
Epoxidation, and Trichloroacetimidate-
Mediated Epoxide Opening
Zheng Liu, Hoe-Sup Byun, and Robert Bittman*
Department of Chemistry and Biochemistry, Queens College of The City UniVersity of
New York, Flushing, New York 11367-1597
robert.bittman@qc.cuny.edu
Received April 30, 2010
ABSTRACT
Stereocontrolled syntheses of r-C-GalCer (2) and its r-C-acetylenic analogue 6 were accomplished in high efficiency by a convergent construction
strategy from 1-hexadecene and D-galactose. The key transformations include Sonogashira coupling, Sharpless asymmetric epoxidation, and
Et2AlCl-catalyzed cyclization of an epoxytrichloroacetimidate to generate protected dihydrooxazine 21.
KRN7000 [(2S,3S,4R)-1-O-(R-D-galactopyranosyl)-2-(N-
hexacosanoylamino)-1,3,4-octadecanetriol, R-GalCer, 1, Fig-
ure 1] is a synthetic analogue of a glycolipid extracted from
the marine sponge1 Agelas mauritianus during a screen of
natural products possessing antitumor properties in mice by
Kirin Pharmaceuticals.2 R-GalCer forms a complex with a
glycoprotein in antigen-presenting cells known as CD1d.3
Glycolipid presentation in CD1d-ligand complexes to the
T cell receptor of invariant natural killer T (iNKT) cells gives
a high-affinity ternary complex4 that activates iNKT cells
in mice and humans to secrete a complex mixture of
cytokines. The production of pro-inflammatory T helper
(Th1) type cytokines such as interferon γ (IFN-γ) is
correlated with antitumor, antiviral/antibacterial, and adjuvant
activities, whereas anti-inflammatory Th2 type cytokines
(such as interleukins 4, 5, 10, and 13) are regulators of some
autoimmune and inflammatory diseases.5 However, the
simultaneous production of both types of conflicting cytokine
activities comprises the therapeutic potential of activated
(3) CD1d molecules, which are expressed by professional antigen-
presenting cells such as dendritic cells, macrophages, and B cells, recognize
various lipid and glycolipid antigens. Bendelac, A.; Savage, P. B.; Teyton,
L. Annu. ReV. Immunol. 2007, 35, 297.
(1) Since mammalian and plant glycosphingolipids generally are ꢀ-ano-
mers, it is surprising that sponges would produce R-glycosphingolipids. It
is likely that 1 was actually derived from NoVosphingobium bacteria that
colonized A. mauritianus.
(2) (a) Natori, A.; Koezuka, Y.; Higa, T. Tetrahedron Lett. 1993, 34,
5591. (b) Kobayshi, E.; Motoki, K.; Uchida, T.; Fukushima, H.; Koezuka,
Y. Oncol. Res. 1995, 7, 529.
(4) Invariant NKT cells are a subset of T lymphocytes that recognize
glycolipid antigens and are dependent on CD1d as the antigen-presenting
molecule to their T cell receptor (TCR). In the glycolipid-CD1d complex,
the two long hydrocarbon chains of R-GalCer are buried in the two long
apolar channels of CD1d, and the carbohydrate headgroup protrudes above
the surface toward the TCR of iNKT cells.7
(5) Savage, P. B.; Teyton, L.; Bendelac, A. Chem. Soc. ReV. 2006, 35, 771.
10.1021/ol1009976  2010 American Chemical Society
Published on Web 06/02/2010

induced a potent stimulatory activity against human iNKT
cells, which was ascribed to the preservation of an ∼170°
dihedral angle in the linker region between the galactose and
the ceramide (Gal-C1-O1-phytosphingosine C1′-phytosph-
ingosine C2′).10 These studies indicate that R-C-GalCer
analogues are useful for presentation by CD1d to iNKT cells
and have potential immunotherapeutical applications com-
pared with 1, the most commonly used ligand.
In order to make larger quantities of 2 available to the
immunology community,11 diverse synthetic approaches
toward this important synthetic target have been devel-
oped.8,12 However, there remains a need for efficient
stereoselective methods for the preparation of 2. We report
a concise convergent synthesis of 2 from readily available,
inexpensive starting materials. In addition, the synthetic route
to 2 reported here permits modification of the linker region,
which appears to be critical for Th1 vs Th2 selectivity. We
also report the synthesis of 6, which contains an acetylenic
moiety and also preserves an ∼170° dihedral angle in the
linker.
Figure 1. Structures of glycolipids 1-6.
As shown in the retrosynthetic analysis (Scheme 1), we
envisioned that the three contiguous stereogenic centers in
iNKT cells and interferes with a concerted biological
outcome.6 The potential clinical utility of 1 is also limited
by the long-term unresponsiveness (anergy) of iNKT cells
when multiple doses of 1 are administered.
R-C-Glycoside analogues of R-GalCer are expected to
be long-lived because they are resistant to R-glycosidase
activity. Moreover, replacing the glycosidic oxygen atom
with a methylene group removes a hydrogen-bonding ac-
ceptor site.7 Thus, R-C-GalCer analogues may bind less
tightly to CD1d, which may be a factor in determining the
type of cytokine release. An isosteric C-glycoside analogue
(2, Figure 1) was found to be active in mice in vivo, with a
biased induction of Th1 responses compared to 1.8 In
addition, 2 produced a long-term production of IFN-γ in
mice, suggesting that the R-C-GalCer/CD1d complex is more
stable in antigen-presenting cells in vivo than the KRN7000/
CD1d complex.8e,f We found that nonisosteric R-C-GalCer
analogue 3, in which the glycosidic oxygen was deleted,
induced an even higher Th1-type cytokine response than 1
and 2 in human iNKT cells in vitro.9 Interestingly, other
R-C-glycoside homologues that contain a three-carbon linker
(4) were inactive.8b GCK127 (5), an analogue with an
E-alkene linker, not only exhibited activity in mice but also
Scheme 1. Retrosynthetic Plan
(6) (a) Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413,
531. (b) Van Kaer, L. Nat. ReV. Immunol. 2005, 5, 31.
(7) (a) Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.;
Pellicci, D. G.; Koh, R.; Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.;
McCluskey, J.; Rossjohn, J. Nature 2007, 448, 44. (b) Koch, M.; Stronge,
V. S.; Shepherd, D.; Gadola, S. D.; Mathew, B.; Ritter, G.; Fersht, A. R.;
Besra, G. S.; Schmidt, R. R.; Jones, E. Y.; Cerundolo, V. Nat. Immunol.
2005, 6, 819. (c) Schiefner, A.; Fujio, M.; Wu, D.; Wong, C.-H.; Wilson,
I. A. J. Mol. Biol. 2009, 394, 71.
the phytosphingosine moiety can be accessed from epoxy
alcohol 7 after reaction with trichloroacetonitrile to give 8,
followed by a Lewis acid catalyzed epoxide opening at the
propargylic carbon. The requisite epoxide 7 could be
(8) (a) Yang, G.; Schmieg, J.; Tsuji, M.; Franck, R. W. Angew. Chem.,
Int. Ed. 2004, 43, 3818. (b) Chen, G.; Schmieg, J.; Tsuji, M.; Franck, R. W.
Org. Lett. 2004, 6, 4077. (c) Pu, J.; Franck, R. W. Tetrahedron 2008, 64,
8618. (d) Franck, R. W.; Tsuji, M. Acc. Chem. Res. 2006, 39, 692. (e)
Schmieg, J.; Yang, G.; Franck, R. W.; Tsuji, M. J. Biomed. Biotechnol.
2010, 2010, 283612. (f) Sullivan, B. A.; Nagarajan, N. A.; Wingender, G.;
Wang, J.; Scott, I.; Tsuji, M.; Franck, R. W.; Porcelli, S. A.; Zajonc, D. M.;
Kronenberg, M. J. Immunol. 2010, 184, 141.
(10) (a) Chen, G.; Chien, M.; Tsuji, M.; Franck, R. W. ChemBioChem
2006, 7, 1017. (b) Li, X.; Chen, G.; Garcia-Navarro, R.; Franck, R. W.;
Tsuji, M. Immunology 2009, 127, 216. (c) Li, X.; Shiratsuchi, T.; Chen,
G.; Dellabona, P.; Casorati, G.; Franck, R. W.; Tsuji, M. J. Immunol. 2009,
183, 4415.
(11) The Tetramer Core Facility funded by the NIH provides reagents
for NKT cell activation, including CD1d ligands, to approved investi-
gators.
(9) Lu, X.; Song, L.; Metelitsa, L. S.; Bittman, R. ChemBioChem 2006,
7, 1750.
(12) Wipf, P.; Pierce, J. G. Org. Lett. 2006, 8, 3375
.
Org. Lett., Vol. 12, No. 13, 2010
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furnished by Sharpless asymmetric epoxidation (SAE)13 of
9, which in turn could be obtained from 10 via Sonogashira
cross-coupling14 between two building blocks, 11 and 12 (or
13). Compound 11 can be assembled via R-C-ethynylation of
14a (accessible from D-galactose; see the Supporting Informa-
tion), and 12 can be prepared via Takai olefination15 of aldehyde
15, which can be synthesized from 1-hexadecene (16).
with iodoform in the presence of chromium(II) chloride
yielded the expected (E)-vinyl iodide 13 in 70% yield when
the Evans modification21 was used. Deprotection of the PMB
group using I2 in MeOH22 afforded vinyl iodide 12 (75%).
Initially, R-C-ethynylgalactoside 11 was prepared by
reaction of 1-acetoxy-2,3,4,6-tetra-O-benzyl-D-galactopyran-
oside (14b,c) with ethynyl precursor 19 in the presence of
TMSOTf followed by desilylation of 20.23 However, we
subsequently found that methyl ꢀ-D-galactosylpyranoside
(14a), which is crystalline, can also react with 19 under the
same conditions (Scheme 3). This reaction proceeded with
very high R-stereoselectivity; no corresponding ꢀ-anomer
Scheme 2. Synthesis of Vinyl Iodide 12
1
was found by H NMR. Its efficiency in the preparation of
11 is comparable to that of acetate 14c. The two-step yield
of 11 from 14c was 54%.23a,c Furthermore, 14c must be
prepared from 14a in two additional steps (∼67% overall
yield).24 Thus our two-step yield of 11 from 14a (37%) is
not only comparable to that from 14c but also offers the
advantage that 14a can be prepared from the very inexpen-
sive D-galactose as reported in the Supporting Information.
With an efficient synthesis of the two building blocks 11
and 12 established, we directed our efforts toward Sono-
gashira coupling (Scheme 3).14 An initial trial of cross-
coupling [Pd(PPh3)4, CuI (0.5 equiv), i-Pr2NEt (6 equiv),
THF] between PMB-protected alcohol 13 and alkyne 11 in
THF provided enyne 10 in 56% yield. During deprotection
of the PMB group of 10 with DDQ, the hydroxy group was
oxidized to the corresponding ketone. However, when free
alcohol 12 was coupled with 10 in the presence of Pd(Ph3P)4
and CuI in CH3CN/Et3N (5:1) the yield of enynol 9 improved
to 88%. Use of Pd(PPh3)2Cl2 as a precatalyst [Pd(PPh3)2Cl2,
CuI, CH3CN/Et3N (5:1)] afforded 9 in about the same yield
as obtained with Pd(Ph3P)4.
As shown in Scheme 2, Sharpless asymmetric dihydroxyl-
ation of 16 with AD-mix-ꢀ provided the desired diol 17 (85%
ee) in almost quantitative yield.16 Alternatively, the use of
the ligand (DHQD)2AQN, which was reported to have a
higher enantioselectivity than the PHAL-based ligand in an
aliphatic system,17 delivered 17 in a lower yield (71%) and
slightly higher ee value (89% ee). Diol 17 was converted to
its p-methoxybenzylidene (PMB) acetal, which was reduced
with DIBAL-H to give alcohol 18 (85%, two steps).18 The
use of a protocol with sodium hypochlorite catalyzed by
TEMPO19 gave the desired aldehyde 15 in 62% yield without
any erosion of the ee value.20 Since an (E)-l-iodoalkene was
required, we used the Takai reaction,15 which is known to
be highly E stereoselective. Condensation of aldehyde 15
Catalytic or substoichometric SAE of 9 was ineffective,
producing little conversion after 20 h at -20 °C. The reaction
using 4 equiv of cumene hydroperoxide as the epoxidizing
agent catalyzed by 2.5 equiv of Ti(O-i-Pr)4 and 2.6 equiv of
D-(-)-DIPT provided propargylic epoxy alcohol 7 in high
yield (84%) and excellent diastereoselectivity (>95%).25
Chelation-controlled opening of 2,3-epoxy alcohol 7 with
NaN3 and in the presence of NH4Cl in aqueous MeOH under
reflux failed to provide the desired azido diol, delivering
instead a complex mixture.26 Et2AlCl-catalyzed cyclization27
of trichloroacetimidate 8, prepared by reaction of 7 with 6.0
equiv of trichloroacetonitrile in the presence of 3.5 equiv of
DBU,27d gave dihydrooxazine 21 in a two-step yield of 67%.
It is noteworthy that BF3·Et2O also catalyzed cyclization of
8 to 21; however, we obtained a mixture of 21 and its
hydrolysis product 22 in a ratio of 1:1 (30% vs 27%,
respectively).
(13) For a review of SAE, see: Katsuki, T.; Martin, V. S. Org. React.
1996, 48, 1.
(14) For a review, see: Sonogashira, K. J. Organomet. Chem. 2002, 653,
46.
(15) Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986, 108,
7408.
(16) The ee of 17 was enriched to >95% by recrystallization from EtOAc,
and this highly enantiopure diol was used in the following reaction. See:
He, L.; Byun, H.-S.; Bittman, R. J. Org. Chem. 2000, 65, 7618.
(17) Eecker, H.; Sharpless, K. B. Angew. Chem., Int. Ed. Engl. 1996,
35, 448.
Acid hydrolysis of 21 provided trichloroacetimide 22,
which was treated with ethanolic NaOH to deliver amine
23 in 68% overall yield. Reaction of amine 23 with
n-hexacosanoyl chloride12 gave amide 24 in 73% yield.
Catalytic hydrogenation (Pd/C, H2, EtOH/TFA)10a of the
linking triple bonds, together with global removal of the
benzyl protecting groups, afforded the target R-C-glycoside
2.28 However, attempted reduction using Pearlman’s catalyst
(18) Synthesis of primary alcohol 18 was readily achieved by following
a reported synthesis of a shorter chain analogue from 1-heptene. Smith,
A. B.; Chen, S. S. Y.; Nelson, F. C.; Reichert, J. M.; Salvatore, B. A. J. Am.
Chem. Soc. 1997, 119, 10935.
(19) Jurczak, J.; Gryko, D.; Kobrzycka, E.; Gruza, H.; Prokopowiczit,
P. Tetrahedron 1998, 54, 6051.
(20) Compound 15 was reduced back to 18 by NaBH4 and subsequently
converted to the (S)-MTPA ester using (R)-MTPA chloride. The 1H NMR
spectrum of the MTPA ester exhibited no signals of the other isomer; see
the Supporting Information.
2976
Org. Lett., Vol. 12, No. 13, 2010

Scheme 3. Synthesis of 2 and 6
[H2, Pd(OH)2, CH2Cl2/MeOH] resulted in incomplete satura-
tion of the acetylenic group. The preparation of 6 from 24
was achieved by BF3·OEt2/EtSH deprotection of the benzyl
groups,29 leaving the acetylenic moiety intact, in almost
quantitative yield.
In conclusion, a convergent and stereoselective synthetic
route to R-C-GalCer (2) and its analogue 6 containing an
acetylenic linker was accomplished. Notable features include
the concise formation of three contiguous stereogenic centers
in the phytosphingosine moiety by Sonogashira cross-
coupling followed by Sharpless asymmetric epoxidation and
Et2AlCl-catalyzed cyclization of an epoxytrichloroacetimidate
intermediate. This convergent construction from simple
starting materials (10 steps from 14a with 6.5% overall yield)
permits the preparation of analogues with variations in the
linker area.
(21) Evans, D. A.; Ng, H. P. Tetrahedron Lett. 1993, 34, 2229.
(22) Vaino, A. R.; Szarek, W. A. Synlett 1995, 1157.
(23) (a) Nishikawa, T.; Koide, Y.; Kajii, S.; Wada, K.; Ishikawa, M.;
Isobe, M. Org. Biomol. Chem. 2005, 3, 687. (b) Dondoni, A.; Moriotti, G.;
Marra, A. J. Org. Chem. 2002, 67, 4475. (c) The stereoselectivity of the
C-glycosidation was found to be independent of the anomeric configuration
of the acetate.23a,b
(24) Kulkarni, S. S.; Gervay-Hague, J. Org. Lett. 2006, 8, 5765.
(25) Compound 7 was converted to the (S)-MTPA ester using using
(R)-MTPA chloride. The de value was determined by analyzing the 1H NMR
spectrum of the (S)-MTPA ester of 7.
(26) Two mechanisms may be responsible for the failure: (1) intramo-
lecular 1,3-dipolar cycloaddition of the propargyl azide followed by MeOH
attack on the resulting strained bicyclic triazole or (2) a triaza-Cope
rearrangement followed by cyclization of the resulting allenyl azide to
triazafulvene and reaction with MeOH. See: (a) Banert, K. Chem. Ber. 1989,
122, 911. (b) Banert, K. Chem. Ber. 1989, 122, 1963.
Acknowledgment. This work was supported in part by
National Institutes of Health Grant HL-083187. Z.L. ac-
knowledges a Doctoral Student Research Grant from the
CUNY Graduate Center.
Supporting Information Available: Experimental pro-
1
cedures as well as H and 13C NMR spectra for all new
(27) For Lewis acid catalyzed cyclization of 2,3-epoxytrichloroacetimi-
dates, see: (a) Schmidt, U.; Respondek, M.; Lieberknecht, A.; Werner, J.;
Fisher, P. Synthesis 1989, 256. (b) Hatakeyama, S.; Matsumoto, H.;
Fukuyama, H.; Mukugi, Y.; Irie, H. J. Org. Chem. 1997, 62, 2275. (c) Lu,
X.; Sun, C.; Valentine, W. J.; E, S.; Liu, J.; Tigyi, G.; Bittman, R. J. Org.
Chem. 2009, 74, 3192. (d) The preparation of 8 in the presence of a catalytic
amount of DBU27b was ineffective.
compounds and synthetic 2. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL1009976
(28) The spectral data of 2 matched well with the previously reported
material in 500 MHz 1H and 125 MHz 13C NMR spectra and optical rotation
(29) Xu, J.; Egger, A.; Bernet, B.; Vasella, A. HelV. Chim. Acta 1996,
79, 2004.
(see Table S1, Supporting Information).8,12
.
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