The first synthesis of a thioglycoside analogue of the immunostimulant KRN7000

Article abstract of DOI:10.1021/ol8019555

(Chemical Equation Presented) The first total synthesis of a thioglycoside analogue of KRN7000, a potential immunostimulant, is described. Two key intermediates are α-galactosyl thiol 4 and phytosphingosine derivative 5, which were both prepared from D-galactose.

Full text of DOI:10.1021/ol8019555

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4641-4644
The First Synthesis of a Thioglycoside
Analogue of the Immunostimulant
KRN7000
Ravindra T. Dere and Xiangming Zhu*
Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical
Biology, UniVersity College Dublin, Belfield, Dublin 4, Ireland
xiangming.zhu@ucd.ie
Received August 21, 2008
ABSTRACT
The first total synthesis of a thioglycoside analogue of KRN7000, a potential immunostimulant, is described. Two key intermediates are
r-galactosyl thiol 4 and phytosphingosine derivative 5, which were both prepared from D-galactose.
Glycosphingolipids have been a target of increasing interest
in immunostimulant research since the discovery of the
therapeutic potential of R-galactosylceramides.1 To date, very
few compounds have been found that specifically stimulate
natural killer T (NKT) cells, the unique lymphocytes defined
by their coexpression of surface marker associated with NK
cells along with a T cell antigen receptor (TCR). The best-
characterized is the glycolipid, R-galactosylceramide, which
stimulates the production of large amounts of cytokines, such
as interferon IFN-γ and interleukin IL-4, by these cells within
2 h of injection.2,3 These cytokines are recognized subse-
quently by other cells of the immune system and may have
a widespread influence on immune responses, including
protection against autoimmune diseases, the host response
to parasites and bacteria, and antitumor responses. Very
recently, several excellent reviews have been published
describing the immunostimulatory role of glycolipids and
the potential for use of NKT cell responses for intervention
in human diseases.4 Molecules belonging to the R-galacto-
sylceramide family may thus evolve into useful anticancer
drugs or immunomodulatory drugs.
The first isolation of R-galactosylceramides was reported
by a research group at Kirin Pharmaceuticals in 1993.5 The
glycolipid that they named agelasphins6 (Figure 1) demon-
strated potent antitumor and immunostimulatory activities.
Further experiments also showed that these molecules were
potent in vivo active agents against the murine B16
melanoma. Encouraged by these results, various derivatives
were synthesized,1a culminating in the production of
KRN7000.7 The biological action of KRN7000 comprises
several consecutive steps. First, the glycolipid is bound to
(1) For some examples, see: (a) Morita, M.; Motoki, K.; Akimoto, K.;
Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.;
Fukushima, H. J. Med. Chem. 1995, 38, 2176–2187. (b) Vankar, Y. D.;
Schmidt, R. R. Chem. Soc. ReV. 2000, 29, 201–216. (c) Schmieg, J.; Yang,
G.; Franck, R. W.; Tsuji, M. J. Exp. Med. 2003, 198, 1631–1641. (d) Wu,
D.; Xing, G.; Poles, M. A.; Horowitz, A.; Kinjo, Y.; Sullivan, B.; Bodmer-
Narkevitch, V.; Plettenburg, O.; Kronenberg, M.; Tsuji, M.; Ho, D. D.;
Wong, C. H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1351–1356. (e) Kinjo,
Y.; Wu, D.; Kim, G.; Xing, G.; Poles, M. A.; Ho, D. D.; Tsuji, M.;
Kawahara, K.; Wong, C. H.; Kronenberg, M. Nature 2005, 434, 520–525.
(f) Chen, W.; Xia, C.; Wang, J.; Thapa, P.; Li, Y.; Nadas, J.; Zhang, W.;
Zhou, D.; Wang, P. G. J. Org. Chem. 2007, 72, 9914–9923.
(4) (a) Yu, K. O. A.; Porcelli, S. A. Immunol. Lett. 2005, 100, 42–55.
(b) Savage, P. B.; Teyton, L.; Bendelac, A. Chem. Soc. ReV. 2006, 35,
771–779. (c) Wu, D.; Fujio, M.; Wong, C. H. Bioorg. Med. Chem. 2008,
16, 1073–1083.
(2) Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki,
K.; Ueno, H.; Nakagawa, R.; Sato, H.; Kondo, E.; Koseki, H.; Taniguchi,
M. Science 1997, 278, 1626–1629
.
(5) Natori, T.; Koezuka, Y.; Higa, T. Tetrahedron Lett. 1993, 34, 5591–
5592.
(3) Stetson, D. B.; Mohrs, M.; Reinhardt, R. L.; Baron, J. L.; Wang,
Z. E.; Gapin, L.; Kronenburg, M.; Locksley, R. M. J. Exp. Med. 2003,
(6) Natori, T.; Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994,
50, 2771–2784.
198, 1069–1076
.
10.1021/ol8019555 CCC: $40.75
Published on Web 09/23/2008
2008 American Chemical Society

production of the TH1 cytokines and decreased production
of TH2 cytokines, which suggests that the C-glycoside
analogue may be an excellent therapeutic option for diseases
resolved by TH1-type responses. An underlying cause for
the dramatic difference between C- and O-glycosides remains
to be clarified. Other different procedures for the synthesis
of the C-glycoside analogue of KRN7000 have also been
reported.12 Also, the C-glycoside version of a truncated
analogue of KRN7000 termed OCH13a and a truncated
nonisosteric R-C-galactosylceramide13b have also been syn-
thesized.
Recently, Cerundolo and his co-workers reported the X-ray
crystal structures of KRN7000 bound to human CD1d
molecules,14 which shows that the lipid chains of the
glycolipid are buried in the two grooves in CD1d composed
largely of hydrophobic amino acids, while the galactose
residue is largely exposed for the recognition by the T cell
receptor. From the structure, it is not immediately apparent
how lipid chain length and sugar moiety influence NKT cell
responses. However, several hydrogen bonds identified
between human CD1d and KRN7000 at the junction of the
two alkyl chains and the polar headgroup did answer
theoretically some of the structure-activity data derived from
R-galactosylceramide analogues.14
Figure 1. Structures of agelasphins (1), KRN7000 (2), and
S-glycoside analogue (3).
the CD1d protein on antigen-presenting cells. The KRN7000/
CD1d complex is then recognized by the TCR on NKT cells,
stimulating rapid T helper type 1 (TH1) and type 2 (TH2)
cytokine production. Release of TH1-biased cytokines, like
IFN-γ, is believed to be responsible for the antitumor,
antiviral, and antibacterial effects of R-galactosylceramide,
while TH2-biased cytokines, like IL-4, can attenuate proin-
flammatory responses, and thereby prevent the onset of some
autoimmune diseases.
Nevertheless, the efficacy of KRN7000 has been limited
because of the reciprocal inhibition exhibited by TH1 and
TH2 cytokines.8 Hence, many efforts have been devoted in
the past decade to synthesize KRN7000 analogues with the
hope of developing novel lead compounds with better
cytokine-inducing selectivities and greater potency as im-
munostimulatory agents.9 A series of structural modifications
toward both the galactose residue and the lipid chains of the
galactosylceramide have been conducted.9,10 In addition, due
to the inherent in vivo instability of O-glycosides, Franck
and his co-workers synthesized a C-glycoside analogue of
KRN7000,11 and examined its biological activities. Surpris-
ingly, the anomeric analogue exhibited a 1000-fold more
potent antimalaria activity and a 100-fold more potent
antimetastatic activity than KRN7000.1c Compared with
KRN7000, the C-glycoside consistently stimulated prolonged
As part of our ongoing program on the synthesis of
catabolically stable glycosides,15,16 particularly S-linked
glycoconjugates,16 we wish to report here the first total
synthesis of a thioglycoside analogue of KRN7000 3 as a
potential immunostimulant. Thioglycosides17 in which the
(10) (a) Du, W.; Gervay-Hague, J. Org. Lett. 2005, 7, 2063–2065. (b)
Ndonye, R. M.; Izmirian, D. P.; Dunn, M. F.; Yu, K. O. A.; Porcelli, S. A.;
Khurana, A.; Kronenberg, M.; Richardson, S. K.; Howell, A. R. J. Org.
Chem. 2005, 70, 10260–10270. (c) Murata, K.; Toba, T.; Nakanishi, K.;
Takahashi, B.; Yamamura, T.; Miyake, S.; Annoura, H. J. Org. Chem. 2005,
70, 2398–2401. (d) Yu, K. O. A.; Im, J. S.; Molano, A.; Dutronc, Y.;
Illarionov, P. A.; Forestier, C.; Fujiwara, N.; Arias, I.; Miyake, S.;
Yamamura, T.; Chang, Y. T.; Besra, G. S.; Porcelli, S. A. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 3383–3388. (e) Wu, D.; Zajonc, D. M.; Fujio, M.;
Sullivan, B. A.; Kinjo, Y.; Kronenberg, M.; Wilson, I. A.; Wong, C. H.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3972–3977. (f) Long, X.; Deng,
S.; Mattner, J.; Zang, Z.; Zhou, D.; McNary, N.; Goff, R. D.; Teyton, L.;
Bendelac, A.; Savage, P. B. Nat. Chem. Biol. 2007, 3, 559–564. (g) Du,
W.; Kulkarni, S. S.; Gervay-Hague, J. Chem. Commun. 2007, 233, 6–2338.
(h) Tashiro, T.; Nakagawa, R.; Hirokawa, T.; Inoue, S.; Watarai, H.;
Taniguchi, M.; Mori, K. Tetrahedron Lett. 2007, 48, 3343–3347. (i)
Trappeniers, M.; Goormans, S.; Beneden, K. V.; Decruy, T.; Linclau, B.;
Al-Shamkhani, A.; Elliott, T.; Ottensmeier, C.; Werner, J. M.; Elewaut,
D.; Calenbergh, S. V. ChemMedChem 2008, 3, 1061–1070.
(7) For some recent examples of the synthesis of KRN7000, see: (a)
Figueroa-Pe´rez, S.; Schmidt, R. R. Carbohydr. Res. 2000, 328, 95–102.
(b) Plettenburg, O.; Bodmer-Narkevitch, V.; Wong, C. H. J. Org. Chem.
2002, 67, 4559–4564. (c) Fan, G. T.; Pan, Y.; Lu, K. C.; Cheng, Y. P.;
Lin, W. C.; Lin, S.; Lin, C. H.; Wong, C. H.; Fang, J. M.; Lin, C. C.
Tetrahedron 2005, 61, 1855–1862. (d) Risseeuw, M. D. P.; Berkers, C. R.;
Ploegh, H. L.; Ovaa, H. Tetrahedron Lett. 2006, 47, 3677–3679. (e) Lee,
A.; Farrand, K. J.; Dickgreber, N.; Hayman, C. M.; Ju¨rs, S.; Hermans, I. F.;
Painter, G. F. Carbohydr. Res. 2006, 341, 2785–2798. (f) Luo, S. Y.;
Kulkarni, S. S.; Chou, C. H.; Liao, W. M.; Hung, S. C. J. Org. Chem.
2006, 71, 1226–1229. (g) Tsujimoto, T.; Ito, Y. Tetrahedron Lett. 2007,
48, 5513–5516.
(11) (a) Franck, R. W.; Tsuji, M. Acc. Chem. Res. 2006, 39, 692–701.
(b) Yang, G.; Schmieg, J.; Tsuji, M.; Franck, R. W. Angew. Chem., Int.
Ed. 2004, 43, 3818–3822. (c) Chen, G.; Schmieg, J.; Tsuji, M.; Franck,
R. W. Org. Lett. 2004, 6, 4077–4080. (d) Chen, G.; Chien, M.; Tsuji, M.;
Franck, R. W. ChemBioChem 2006, 7, 1017–1022.
(12) Wipf, P.; Pierce, J. G. Org. Lett. 2006, 8, 3375–3378, and references
cited therein.
(13) (a) Toba, T.; Murata, K.; Yamamura, T.; Miyake, S.; Annoura, H.
Tetrahedron Lett. 2005, 46, 5043–5047. (b) Lu, X.; Song, L.; Metelitsa,
L. S.; Bittman, R. ChemBioChem 2006, 7, 1750–1756.
(14) 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, 819–826.
(8) Berkers, C. R.; Ovaa, H. Trends Pharmacol. Sci. 2005, 26, 252–
257.
(9) (a) Sakai, T.; Morita, M.; Matsunaga, N.; Akimoto, K.; Yokoyama,
T.; Iijima, H.; Koezuka, Y. Bioorg. Med. Chem. Lett. 1999, 9, 697–702.
(b) Goff, R. D.; Gao, Y.; Mattner, J.; Zhou, D.; Yin, N.; Cantu, C., Jr.;
Teyton, L.; Bendelac, A.; Savage, P. B. J. Am. Chem. Soc. 2004, 126,
13602–13603. (c) Xing, G. W.; Wu, D.; Poles, M. A.; Horowitz, A.; Tsuji,
M.; Ho, D. D.; Wong, C. H. Bioorg. Med. Chem. 2005, 13, 2907–2916. (d)
Fujio, M.; Wu, D.; Garcia-Navarro, R.; Ho, D. D.; Tsuji, M.; Wong, C. H.
J. Am. Chem. Soc. 2006, 128, 9022–9023.
(15) Zhu, X.; Jin, Y.; Wickham, J. J. Org. Chem. 2007, 72, 2670–2673.
(16) (a) Zhu, X.; Pachamuthu, K.; Schmidt, R. R. Org. Lett. 2004, 6,
1083–1085. (b) Zhu, X.; Schmidt, R. R. Tetrahedron Lett. 2003, 44, 6063–
6067. (c) Zhu, X.; Stolz, F.; Schmidt, R. R. J. Org. Chem. 2004, 69, 7367–
7370. (d) Zhu, X.; Pachamuthu, K.; Schmidt, R. R. J. Org. Chem. 2003,
68, 5641–5651. (e) Zhu, X.; Haag, T.; Schmidt, R. R. Org. Biomol. Chem.
2004, 2, 31–33
.
(17) Driguez, H. Top. Curr. Chem. 1997, 187, 85–116.
4642
Org. Lett., Vol. 10, No. 20, 2008

glycosidic oxygen atom has been replaced by a sulfur atom
are tolerated by most biological systems; moreover, this
replacement increases the stability of the sugar-aglycon
linkage against enzymatic cleavage as well as chemical
degradation. Thus, the target molecule 3 might remain active
in the biological systems for a longer period of time. Also,
based on the biological results of R-C-galactosylceramides,11a
the new S-glycolipid may have better binding affinity with
CD1d, favoring more effective signaling cascades in the
immune systems.
Our synthesis started with the preparation of thiol 4.19 1,6-
Anhydrosugar 7 was first prepared from D-galactose by
literature procedures20and then selectively ring-opened with
commercially available bis(trimethylsilyl)sulfide following
our own procedure19 to give the desired R-galactosyl thiol
4 in 88% yield (Scheme 2). The anomeric configuration of
Scheme 2. Synthesis of Galactosyl Thiol 4
Currently, glycosyl thiols are the key building blocks for
the construction of thioglycosides,18 although thioglycosides
can also be synthesized conventionally from normal glycosyl
donors and the corresponding sulfur-containing acceptors.
To a great extent, this nonconventional approach became
popular owing to the chemical stability of glycosyl thiols.
Unlike sugar hemiacetals, glycosyl thiols are quite stable,
and the thioglycosyl anions do not mutarotate even under
basic conditions.16 In other words, the anomeric configuration
of a glycosyl thiol can be maintained during the formation
of its corresponding thioglycoside products. To circumvent
the conventional glycosidation strategy that could suffer from
poor R/ꢀ selectivity, and to take advantage of the glycosyl
thiol chemistry, a synthetic strategy involving the galactosyl
thiol 4 and electrophilic lipid building block 5 was envisaged
for the construction of the target compound 3, as outlined
in Scheme 1. A base-promoted coupling between 4 and 5
4 was readily determined from the coupling constant: 3JH1-H2
) 4.5 Hz, whereas analogous ꢀ-glycosyl thiols usually have
3JH1-H2 ) 7-10 Hz. This ring-opening procedure is a
significant advance in glycosyl thiol chemistry because there
have not been any reports on direct stereoselective prepara-
tion of R-glycosyl thiols prior to our work. Furthermore, 4
was produced exclusively as R-anomer, which made the
purification very simple and straightforward.
In the meantime, phytosphingosine derivative 8 (Scheme
3) was also synthesized from D-galactose following Schmidt’s
procedure,7a and then subjected to the normal isopropylide-
nation conditions (2,2-dimethoxypropane/p-TsOH) to give
the intermediate 921 in 80% yield. Compound 9 was
subsequently mesylated with methanesulfonyl chloride in
pyridine to afford an 87% yield of compound 10, which was
converted into the iodide 5 in excellent yield by treatment
with LiI in DMF.
Scheme 1. Retrosynthetic Analysis of the Target Molecule 3
With the requisite building blocks 4 and 5 in hand, the
task that now confronted us was to find suitable conditions
to achieve their ligation. Recently, S-linked glycopeptides
have been successfully assembled from glycosyl thiols and
ꢀ-bromoalanine-containing peptides under phase transfer
conditions.22 Notably, in this work other nucleophilic
functional groups present on both sugars and peptides, such
as hydroxyl groups, did not participate in the ligation due to
the highly nucleophilic thiolate group and the extremely mild
conditions. On the basis of this work, coupling between thiol
4 and iodide 5 was performed in the presence of tetrabuty-
lammonium hydrogen sulfate (TBAHS) in ethyl acetate and
an aqueous solution of NaHCO3 at pH 8.5, i.e. phase transfer
conditions, as shown in Scheme 3. As expected, the desired
product 11 was obtained in very good yield (73%) after SiO2
was expected to provide the precursor to the S-glycolipid 3,
and both 4 and 5 could be derived from D-galactose.
(19) Dere, R. T.; Wang, Y.; Zhu, X. Org. Biomol. Chem. 2008, 6, 2061–
2063.
(20) Åberg, P. M.; Ernst, B. Acta Chem. Scand. 1994, 48, 228–233.
(21) Schmidt, R. R.; Maier, T. Carbohydr. Res. 1988, 174, 169–179.
(22) Zhu, X.; Schmidt, R. R. Chem. Eur. J. 2004, 10, 875–887.
(18) Zhu, X. Tetrahedron Lett. 2006, 47, 7935–7938, and references
cited therein.
Org. Lett., Vol. 10, No. 20, 2008
4643

Scheme 3. Synthesis of R-S-Galactosylceramide 3
flash chromatography, and the 6-OH group of 4 also
remained inert under the conditions.
yield, and to the best of our knowledge, this is the first total
synthesis of a thioglycoside analogue of the immunostimulant
KRN7000. An advantage of this protocol is that the anomeric
stereochemistry was introduced at an early stage in the
synthesis by use of a glycosyl thiol building block. In view
of the enhanced catabolic stability of S-linked glycoconju-
gates, the resultant structure of 3 is of particular interest for
studying its biological properties, and testing of 3 as
immunostimulant is in progress.
The conversion of azide 11 into the corresponding amine
was effected by Staudinger reduction, using a 1 M solution
of trimethylphosphine in THF,9c and the amine was used
directly in the acylation reaction without further purification.
Coupling of the amine with hexacosanoic acid in the presence
of 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochlo-
ride (EDC) afforded the desired compound 12 in 84% overall
yield. Subsequently, deprotection of 12 to convert into the
target compound 3 was achieved in two steps: removal of
the isopropylidene protecting group was first conducted by
treatment with 4 M HCl in dioxane to give rise to the partially
protected S-glycolipid 13 in 68% yield; 13 was then subjected
to Birch reduction to furnish the R-S-galactosylceramide 3
in 63% yield. The synthetic sample was purified by flash
chromatography on silica gel (eluant: CHCl3/MeOH 10:1),
and characterized by NMR and HR-ESIMS
Acknowledgment. This work was supported by Research
Frontier Program of Science Foundation Ireland. We thank
Johannes Klein (School of Chemsitry and Chemical Biology,
University College Dublin) for help with the Birch reduction.
Supporting Information Available: Experimental pro-
1
cedures and H and 13C data for the new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, we have developed a convenient and
efficient protocol for the synthesis of R-S-galactosylceramide
3 involving 14 steps starting from D-galactose in 3% overall
OL8019555
4644
Org. Lett., Vol. 10, No. 20, 2008