Efficient synthesis of a C-analogue of the immunogenic bacterial glycolipid BbGL2

Article abstract of DOI:10.1021/ol062354m

(Diagram presented) Synthesis of a C-analogue of bacterial glycolipid BbGL2 is reported using Grignard reaction of in situ generated β-galactosyl iodide and subsequent olefin cross metathesis reaction of C-vinyl galactoside as key steps.

Full text of DOI:10.1021/ol062354m

ORGANIC
LETTERS
2006
Vol. 8, No. 25
5765-5768
Efficient Synthesis of a C-Analogue of
the Immunogenic Bacterial Glycolipid
BbGL2
Suvarn S. Kulkarni and Jacquelyn Gervay-Hague*
Department of Chemistry, UniVersity of California, DaVis, One Shields AVenue,
DaVis, California 95616
gerVay@chem.ucdaVis.edu
Received September 25, 2006
ABSTRACT
Synthesis of a C-analogue of bacterial glycolipid BbGL2 is reported using Grignard reaction of in situ generated
subsequent olefin cross metathesis reaction of C-vinyl galactoside as key steps.
â-galactosyl iodide and
Two major glycolipids have been isolated from Borrelia
burgdorferi, the etiological agent of Lyme disease, which is
a multisystemic disorder that affects the skin, nervous system,
heart, and joints.1 The structures of these highly immunore-
active glycolipids have recently been elucidated as cholesteryl
6-O-acyl-â-D-galactopyranoside (BbGL1) 1 and 1,2-di-O-
acyl-3-O-R-D-galactopyranosyl-sn-glycerol (BbGL2) 2 (Fig-
ure 1).2 The major fatty acids were palmitate and oleate.
Glycolipids 1 and 2, being the only antigenic lipid compo-
nents of B. burgdorferi, are looked upon as valuable
candidates for diagnosis and potential vaccination for Lyme
disease. Moreover, BbGL2 2 bears a structural resemblance
to the powerful immunostimulant R-galactosyl ceramide (R-
GalCer) KRN7000 3,3 which is a slightly simplified analogue
of glycosphingolipids derived from marine origin (Agelas-
phins), capable of activating natural killer T cells (NKT
cells).4 The B. burgdorferi O-glycosides 1 and 2 have been
synthesized;5 and BbGL2 2 and its various lipid analogues
have been shown to activate mouse and human NKT cells,
whereas 1 is inactive.5b
Over the years, C-glycosides, wherein an acetal linkage
is replaced by a methylene group, have been introduced as
(2) Ben-Menachem, G.; Kubler-Kielb, J.; Coxon, B.; Yergey, A.;
Schneerson, R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7913-7918.
(3) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa,
E.; Yamaji, K.; Kobayashi, E.; Fukushima, H.; Koezuka, Y. J. Med. Chem.
1995, 38, 2176-2187.
(4) Kumar, H.; Belperron, A.; Barthold, S. W.; Bockenstedt, L. K. J.
Immunol. 2000, 165, 4797-4801.
(5) (a) Wu, D.; Xing, G.-W.; Poles, M. A.; Horowitz, A.; Kinjo, Y.;
Sullivan, B.; Bodmer-Narkevitch, V.; Plattenberg, O.; Kronenberg, M.; Tsuji,
M.; Ho, D. D.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1351-
1356. (b) Kinjo, Y.; Tupin, E.; Wu, D.; Fujio, M.; Garcia-Navarro, R.;
Benhia, M. R.; Zanonc, D. M.; Ben-Menachem, G.; Ainge, G. D.; Painter,
G. F.; Khurana, A.; Hoebe, K.; Behar, S. M.; Beutler, B.; Wilson, I. A.;
Tsuji, M.; Sellati, T. J.; Wong, C.-H.; Kronenberg, M. Nat. Immunol. 2006,
7, 978-986.
(1) Hossain, H.; Wellensiek, H.-J.; Geyer, R.; Lochnit, G. Biochimie 2001,
83, 683-692.
10.1021/ol062354m CCC: $33.50
© 2006 American Chemical Society
Published on Web 11/10/2006

Figure 1. Structures of various biologically significant glycolipids.
stable analogues of O-glycosides.6 Recently, a synthetic
C-glycoside analogue of KRN7000 (4) was shown to exhibit
remarkably enhanced activity.7 In light of these results,
C-analogues of the bacterial glycolipids became attractive
synthetic targets. Recently, we demonstrated the utility of
glycosyl iodides in the synthesis of R-O-glycosides8 and we
sought to extend this methodology to the construction of
C-vinyl glycosides. We envisioned that olefin cross metath-
esis (CM) of the formed C-vinyl galactoside 7 would lead
to the target molecule 5.
derivative 7 with (S)-3-butene-1,2-diol derived 8. Franck and
co-workers have used a similar strategy, for their second-
generation synthesis of the C-analogue of KRN7000, em-
ploying ethylene-promoted CM of C-vinyl and C-propenyl
galactosides with vinyl derivatives of phytosphingosine.9 The
Franck protocol offers marked improvement over earlier
attempts; however, the preparation of C-vinyl galactoside
was lengthy and indirect.
The deceptively simple 7 has previously been prepared
using two routes either by controlled hydrogenation of the
corresponding C-ethynyl sugar (30%, five steps) using the
Dondoni and Isobe10 protocol or through Pd-catalyzed double
bond isomerization of the corresponding C-allyl sugar, gener-
ated essentially along Kishi’s C-glycosylation route,11 followed
by olefin cross metathesis with ethylene (five steps, 50%).
Another indirect method to prepare a related glucoside starts
from expensive tri-O-benzyl D-glucal and affords the corres-
ponding 2-OH gluco derivative via epoxidation of the double
bond and its concomitant syn opening with trivinylalumi-
num.12 On the other hand, vinyl Grignard reactions of R-D-
glucosyl13 and R-D-mannosyl13b bromides have been reported
to proceed with low-moderate yields (30%-45%) and selec-
tivity, generating R/â mixtures that favor the â-isomer. We
envisioned that a direct nucleophilic SN2 displacement of
tetra-O-benzyl â-galactosyl iodide with vinyl magnesium bro-
mide would be the most direct route to R-vinyl galactoside 8.
We first streamlined a highly efficient and straightforward
route for the preparation of 2,3,4,6-tetra-O-benzyl D-galac-
topyranosyl acetate 10, amenable to scale-up. As shown in
Scheme 2, commercially available R-methyl-D-galactoside
Our convergent retro-synthesis of 5 entails two building
blocks 7 and 8 as delineated in Scheme 1. The target
Scheme 1. Retrosynthesis of C-Glycolipid Dihydro-BbGL2 5
molecules can be accessed from di-O-acetate 6, which in
turn can be synthesized via CM of C-vinyl galactose
(6) Books and reviews on C-glycosides: (a) Levy, D. E.; Tang, C. The
Chemistry of C-Glycosides; Pergamon: Oxford, 1995. (b) Bertozzi, C. R.;
Bednarski, M. D. In Modern Methods In Carbohydrate Synthesis; Harwood
Academic Publishers: Reading, 1996; pp 316-351. (c) Postema, M. H.
D.; Piper, J. L.; Betts, R. L. Synlett 2005, 1345-1358. (d) Yuan, X.;
Linhardt, R. J. Curr. Top. Med. Chem. 2005, 5, 1393-1430.
(7) Yang, G.; Schmieg, J.; Tsuji, M.; Franck, R. W. Angew. Chem., Int.
Ed. 2004, 43, 3818-3822.
(8) (a) Hadd, M. J.; Gervay, J. Carbohydr. Res. 1999, 320, 61-69. (b)
Lam, S. N.; Gervay-Hague, J. Carbohydr. Res. 2002, 337, 1953-1965. (c)
Lam, S. N.; Gervay-Hague, J. Org. Lett. 2002, 4, 2039-2042. (d) Du, W.;
Gervay-Hague, J. Org. Lett. 2005, 7, 2063-2065.
(9) Chen, G.; Schmieg, J.; Tsuji, M.; Franck, R. W. Org. Lett. 2004, 6,
4077-4080.
(10) (a) Dondoni, A.; Mariotti, G.; Marra, A. J. Org. Chem. 2002, 67,
4475-4486. (b) Isobe, M. J. Synth. Org. Chem. Jpn. 1994, 52, 968-979.
(11) (a) Giannis, A.; Sandhoff, K. Tetrahedron Lett. 1985, 26, 1479-
1482. (b) Lewis, M. D.; Cha, J. K.; Kishi, Y. J. Am. Chem. Soc. 1982, 104,
4976-4978.
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(13) (a) Shulman, M. L.; Shiyan, S. D.; Khorlin, A.Y. Carbohydr. Res.
1974, 33, 229-235. (b) Ju¨rs, S.; Thiem, J. Tetrahedron: Asymmetry 2005,
16, 1631-1638.
5766
Org. Lett., Vol. 8, No. 25, 2006

We initially tried the reaction without diluting in THF and
at 0 °C (entry 1); the requisite C-vinyl glycoside 7 was
obtained in 47% yield as an R/â mixture with the ratio
slightly in favor of the R-isomer. Conducting the same
reaction at room temperature resulted in a higher proportion
of the R-isomer (R/â ) 2:1, 49%, entry 2). This suggested
that the reaction was proceeding by a mixed SN1/SN2
mechanism owing to the low reactivity of the vinyl Grignard
reagent. We believe the â-isomer results from SN2-like attack
on the R-iodide, whereas under conditions of prevailing free
iodide ions in solution, the R-iodide anomerizes to the
â-iodide, which being more reactive preferentially undergoes
attack by the C-nucleophile giving the R-C-vinyl compound.
This anomerization of glycosyl iodide by iodide ions is
favored at higher temperature (thermal effect).16 We then
tried “in situ anomerization” using an external iodide source
such as tetrabutylammonium iodide (TBAI) in THF. This
reaction gave a higher proportion of R-isomer (3.2:1, 50%)
when the Grignard addition was conducted at room temper-
ature (entry 3). On the other hand, conducting the reaction
in THF at reflux had an adverse effect on the yield (entry
4), although the selectivity was maintained. At this juncture,
we considered the possibility that a nonpolar solvent such
as benzene would be a better choice to achieve R-selectivity
in the reaction. To this end, we tried slow addition of vinyl
Grignard at 65 °C in the presence of 1.75 equiv of TBAI in
benzene. As shown in entry 5, the reaction was quenched in
1.5 h and the C-galactoside 7 was obtained in 60% yield
with an R/â ratio of 5:1. A slight increase in the temperature
with a 2-fold increase in TBAI (3.5 equiv, entry 6) resulted
in higher yield and selectivity (6.2:1, 65%). This situation
was further improved when the reaction was conducted in a
toluene/benzene solvent mixture (4:1) at 100 °C affording 7
in 75% isolated yield over two steps with an R/â ratio of
7.5:1 (entry 7). The best results were obtained in toluene at
110 °C; this reaction rapidly offered 7 in 79% over two steps
(R/â ) 12:1, entry 8). The reaction worked equally well on
a 1 g scale, and the R-isomer could be separated upon careful
column chromatography. The data of C-R 79 and its
â-isomer17 matched well with previously reported values.
Scheme 2. Efficient Preparation of Anomeric Acetate 10
9 was per-O-benzylated (NaH/DMF/BnBr). Upon completion
of this reaction, DMF was evaporated and the crude product
was treated with 1 M H2SO4 in 80% AcOH14 in the same
flask and refluxed gently for 8 h. Upon neutralization, the
so formed 1-OH hemiacetal was extracted into dichlo-
romethane and acetylated without any further purification
using Ac2O/Et3N.15 The crude product, obtained by evapora-
tion, was purified by recrystallization to afford the pure
â-isomer (10) in 67% overall yield over three steps. Notably,
no intermediate column chromatography purifications were
necessary. The efficient preparation of starting material
enabled further studies of C-nucleophilic additions to gly-
cosyl iodides.
Vinyl Grignard reactions with glycosyl iodides are un-
precedented in the literature. We conducted a systematic
study of Grignard additions to establish optimum reaction
conditions including solvents, temperature, and reagents, as
summarized in Table 1. The R-galactosyl iodide is generated
Table 1. Vinyl Grignard Reaction of Galactosyl Iodide
entry TBAI
solvent
temp
time (h) R/â (% yield)
1
2
0 °C to rt
rt
rt
60 °C
65 °C
70 °C
20
20
20
3
1.5
1.5
0.5
1.25:1 (47)
2:1 (49)
3.2:1 (50)
3.2:1 (32)
5:1 (60)
3
4
5
6
7
1.5
1.5
THF
THF
1.75 benzene
3.5
3.5
With the key synthon C-vinyl galactoside 7 in hand, we
proceeded to synthesize a simplified C-glycoside analogue
of BbGL2 (Scheme 3). Our synthetic strategy involved olefin
cross metathesis of 7 with 8 as the key step. CM reactions
involving vinyl glycosides are reported to proceed in low to
modest yields mostly due to steric effects.18 A recent report
by Franck and co-workers showed the first example of
benzene
6.2:1 (70)
7.5:1 (75)
toluene/benzene 100 °C
(4:1)
8
3.5
toluene
110 °C
0.5
12:1 (79)
by addition of trimethylsilyliodide (TMSI) into a dichlo-
romethane solution of 10 at 0 °C according to a procedure
reported earlier by our laboratory.8a The reaction was
completed in an hour, and the TMSOAc byproduct was
removed by repeated azeotroping with dry benzene. The
resulting crude R-galactosyl iodide was subsequently treated
with vinyl magnesium bromide (1 M in THF).
(16) El-Badry, M. H.; Gervay-Hague, J. Tetrahedron Lett. 2005, 46,
6727-6728.
(17) Xie, J.; Durrat, F.; Valery, J.-M. J. Org. Chem. 2003, 68, 7896-
7898.
(18) (a) Roy, R.; Das, S. K.; Dominique, R.; Trono, M. C.; Hernandez-
Mateo, F.; Santoyo-Gonzalez, F. Pure Appl. Chem. 1999, 71, 565-571.
(b) Godin, G.; Compain, P.; Martin, O. R. Org. Lett. 2003, 5, 3269-3272.
(c) Dondoni, A.; Giovannini, P. P.; Marra, A. J. Chem. Soc., Perkin Trans.
1 2001, 2380-2388. (d) Nolen, E. G.; Kurish, A. J.; Wong, K. A.; Orlando,
M. D. Tetrahedron Lett. 2003, 44, 2449-2453. (e) Oguri, H.; Sasaki, S.-
y.; Oishi, T.; Hirama, M. Tetrahedron Lett. 1999, 40, 5405-5408. (f)
Connon, S. J.; Blechert, S. Angew. Chem., Int. Ed. 2003, 42, 1900-1923.
(14) Arya, P.; Barkley, A.; Randell, K. D. J. Comb. Chem. 2002, 4, 193-
198.
(15) Luo, S.-Y.; Kulkarni, S. S.; Chou, C.-H.; Liao, W.-M.; Hung, S.-
C. J. Org. Chem. 2006, 71, 1226-1229.
Org. Lett., Vol. 8, No. 25, 2006
5767

ethylene gas under an argon atmosphere using less dichlo-
romethane. The reaction was refluxed for 4 days with
periodic addition of fresh catalyst (25% total). From the onset
of the reaction, the formation of the dimer of 8 was observed
and its gradual consumption with the increased formation
of the product was followed on TLC. Under these conditions,
the reaction cleanly afforded the desired olefin 11 in 82%
yield as the E-isomer. Di-O-acetate 11 was deacetylated using
NaOMe, and the corresponding crude diol was selectively
acylated using palmitic acid, DCC, and DMAP at 0 °C to
provide monoacylated 12 as the sole product in 69% overall
yield. The remaining secondary hydroxyl was acylated using
stearic acid, DCC, and DMAP to produce 13 (95%), which
upon hydrogenolysis at 54 psi cleanly afforded the target
molecule 5 in 92% yield.
Scheme 3. Synthesis of the C-Analogue of Dihydro-BbGL2
In conclusion, a stable C-glycoside analogue of BbGL2
has been prepared in a straightforward manner employing
direct C-vinylation of a galactosyl iodide and subsequent
olefin cross metathesis as key steps. In this endeavor, we
established reaction conditions for the R-stereoselective
addition of vinyl Grignard to galactosyl iodide using an “in
situ anomerization” process and thereby explored the general
principles of the stereoselective C-C bond formation reac-
tion. These general findings should serve as valuable tools
for future syntheses of analogues of biologically important
O-glycolipids. The synthesis of other significant C-analogues
of BbGL2 with diverse lipid chains, using the above-
established methodology, and their biological evaluation are
currently underway in our laboratories.
successful cross metathesis using a C-vinyl galactoside under
modified conditions of ethylene promotion.9 However, reac-
tion of the diacetate 8 (3.4 equiv), obtained by acetylation
of the corresponding diol, with 7 (1 equiv) under an ethylene
atmosphere failed in our hands. The starting materials were
recovered after refluxing the reaction in CH2Cl2 for 2 days
in the presence of Grubbs’ catalyst (second generation). We
rationalized that the dilution19 that is required under the
ethylene bubbling conditions and/or the presence of ethylene
itself may have been detrimental to the success of the
reaction. So, we conducted a reaction in the absence of
Acknowledgment. This work was supported by NSF
Grant 0518010.
Supporting Information Available: General experimen-
1
tal details, experimental data, and H NMR and 13C NMR
spectra for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
(19) Hoveyda, H. R.; Ve´zina, M. Org. Lett. 2005, 7, 2113-2116.
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