Synthesis of tritium labeled KRN7000

Article abstract of DOI:10.1016/j.tetlet.2006.03.131

The synthesis of a multiple tritiated analog of KRN7000 (α-galactosyl ceramide) is described, enabling further studies to quantitate the affinity of KRN7000 for its receptor and to study its pharmacokinetic properties.

Full text of DOI:10.1016/j.tetlet.2006.03.131

Tetrahedron Letters 47 (2006) 3677–3679
Synthesis of tritium labeled KRN7000
Martijn D. P. Risseeuw,ꢀ Celia R. Berkers,ꢁ Hidde L. Ploegh§ and Huib Ovaa*
Harvard Medical School, Department of Pathology, 77 avenue Louis Pasteur Boston, MA 02115, USA
Received 26 January 2006; revised 17 March 2006; accepted 22 March 2006
Available online 12 April 2006
Abstract—The synthesis of a multiple tritiated analog of KRN7000 (a-galactosyl ceramide) is described, enabling further studies to
quantitate the affinity of KRN7000 for its receptor and to study its pharmacokinetic properties.
Ó 2006 Elsevier Ltd. All rights reserved.
O
OH
Presentation of antigens to T cells is a key feature of cell-
mediated immunity. Whereas peptide antigens are pre-
sented by major histocompatibility complexes (MHC)
class I or class II, lipid antigens are presented by CD1
complexes,1 heterodimeric protein complexes with a high
degree of structural analogy to MHC complexes. The
CD1 family is expressed on dendritic cells, B-cells and
macrophages and comprises five members, CD1a–e, each
of which have been shown to bind and present different
lipids. Lipid antigen presentation by members of the
CD1 family has recently gained interest and holds poten-
tial for the development of novel lipid-based immuno-
therapeutics. Despite the recent gain in interest, few
ligands have been described.
HO
HO
C24H49
OH
O
HN
HO
O
C13H27
OH
1
Figure 1. Structure of KRN7000 (a-galactosyl ceramide) 1.
D-galactose and cerotic acid (hexacosanoic acid)
(Fig. 1). Binding of the linear hydrocarbon moieties,
into two deep hydrophobic binding grooves aligned
by two a-helices in the CD1d heavy chain,5 results in
activation of NKT cells through interaction of glyco-
lipid-loaded CD1d and the appropriate T-cell receptor.
KRN7000, also known as a-galactosyl ceramide or a-
GalCer (1),2 is a synthetic glycolipid originally described
as an anti-tumor agent. It was shown to act as a ligand
for CD1d3 with the ability to activate natural killer T
(NKT) cells. a-GalCer, is a simplified analog of a
glycolipid extracted from the marine sponge Agelas
mauritianus4 and is constructed from phytosphingosine,
Although a-GalCer has enabled the immunological
community to extensively study the role of CD1d in
cell-mediated immunity, it has been difficult to study
a-GalCer pharmacokinetics or to quantitate the extent
of interaction between a-GalCer and CD1d. The bind-
ing half-life of the interaction of a-GalCer with CD1d
has indirectly been determined by different means, and
is estimated to range anywhere from 6 min to 24 h. Sev-
eral groups have reported on a-GalCer derivatives that
allow tracking of the glycolipid, none of them using
radiolabelling. a-GalCer has been derivatized by label-
ing with fluorescent tags or with biotin.6 In most of these
cases the tracer molecule has been incorporated in the
acyl side chain, that binds into one of the two CD1d
lipid binding grooves.
Keywords: Glycolipids; Ceramides; CD1 ligands; Tritiated compounds.
*
Corresponding author at present address: Netherlands Cancer
Institute, Department of Cellular Biochemistry, Plesmanlaan 121,
1066 CX Amsterdam, The Netherlands. Tel.: +31 20 5121979; fax:
+31 20 5121989; e-mail: h.ovaa@nki.nl
ꢀ Present address: Leiden Institute of Chemistry, Gorlaeus Laborato-
ries, PO Box 9502, 2300 RA, Leiden, The Netherlands.
ꢁ Present address: Netherlands Cancer Institute, Department of Cellu-
lar Biochemistry, Plesmanlaan 121, 1066 CX Amsterdam, The
Netherlands.
§ Present address: Whitehead Institute of Biomedical Research, Mas-
sachusetts Institute of Technology, Nine Cambridge Centre, Room
345, MA 02142, Cambridge, USA.
Since chemical modifications of a-GalCer are likely to
alter its binding properties and thus limit their use, it
was decided to track a-GalCer by the incorporation of
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.03.131

3678
M. D. P. Risseeuw et al. / Tetrahedron Letters 47 (2006) 3677–3679
radio nuclides. The advantage of radiolabeling is
several-fold: it allows for both qualitative analysis and
for quantitation. We decided to construct the radio-
active derivative in such a way that the dimensions and
elemental composition are very similar to the unmodified
counterpart.
Ph
Ph
O
O
O
O
O
i-ii
O
BnO
N3 OBn
BnO
10
BnO
BnO OH
N3 OBn
O
C
14H29
OBn
12
3
HO
11
It was envisaged that tritium (T, H) could be incorpo-
s
C14H29
iii-iv
rated by catalytic reduction of unsaturations using tri-
tium gas, the most obvious site for the introduction of
tritium atoms being the fatty acyl chain of 1. This chain
contains neither heteroatoms nor chiral centers, poten-
tially sensitive to the effects of radioactive decay, unlike
the carbohydrate and the phytosphingosine moieties.
The introduction of tritium into the fatty acid requires
an unsaturated analog of cerotic acid. Bisacetylene fatty
acid 9 was constructed according to Scheme 1. The
synthesis was initiated from commercially available
16-hydroxypalmitic acid (2), which was converted into
the methyl ester 3 under acidic conditions.
OBn
6
Ph
O
O
O
HN
15
O
v
BnO
1
OBn
BnO
O
C14H29
13
OBn
Scheme 2. Reagents and conditions: (i) Cl3CN, DBU, CH2Cl2, 81%;
(ii) compound 11, BF3ÆOEt2, THF, Et2O, powdered molecular sieves
˚
4 A, ꢀ20 °C, 70%; (iii) Me3P, THF, 0 °C ! rt; (iv) compound 9
PyBOP, DIPEA, CH2Cl2, 65%, two steps; (v) H2 (1 atm), Pd black,
EtOAc, rt quant.
Protection of the free hydroxyl group as a silyl ether
(TBDMS) followed by reduction with LiAlH4 yielded
alcohol 5. Treatment of this alcohol with methanesulfon-
yl chloride yielded mesylate 6, which was converted
into iodide 7 under Finkelstein conditions in 82% overall
yield. Elongation of the iodide with 1,9-decadiyne afford-
ed compound 8. Desilylation (TBAF) followed by
Jones oxidation yielded bisacetylene fatty acid 9 in
48% (two steps).
Glycolipid intermediate 13 was transformed into the tri-
tiated a-GalCer (14) by catalytic reduction with tritium
gas over palladium black in ethyl acetate followed by
alcohol T/H exchange and silica gel chromatography
(5–10% MeOH in CH2Cl2).
This transformation not only achieves the simultaneous
incorporation of eight tritium nuclei in the acyl side
chain, but also conveniently removes all the protective
groups (Scheme 3) in the same transformation. The
reduction was carried out using palladium black in ethyl
acetate since hydrogenation trials using different sol-
vents and catalysts (palladium carbon formulations,
alcoholic solvents, addition of carboxylic acids) had
yielded impure, partially decomposed products.
Having the fatty acid 9 in hand, stable intermediate 13
was synthesized according to Scheme 2. Known D-
galactose derivative 107 was coupled to the azido-phyto-
sphingosine derivative 118 using Schmidt’s trichloro-
acetimidate method.9 NMR analysis indicated that, in
accordance with analogous glycosylations using the
benzylidene protected galactose donor, only the a
isomer 12 was formed.10,11 Staudinger reduction using
Me3P in THF yielded a labile amine, which was
immediately condensed with bisacetylene fatty acid 9
to afford compound 13.12
3
The reductive tritiation yielded pure H8-a-GalCer as
judged by analysis of the product formed by autoradio-
graphy and thin layer chromatography. Reductive
hydrogenation under otherwise identical conditions
afforded a-GalCer (1), which proved in all aspects
identical to a-GalCer, obtained by elongation of 12 with
cerotic acid followed by reductive hydrogenation.
O
OR2
iii
OTBDMS
R1O
RO
13
13
2 R1 = R2 = H
5 R = H
6 R = OMs
iv
i
3 R1 = Me R2 = H
ii
4 R1 = Me R2 = TBDMS
v
13
T2
Pd black
EtOAc
then
T/H exchange
15
OTBDMS
OTBDMS
vi
I
13
8
6
7
vii-viii
15
O
OH
OH
C
24H41T8
HO
HO
HN
O
6
O
9
OH
HO
O
C13H27
14
Scheme 1. Reagents and conditions: (i) AcCl, MeOH quant.; (ii)
TBDMSCl, Imidazole, DMF; (iii) LiAlH4, THF; (iv) MsCl, TEA,
CH2Cl2; (v) NaI, Acetone, D, 82% four steps; (vi) 1,9-decadiyne, n-
BuLi, THF, DMPU, 32%; (vii) TBAF, THF; (viii) Jones’ reagent,
acetone (48%, two steps).
OH
Scheme 3. Catalytic reduction of intermediate 13 with 3H2 to afford
tritiated ceramide 14.

M. D. P. Risseeuw et al. / Tetrahedron Letters 47 (2006) 3677–3679
3679
4. (a) Natori, T.; Koezuka, Y.; Higa, T. Tetrahedron Lett.
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Koezuka, Y. Tetrahedron 1994, 50, 2771.
Through the construction of an unsaturated analog of
the fatty acid cerotic acid it was possible to synthesize
protected glycolipid 13, which provided homogeneous
3H8-a-GalCer upon reduction with tritium gas over pal-
ladium black. As a result of the high specific activity
5. Zheng, Z.-H.; Castano, A. R.; Segelke, B. W.; Stura, E.
˜
A.; Peterson, P. A.; Wilson, I. A. Science 1997, 277,
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3
(200 Ci/mmol) obtained, H8-a-GalCer (14) will enable
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facile quantitation of interactions with this glycolipid
or with glycolipid-loaded CD1d complexes to study
in vivo a-GalCer pharmacokinetic properties, on which
we will report in due course.
Acknowledgements
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This work was financially supported by NIH (H.L.P.) the
Netherlands Organization for Scientific Research (H.O.),
the Koninklijke Hollandse Maatschappij der Wetensc-
happen (M.D.P.R. and C.R.B.) and by the Stichting
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500 MHz) d: 7.46–7.28 (m, 25H); 5.93 (d, 1H, J = 16.5 Hz
NH); 5.51 (s, 1H PhCH); 5.00 (d, 1H, J = 3.0 Hz); 4.96–
4.53 (m, 13H); 4.27–3.58 (m, 17H); 2.23–1.90 (m, 3H),
1.70–1.30 (m, 52H); 0.94 (m, 6H). 13C NMR (CDCl3
125 MHz) d: 173.2; 138.8; 138.7; 138.1; 129.1; 128.7; 128.7;
128.6; 128.4; 128.1; 128.0; 127.8; 126.6; 101.3; 100.0;
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14.4.
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