Synthesis of threitol ceramide and [14C]threitol ceramide, non-glycosidic analogues of the potent CD1d antigen α-galactosyl ceramide

Article abstract of DOI:10.1016/j.tetasy.2009.02.020

The synthesis of threitol ceramide, which is a non-glycosidic analogue of the potent CD1d antigen α-galactosyl ceramide, is described. The synthesis of a ¹⁴C-labelled threitol ceramide analogue is also presented. This radiolabelled analogue wil

Full text of DOI:10.1016/j.tetasy.2009.02.020

Tetrahedron: Asymmetry 20 (2009) 747–753
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of threitol ceramide and [14C]threitol ceramide, non-glycosidic
analogues of the potent CD1d antigen
a-galactosyl ceramide
b,
Yoel R. Garcia Diaz a,b, Justyna Wojno a,b, Liam R. Cox a, , Gurdyal S. Besra
*
*
a School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
b School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of threitol ceramide, which is a non-glycosidic analogue of the potent CD1d antigen
a-
galactosyl ceramide, is described. The synthesis of a 14C-labelled threitol ceramide analogue is also pre-
sented. This radiolabelled analogue will allow the intracellular trafficking pattern/itinerary of this iNKT-
CD1d cell agonist to be studied.
Received 7 January 2009
Accepted 10 February 2009
Available online 9 March 2009
Ó 2009 Published by Elsevier Ltd.
This paper is dedicated to Professor George
Fleet on the occasion of his 65th birthday
and in recognition of his great contributions
to carbohydrate chemistry
1. Introduction
in different situations display both tolerogenic and immunostimula-
tory functions in vivo following
The therapeutic potential of
explored, but the induction of both Th1 and Th2 cytokines by this
agent may limit its usefulness. Importantly, analogues of GalCer,
such as OCH 2 (Fig. 1), whichdiffers from GalCer in possessing trun-
a
GalCer administration.13–19
CD1 molecules are cell-surface glycoproteins related in structure
and evolutionary origin to MHC class 1 antigen-presenting mole-
cules.1–3 The CD1 molecules comprise a multi-gene family, but
unlike MHC class 1 molecules lack allotypic polymorphism.1–3 Var-
iable numbers of CD1 isoforms are found in different mammalian
species. Genes encoding five distinct CD1 proteins (CD1a, -b, -c, -d
and -e) are present on chromosome 1 in humans,4–7 and two genes
encoding proteins closely homologous to human CD1d (mCD1d1
and d2) are located on chromosome 3 in mice.4–7 In common with
MHC class I proteins, the CD1 proteins are expressed on the surface
of cells as polypeptides associated non-covalently with b2-micro-
globulin. The three-dimensional structures of three different CD1
proteins show a striking similarity in structure to MHC class I mole-
cules.8–10 The protein sequences of CD1 molecules suggest that they
can be classified into two groups, with CD1a, -b and -c forming group
1, and CD1d defining group 2.11 Molecules in group 1 appear to be
involved in the presentation of specific antigens, which constitutes
a novel antigen recognition pathway that is likely to be important
for host defence against infections. In contrast, group 2 molecules,
that is, CD1d, appear to have a regulatory role in innate and adaptive
immune responses.12
aGalCer 1 is currently being
a
a
cated aliphatic chains, can induce NKT cell-derived cytokines more
selectively.20 Thus, OCH 2 strongly induces the secretion of IL-4
while having little effect on IFN
analogues are more effective at inducing both IFN
NKTcellsthan
GalCer.21 Bydesigninganaloguesthatcaneffectively
c
levels.20 Conversely, other
aGalCer
c
and IL-4 from
a
promote the appropriate functions of CD1d-restricted T-cells, it may
be possible to treat diseases in which it is important to alter Th1 or
Th2 polarisation. To achieve this aim, we have recently developed
a non-glycosidic threitol ceramide (ThrCer) 3 analogue of aGalCer
as a lead compound.22 In this report, we describe the chemical syn-
thesis of ThrCer 3. We also describe the synthesis of radiolabelled
Thr[14C]Cer, [14C]-3, which provides a chemical probe for examining
the intracellular trafficking pattern/itinerary of this iNKT-CD1d cell
agonist. Results from such studies should provide a more complete
understanding of how iNKT responses are modified by such agents,
which in turn should help realise the full potential of this novel ther-
apeutic approach.
a
-Galactosyl ceramide(
CD1d-restricted Natural Killer T (NKT) cells specifically to produce
high levels of both IL-4 and IFN
in vitro and in vivo.13–19 NKT cells
aGalCer)1 (Fig. 1) hasthe ability to induce
2. Results and discussion
c
2.1. Synthesis of threitol ceramide
* Corresponding authors. Tel.: +44 (0) 121 4143524; fax: +44 (0) 121 4144403
(L.R.C.); tel.: +44 (0) 121 4158125; fax: +44 (0) 121 4145925 (G.S.B.).
E-mail addresses: l.r.cox@bham.ac.uk (L.R. Cox), g.s.besra@bham.ac.uk (G.S.
Besra).
The retrosynthesis of ThrCer 3 is outlined in Figure 2. Discon-
nection of the amide bond in ThrCer 3 provided amine 4 and the
corresponding commercially available fatty acid 5. Introducing
0957-4166/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.tetasy.2009.02.020

748
Y. R. Garcia Diaz et al. / Tetrahedron: Asymmetry 20 (2009) 747–753
OH
HO
HO
O
O
O
23
OH
m
OH
NH
NH
OH
HO
O
O
HO
n
13
OH
OH
OH
3
ThrCer,
1
n = 13, m = 23, αGalCer,
2
n = 4, m = 21, OCH,
Figure 1. CD1d agonists.
NH2 OH
OH
the fatty acid chain in the final step in the synthesis would have a
number of advantages. First, it would incorporate a late-stage point
of diversity into the synthesis, allowing us to introduce a selection
of fatty acids, including unsaturated derivatives, for studying the
effect of how such structural changes affect the biological
response.23,24 From a more practical viewpoint, introducing the
long-chain fatty acid in the final step would also minimise the
manipulations of a difficult-to-handle amphiphilic dialkyl glyco-
lipid. Amine 4 would be accessed from the corresponding azide
6. Disconnecting the ether bond in azide 6 provided the two cou-
pling partners for a Williamson ether synthesis, namely triflate
7,25 which would be prepared from commercially available
N3
OH
TfN3, cat. CuSO4
HO
HO
11
13
13
OH
10
, 98%
cat. Sc(OTf)3
Me2C(OMe)2
N3
O
N3
O
HO
O
O
13
TFA (aq), CH2Cl2
13
benzylidene-protected
L
-threitol derivative 8,26 and alcohol 9,
O
O
9, 87%
12
O
23
Scheme 1. Synthesis of requisite azido alcohol.
NH
OH
OH
O
which would be accessed from phytosphingosine 10 (Fig. 2).
Reversing the electrophile and nucleophile in the ether coupling
would also be straightforward using this strategy should this be
necessary.
HO
13
OH
OH
3
The synthesis of the suitably protected azido alcohol 9 was
accomplished in two steps from phytosphingosine 10 (Scheme
NH2
OH
OH
O
1). Thus, copper-catalysed diazo transfer27,28 with
a freshly
O
prepared solution of TfN3 yielded the corresponding azido-triol
11 in high yield.29 Subsequent treatment of triol 11 with a 1:1 mix-
ture of acetone and 2,2-dimethoxy propane in the presence of
Sc(OTf)3 provided a mixture of acetals 9 and 12. The acyclic acetal
in 12 was readily hydrolysed chemoselectively by treating the mix-
ture briefly with TFA to provide 1,3-dioxolane 9 exclusively.30
The electrophilic coupling partner required for our Williamson
ether synthesis of azide 6 was next prepared in two steps from diol
8. Monoprotection of diol 8 as its TBDPS ether provided alcohol 13
in 71% yield, from which the corresponding triflate 7 was prepared
by treatment with Tf2O in pyridine (Scheme 2). Owing to its unsta-
ble nature, triflate 7 was used immediately and without purifica-
tion in the subsequent etherification, which proceeded smoothly
on treating 7 with the sodium alkoxide 14 in THF. From azide 6,
ThrCer was synthesised in four steps (Scheme 2). TBAF-mediated
deprotection of the silyl ether provided alcohol 15. Subsequent
Ce(OTf)3-mediated acetal hydrolysis31 and hydrogenolysis of the
azide functionality using Pd/C in acidic MeOH provided amine 4,
which was finally acylated with the acid chloride of hexacosanoic
acid to provide our target ThrCer 3.
HO
HO
13
24
OH
OH
4
5
FGI
N3
O
O
O
TBDPSO
13
Ph
O
O
6
TBDPSO
OTf
N3
O
HO
O
O
13
O
7
9
Ph
FGI
O
FGI
OH
2.2. Synthesis of 14C -labelled threitol ceramide
HO
OH
NH2
We next turned our attention to a radiolabelled analogue of
ThrCer 3. Mindful of the need to minimise any manipulations
involving radioactive compounds, we chose to incorporate a 14C la-
bel into the fatty acid, which would be introduced at a late stage in
the synthesis. Moreover, in our synthesis of non-radiolabelled
O
HO
13
8
10
OH
Ph
Figure 2. Retrosynthetic analysis of threitol ceramide.

Y. R. Garcia Diaz et al. / Tetrahedron: Asymmetry 20 (2009) 747–753
749
of 2 equiv of base to deprotonate both the alcohol and terminal al-
kyne in 16, and HMPA as the solvent in the nucleophilic substitu-
tion, allowed the reaction to proceed without protection of the
primary alcohol.32 Subsequent hydrogenation of the alkyne in the
substitution product 18 provided alcohol 19, which was trans-
formed into bromide 20 by treatment with CBr4/PPh3. One-carbon
homologation33 proceeded efficiently on heating a solution of bro-
mide 20 with Na14C N in DMSO at 85 °C to provide nitrile 21.34 Fi-
nally, base-mediated hydrolysis of nitrile 21 provided 14C-labelled
acid [14C]-5 in quantitative yield.34 Following our established pro-
cedure for acylation, acid [14C]-5 was converted into the corre-
sponding acid chloride by treatment with oxalyl chloride, and
then reacted with amine 4 to provide radiolabelled Thr[14C]Cer
N3
O
TBDPSO
OH
HO
O
O
13
13
9
O
Ph
Tf2O
NaH
O
TBDPSO
OTf
N3
NaO
O
O
13
14
7
O
14C]-3 (Scheme 3).
Ph
[
3. Conclusion
Since it lacks the glycosidic linkage found in aGalCer, ThrCer 3
represents a hydrolytically more stable analogue of this most
potent CD1d agonist. We have described a short route to ThrCer,
and have shown that our approach can be used to incorporate a
radiolabel into the molecule. Thr[14C]Cer provides a useful chemi-
cal probe for examining the intracellular trafficking pattern/itiner-
ary of this novel iNKT-CD1d cell agonist, which will be the focus of
future studies.
N3
O
TBDPSO
O
13
O
O
O
Ph
6
, 92%
TBAF
N3
4. Experimental
O
HO
O
4.1. General experimental procedures
13
O
O
O
Optical rotations were measured using an Optical Activity PolA-
Ar2001 automatic polarimeter. Melting points were determined
using open capillarieson a GallenkampMPD350 melting pointappa-
ratus, and are uncorrected. Infrared spectra were recorded either
neat as thin films between NaCl discs on a Perkin Elmer 1600 FTIR
spectrometer, or neat on a Perkin Elmer Spectrum 100 fitted with
a universal ATR accessory. The intensity of each band is described
as s (strong), m (medium) or w (weak), and with the prefix v (very)
and suffix br (broad) where appropriate. 1H NMR spectra were re-
corded at 500 MHz, 400 MHz or 300 MHz, using Bruker DRX 500,
Bruker AMX 400, Bruker AV 400, Bruker AV 300 and Bruker AC 300
spectrometers. 13C NMR spectra were recorded at 125 MHz,
100 MHz or 75 MHz, respectively, using Bruker DRX 500, Bruker
AMX 400, Bruker AV 400, Bruker AV 300 and Bruker AC 300 spec-
trometers. Chemical shifts are reported as d values (ppm) referenced
to the following solvent signals: CHCl3, dH 7.26; CDCl3, dC 77.0;
CH3OH, dH 3.34; CD3OD, dC 49.9. The term ‘stack’ is used to describe
a region where resonances arising from non-equivalent nuclei are
coincident, and multiplet, m, to describe a region where a resonance
arises from a single nucleus (or equivalent nuclei) but where cou-
pling constants cannot be readily assigned. Mass spectra were re-
corded on a Micromass LCT spectrometer utilising electrospray
ionisation (and a MeOH mobile phase), and are reported as (m/z
(%)). HRMS were recorded on a Micromass LCT spectrometer using
a lock mass incorporated into the mobile phase.
Ph
15
, 95%
i) Ce(OTf)3
ii) Pd/C, H2
OH
NH2 OH
OH
O
HO
13
OH
4, 50%
CH3(CH2)24COCl
O
23
OH
NH
OH
O
HO
13
OH
OH
ThrCer, , 60%
3
Scheme 2. Syntheis of threitol ceramide, 3.
All reagents were obtained from commercial sources, and were
used without further purification unless stated otherwise. Anhy-
drous solvents were purchased from Sigma–Aldrich, UK, stored
over 4 Å molecular sieves and under an Ar atmosphere. All solu-
tions are aqueous and saturated unless stated otherwise.
Reactions were monitored by TLC using pre-coated aluminium-
backed ICN silica plates (60A F254) and visualised by UV detection
(at 254 nm) and staining with 5% phosphomolybdic acid in EtOH
(MPA spray). Column chromatography was performed on Merck
ThrCer 3 (Scheme 2), we had shown that the acylation of amine 4
could be performed in excellent yield even on the small scales that
we would be employing in the synthesis of its radiolabelled ana-
logue. Na14CN was identified as a convenient source of 14C; nucle-
ophilic displacement of a suitable alkyl halide would provide a
straightforward method for generating an alkyl nitrile, and thence
the carboxylic acid upon hydrolysis.
To this end, the required C25-chain alkyl halide was assembled
by coupling undecynol 16 with alkyl bromide 17 (Scheme 3). Use
silica gel (particle size 40–63 lm mesh) or Fluka 60 (40–60 lm

750
Y. R. Garcia Diaz et al. / Tetrahedron: Asymmetry 20 (2009) 747–753
i) nBuLi
THF-HMPA
H2
Pd/C
13
HO
HO
HO
8
24
19
, 95%
Br
8
ii)
16
13
18
, 40%
17
CBr4
Ph
3P
O
NaOH
Na14CN
*
Br
HO
NC
*
23
23
23
14C]-5, 95%
20
, 65%
[
21, 95%
i) (COCl)2
ii) 4
A
B
Front
O
*
23
NH
OH
OH
O
Origin
HO
13
OH
OH
14C]-3
3
3
14
[
3
[
C]-
[
14C]-3, 60%
Scheme 3. Synthesis of [14C]ThrCer, [14C]-3. Panel A: TLC showing 3 (left-hand lane) and purified fractions of [14C]-3 after staining with phosphomolybdic acid spray; panel B
shows the same TLC visualised using autoradiography.
mesh) silica gel. Autoradiograms were produced by 2.5 min expo-
sure of Kodak X-Omat AR films to the TLC plates in the dark.
CHOH), some overlap in the alkyl resonances; m/z (TOF ES+)
366.2 ([M+Na]+, 100%); HRMS m/z (TOF ES+) 366.2728 ([M+Na]+.
C18H37N3O3Na requires 366.2733).
4.2. (2S,3S,4R)-2-Azido-1,3,4-octadecanetriol 11
4.3. (2S,3S,4R)-2-Azido-3,4-O-isopropylidene-1,3,4-
TfN3 was freshly prepared prior to the reaction as follows: NaN3
(12.5 g, 192 mmol) was dissolved in a minimum volume of H2O
(35 mL, solubility of NaN3 in H2O is ꢀ0.4 g mLꢁ1), and was cooled
to 0 °C. CH2Cl2 (35 mL) was added, followed by dropwise addition
of Tf2O over 15 min (15.92 mL, 96 mmol) with vigorous stirring of
the solution. The flask was stoppered and stirring continued at 0 °C.
After 2 h, NaHCO3 solution (30 mL) was carefully added, while stir-
ring was continued until gas evolution had ceased. The reaction
contents were then transferred to a separating funnel, and the
phases were separated. The aqueous phase was washed with
CH2Cl2 (2 ꢂ 25 mL). The combined organic phases were washed
with NaHCO3 solution (1 ꢂ 30 mL).
octadecanetriol 9
Sc(OTf)3 (286 mg, 0.58 mmol) was added to a suspension of triol
11 (2.00 g, 5.82 mmol) in a 1:1 mixture of acetone and 2,2-dime-
thoxy propane (50 mL) at 0 °C, resulting in complete dissolution
of the solid. The reaction mixture was stirred at this temperature
for 20 min, and then neutralised with Et3N. The solvent was re-
moved under reduced pressure, and the residue was dissolved in
CH2Cl2 (50 mL). The solution was washed with NaHCO3 solution
(20 mL), and the aqueous phase was extracted with CH2Cl2
(3 ꢂ 20 mL). The organic fractions were combined, dried (Na2SO4)
and filtered. The solvent was evaporated under reduced pressure
to provide a mixture of acetals 9 and 12, which was dissolved in
CH2Cl2/H2O (10:1; 40 mL) and treated with 50% TFA in H2O
The resulting solution of TfN3 in CH2Cl2 was used in the azida-
tion step without further purification as follows: Amine 10 (10.0 g,
31.5 mmol) and CuSO4ꢃ5H2O (32 mg, 0.13 mmol) were dissolved in
the same volume of H2O as the volume of TfN3 solution to be em-
ployed (85 mL). The CH2Cl2 solution of TfN3 was then added with
vigorous stirring. MeOH (570 mL) was then added over 5 min. After
18 h, the reaction mixture was diluted with H2O (260 mL) and ex-
tracted with EtOAc (3 ꢂ 260 mL). The combined organic phases
were filtered through a silica plug and washed with EtOAc until
complete elution of the product. Removal of the solvent under re-
duced pressure afforded azide 11 as a white solid (10.6 g, 98%):
Rf = 0.7 (eluent: EtOAc); mp 92–94 °C (lit. 91ꢁ92 °C);35
(432 lL, 5.82 mmol). After 10 min, TLC analysis showed that the
acyclic acetal in 12 had been hydrolysed leaving exclusively acetal
9. The reaction mixture was neutralised with Et3N and diluted with
NaHCO3 solution (20 mL). The phases were separated, and the
aqueous phase was extracted with CH2Cl2 (2 ꢂ 20 mL). The com-
bined organic phases were dried (MgSO4) and filtered, and the sol-
vent was removed under reduced pressure. Purification of the
residue by flash column chromatography (eluent: 20% EtOAc in
hexanes) afforded the acetonide 9 as a white, low-melting point,
amorphous solid (1.95 g, 87%): Rf = 0.3 (eluent: 25% EtOAc in hex-
½
a 2D2
ꢄ
¼ þ9:6 (c 1.0, CHCl3) (lit. ½a D25
ꢄ
¼ þ10 (c 1, CHCl3);29 mmax
anes); ½a 2D2
ꢄ
¼ þ23 (c 1.0, CHCl3);
m
max(film)/cmꢁ1 3440br (OH),
(film)/cmꢁ1 3683m (OH), 3390m (OH)) 3328m (OH), 3020s,
2918m, 2847m, 2108m (N3), 1602w, 1521m, 1462w, 1410m,
1355m, 1215s, 1148m, 1071w, 1014w, 929m, 849w; dH
(300 MHz, CDCl3/CD3OD, 2:1) 0.66 (3H, t, J 6.5, CH3CH2), 0.95–
1.22 (24H, stack, CH2), 1.37–1.42 (2H, stack, CH(OH)CH2CH2),
3.34–3.39 (3H, stack), 3.58 (1H, dd, J 6.1, 5.7), 3.72 (1H, dd, J 3.9,
3.7); dC (75 MHz, CD3OD) 14.4 (CH3, CH2CH3), 23.7 (CH2), 26.7
(CH2), 30.5 (CH2), 30.7 (CH2), 30.8 (CH2), 33.1 (CH2), 33.9 (CH2),
62.5 (CH2, CH2OH), 66.6 (CH, CHN3), 72.9 (CH, CHOH), 76.0 (CH,
2925s, 2854s, 2100s (N3), 1465m, 1370m, 1247m, 1217s, 1169m,
1063br, 870w, 758s; dH (300 MHz, CDCl3) 0.88 (3H, t, J 6.0,
CH3CH2), 1.26–1.30 (23H, stack), 1.34 (3H, s, 1 ꢂ C(CH3)2), 1.43
(3H, s, 1 ꢂ C(CH3)2), 1.50–1.63 (3H, stack), 2.08 (1H, t, J 6.0,
CH2OH), 3.44–3.50 (1H, m), 3.83–3.91 (1H, m), 3.94–4.03 (2H,
stack), 4.15–4.21 (1H, m); dC (75 MHz, CDCl3) 14.1 (CH3, CH3CH2),
25.5 (CH3, 1 ꢂ C(CH3)2), 26.5 (CH2), 28.0 (CH3, 1 ꢂ C(CH3)2), 29.35
(CH2), 29.41 (CH2), 29.53 (CH2), 29.58 (CH2), 29.59 (CH2), 29.65
(CH2), 29.68 (CH2), 29.69 (CH2), 31.9 (CH2), 61.2 (CH, CHN3), 63.9

Y. R. Garcia Diaz et al. / Tetrahedron: Asymmetry 20 (2009) 747–753
751
(CH2, CH2OH), 76.6 (CH, CHO), 77.7 (CH, CHO), 108 (quat. C,
C(CH3)2), some overlap in the alkyl resonances; m/z (TOF ES+)
406.2 ([M+Na]+, 100%); HRMS m/z (TOF ES+) 406.3033 ([M+Na]+.
C21H41N3O3Na requires 406.3046).
the addition of MeOH (2 mL) followed by NaHCO3 solution
(10 mL). The phases were separated, and the aqueous phase was
extracted with CH2Cl2 (3 ꢂ 20 mL). The combined organic fractions
were washed with brine (15 mL) and dried (MgSO4), filtered, and
the solvent was removed under reduced pressure. The residue
was purified by flash column chromatography (eluent: 5% EtOAc
in hexanes) to provide ether 6 as a colourless oil (1:1 mixture of
diastereoisomers, 1.13 g, 92%). Data on diastereoisomeric mixture:
4.4. 1-O-(tert-Butyldiphenylsilyl)-2,3-O-benzylidene-
L-threitol 13
A
solution of (ꢁ)-2,3-O-benzylidene- -threitol (1.00 g,
L
2.23 mmol) in THF (7 mL) was added dropwise over 5 min to a sus-
pension of NaH (60% dispersion in mineral oil, 98 mg, 2.45 mmol)
in THF (10 mL) at 0 °C. After 30 min, a solution of tBuPh2SiCl
(674 mg, 2.45 mmol) in THF (7 mL) was added dropwise over
15 min. After stirring at rt for 12 h, the reaction was quenched by
the sequential addition of MeOH (2 mL) and NaHCO3 solution
(20 mL). The phases were separated, and the aqueous phase was
washed with CH2Cl2 (3 ꢂ 20 mL). The combined organic phases
were washed with brine (20 mL), dried (MgSO4), filtered, and the
solvent was evaporated under reduced pressure. The residue was
purified by silica gel flash column chromatography (eluent: 20%
EtOAc in hexanes) to provide silyl ether 13 as a colourless oil
(1:1 mixture of diastereoisomers, 919 mg, 71%); Data on diastereo-
Rf = 0.35 (5% EtOAc in hexanes); m
max(film)/cmꢁ1 3071w, 2926s,
2855s, 2098s (N3), 1589w, 1461m, 1427m, 1379m, 1220m,
1112s, 874w; dH (300 MHz, CDCl3) 0.85 (3H, t, J 6.6, CH3CH2),
1.06 (4.5H, s, (CH3)3CSi), 1.09 (4.5H, s, (CH3)3CSi), 1.24–1.41 (32H,
stack), 3.55–3.63 (1H, stack), 3.67–4.01 (7H, stack), 4.09–4.17
(1.5H, stack), 4.23–4.33 (1H, stack), 4.42–4.46 (0.5H, m), 5.99
(0.5H, s, PhCH), 6.00 (0.5H, s, PhCH), 7.33–7.51 (11H, stack, Ph),
7.66–7.73 (4H, stack, Ph); dC (75 MHz, CDCl3) 14.1 (CH3, CH3CH2),
19.2 (quat. C, (CH3)3CSi), 22.7 (CH2), [25.6 (CH3, 1 ꢂ C(CH3)2),
25.7 (CH3, 1 ꢂ C(CH3)2], 26.4 (CH2), [26.82 (CH3, C(CH3)3, 26.86
(CH3, C(CH3)3], [28.1 (CH3, 1 ꢂ C(CH3)2, 28.2 (CH3, 1 ꢂ C(CH3)2],
29.3 (CH2), 29.41 (CH2), 29.45 (CH2), 29.53 (CH2), 29.57 (CH2),
29.59 (CH2), 29.6 (CH2), 29.7 (CH2), 31.9 (CH2), [59.8 (CH, CHN3),
59.9 (CH, CHN3)], [64.2 (CH2, CH2O), 64.3 (CH2, CH2O)], [72.0
(CH2, CH2O), 72.2 (CH2, CH2O)], [72.89 (CH2, CH2O), 72.94 (CH2,
CH2O)], 75.6 (CH, CHO), [77.7 (CH, CHO), 77.8 (CH, CHO)], [77.9
(CH, CHO), 78.0 (CH, CHO)], [78.7 (CH, CHO), 78.8 (CH, CHO)],
[104.0 (CH, PhCH), 104.3 (CH, PhCH)], [108.2 (quat. C, C(CH3)2),
108.3 (quat. C, C(CH3)2)], [126.7 (CH, Ph), 126.8 (CH, Ph)], [127.70
(CH, Ph), 127.74 (CH, Ph)], [128.2 (CH, Ph), 128.3 (CH, Ph)],
[129.25 (CH, Ph), 129.34 (CH, Ph)], 129.73 (CH, Ph), 129.74 (CH,
Ph), 129.77 (CH, Ph), 133.02 (quat. C, ipsoPh), 133.05 (quat. C,
ipsoPh), 133.06 (quat. C, ipsoPh), 133.09 (quat. C, ipsoPh), 135.6
(CH, Ph), [137.4 (quat. C, ipsoPh), 137.5 (quat. C, ipsoPh), some
overlap in the alkyl chain resonances; m/z (TOF ES+) 836.3
([M+Na]+, 100%); HRMS m/z (TOF ES+) 836.5041 ([M+Na]+.
C48H71N3O6SiNa requires 836.5010).
isomeric mixture: Rf = 0.25 (20% EtOAc in hexanes);
mmax(film)/
cmꢁ1 3432br (OH), 3070m, 2930s, 2858s, 1654w, 1589w, 1471m,
1427m, 1390m, 1361m, 1310m, 1218m, 1112s, 1027m, 998m,
917m; dH (300 MHz, CDCl3) 1.07 (4.5H, s, (CH3)3CSi), 1.09 (4.5H,
s, (CH3)3CSi), 1.96–2.05 (1H, stack, CH2OH), 3.70–3.96 (4H, stack),
4.11–4.25 (1.5H, stack), 4.32–4.37 (0.5H, m), 5.96 (0.5H, s, PhCH),
5.99 (0.5H, s, PhCH), 7.35–7.50 (11H, stack, Ph), 7.65–7.72 (4H,
stack, Ph); dC (75 MHz, CDCl3) [19.1 (quat. C, (CH3)3CSi), 19.2 (quat.
C, (CH3)3CSi)], [26.72 (CH3, (CH3)3C), 26.75 (CH3, (CH3)3C)], [62.7
(CH2, CH2O), 62.8 (CH2, CH2O)], [63.9 (CH2, CH2O), 64.0 (CH2,
CH2O)], [77.8 (CH, CHO), 78.7 (CH, CHO), 79.6 (CH, CHO), 79.9
(CH, CHO)], [103.7 (CH, PhCH), 104.1 (CH, PhCH)], [126.52 (CH,
Ph), 126.58 (CH, Ph), 127.73 (CH, Ph), 127.77 (CH, Ph), 128.26
(CH, Ph), 128.37 (CH, Ph), 129.3 (CH, Ph), 129.5 (CH, Ph), 129.79
(CH, Ph), 129.81 (CH, Ph), 129.82 (CH, Ph), 129.85 (CH, Ph)],
[132.81 (quat. C, ipsoPh), 132.85 (quat. C, ipsoPh)], [135.49 (CH,
Ph), 135.53 (CH, Ph)], [137.2 (quat. C, ipsoPh), 137.4 (quat. C, ip-
soPh)] some overlap in aromatic resonances; m/z (TOF ES+) 471
([M+Na]+, 100%); HRMS m/z (TOF ES+) 471.1972 ([M+Na]+.
C27H32O4SiNa requires 471.1968).
4.6. 1-O-[20,30-O-Benzylidene-
pylidene-1,3,4- -ribo-octadecanetriol 15
L-threitol]-2-azido-3,4-O-isopro-
D
TBAF (1 M solution in THF, 1.4 mL, 1.4 mmol) was added to a
solution of silyl ether 6 (1.10 g, 1.35 mmol) in THF (15 mL) at rt.
After 4 h, NH4Cl solution (10 mL) was added. The phases were sep-
arated, and the aqueous phase was extracted with CH2Cl2
(3 ꢂ 10 mL). The solvent was removed under reduced pressure,
and the residue was purified by flash column chromatography
(eluent: 25% EtOAc in hexanes) to provide alcohol 15 as a colour-
less oil (1:1 mixture of diastereoisomers, 740 mg, 95%). Data on
diastereoisomeric mixture: Rf = 0.35 (30% EtOAc in hexanes);
4.5. 1-O-[10-O-tert-Butyldiphenylsilyl-20,30-O-benzylidene-
2-azido-3,4-O-isopropylidene-1,3,4- -ribo-octadecanetriol 6
L-threitol]-
D
Tf2O (329
solution of alcohol 13 (878 mg, 1.96 mmol) and 2,6-di-tert-butyl-
pyridine (484 L, 2.16 mmol) in CH2Cl2 (20 mL) at 0 °C. After 1 h,
lL, 1.96 mmol) was added dropwise over 10 min to a
l
the reaction mixture was diluted with CH2Cl2 (20 mL), and the
resulting solution washed sequentially with cold H2O (2 ꢂ 50 mL)
and brine (10 mL), dried (MgSO4) and filtered. Removal of the sol-
vent under reduced pressure and purification of the residue by
flash column chromatography (eluent: 5% EtOAc in hexanes con-
taining drops of Et3N) yielded triflate 7 as a colourless oil (1:1 mix-
ture of diastereoisomers, 870 mg, 85%). The triflate was unstable
and used immediately. Selected data on diastereoisomeric mix-
ture: Rf = 0.3 (5% EtOAc in hexanes); dH (300 MHz, CDCl3) 1.07
(4.5H, s, (CH3)3CSi), 1.09 (4.5H, s, (CH3)3CSi), 3.81–3.99 (2H, stack),
4.11–4.17 (1H, stack), 4.41–4.45 (0.5H, m), 4.52–4.61 (1.5H, stack),
4.69–4.75 (1H, stack), 5.95 (0.5H, s, PhCH), 6.03 (0.5H, s, PhCH),
7.36–7.48 (11H, stack, Ph), 7.63–7.70 (4H, stack, Ph). Alcohol 9
(575 mg, 1.5 mmol) in THF (10 mL) was treated with NaH (60% in
mineral oil, 64 mg, 1.6 mmol) at 0 °C. After 1 h, a solution of triflate
7 (870 mg, 1.5 mmol) in THF (5 mL) was added dropwise over
5 min. The resulting solution was stirred at this temperature for
1 h, and then at rt for 12 h. The reaction was then quenched by
m
max(film)/cmꢁ1 3463br (OH), 2924s, 2853s, 2099s (N3), 1459m,
1379m, 1246m, 1220m, 1092m, 1065m, 1027m, 869w; dH (300
MHz, CDCl3) 0.88 (3H, t, J 6.6, CH3CH2), 1.23–1.31 (28H, stack),
1.35–1.41 (3H, stack), 1.45–1.60 (1H, stack), 1.96–2.04 (1H, stack,
OH), 3.53–3.60 (1H, stack), 3.67–3.98 (7H, stack), 4.08–4.31 (3H,
stack), 5.97 (0.5H, s, PhCH), 6.00 (0.5H, s, PhCH), 7.37–7.40 (3H,
stack, Ph), 7.48–7.51 (2H, stack, Ph); dC (75 MHz, CDCl3) 14.0
(CH3, CH2CH3), 22.6 (CH2), [25.5 (CH3, 1 ꢂ C(CH3)2, 25.6 (CH3,
1 ꢂ C(CH3)2], 26.3 (CH2), [28.0 (CH3, 1 ꢂ C(CH3)2, 28.1 (CH3,
1 ꢂ C(CH3)2], 29.27 (CH2), 29.33 (CH2), 29.35 (CH2), 29.46 (CH2),
29.51 (CH2), 29.57 (CH2), 29.6 (CH2), 31.8 (CH2), [59.8 (CH,
CHN3), 59.9 (CH, CHN3)], 62.5 (CH2, CH2O), [71.5 (CH2, CH2O),
71.6 (CH2, CH2O)], [72.78 (CH2, CH2O), 72.84 (CH2, CH2O)], 75.5
(CH, CHO), 76.4 (CH, CHO), 77.2 (CH, CHO), [77.68 (CH, CHO),
77.70 (CH, CHO)], [79.7 (CH, CHO), 79.9 (CH, CHO)], [103.8 (CH,
PhCH), 104.0 (CH, PhCH)], [108.20 (quat. C, (CH3)2C), 108.23 (quat.
C, (CH3)2C)], 126.6 (CH, Ph), [128.2 (CH, Ph), 128.3 (CH, Ph)], [129.3
(CH, Ph), 129.4 (CH, Ph)], [137.2 (quat. C, ipsoPh), 137.4 (quat. C,

752
Y. R. Garcia Diaz et al. / Tetrahedron: Asymmetry 20 (2009) 747–753
ipsoPh), some overlap in alkyl chain region; m/z (TOF ES+) 598.2
([M+Na]+, 100%); HRMS m/z (TOF ES+) 598.3805 ([M+Na]+.
C32H53N3O6Na requires 598.3832).
C(2) or C(3)), 71.5 (CH2, C(10)), 73.0 (CH, C(40)), 73.1 (CH, C(3) or
C(2)), 74.1 (CH2, C(1)), 76.7 (CH, C(30)), 173.0 (quat. C, C@O); m/z
(TOF ES+) 822.7 ([M+Na]+, 100%); HRMS m/z (TOF ES+) 822.7175
([M+Na]+. C48H97NO7Na requires 822.7163).
4.7. 1-O-[L-Threitol]-2-amino-1,3,4-D-ribo-octadecantriol 4
4.9. Pentacos-10-yn-1-ol 18
Ce(OTf)3 (117 mg, 0.20 mmol) was added to a vigorously stirred
solution of acetal 15 (200 mg, 0.32 mmol) in MeNO2 (saturated
with H2O, 3 mL) and CH2Cl2 (2 mL). After 4 h at rt, the reaction
mixture was diluted with CH2Cl2 (5 mL), and NaHCO3 solution
(10 mL) was added. The aqueous phase was extracted with CH2Cl2
(10 mL), and the organic phases were combined and washed with
brine (5 mL), and then dried (Na2SO4). The solvent was removed
under reduced pressure. The residue (yellow solid) was dissolved
nBuLi (2.5 M solution in hexanes, 8.72 mL, 21.8 mmol) was
added dropwise over 40 min to a solution of 10-undecyn-1-ol 16
(1.75 g, 10.4 mmol) in THF (21 mL) containing HMPA (7.68 mL,
41.6 mmol) at ꢁ78 °C. After 15 min, a solution of 1-bromotetradec-
ane 17 (3.17 mg, 11.4 mmol) in THF (2 mL) was added. The reac-
tion mixture was stirred at ꢁ78 °C for 1 h, and then left to warm
to rt overnight. The mixture was then diluted with Et2O (20 mL)
and quenched by slow addition of H2O (20 mL). The phases were
separated, and the aqueous phase was extracted with Et2O
(2 ꢂ 20 mL). The combined organic phases were dried with MgSO4,
and the volatiles were removed under reduced pressure. The resi-
due was purified by column chromatography (eluent: hexanes/
Et2O, 2:1) to afford the alkyne 18 as a white solid (1.57 g, 40%):
in MeOH (5 mL), and Pd/C (30 mg, 32 lmol) and AcOH (40 lL,
0.65 mmol) were added. The reaction vessel was evacuated, and
then placed under an atmosphere of H2. The suspension was stirred
overnight. The reaction mixture was filtered, and the filtrate was
concentrated under reduced pressure. The residue was purified
by column chromatography (eluent: 10% MeOH in CHCl3 to 50%
MeOH in CHCl3) to afford amine 4 as a white foam (72 mg, 50%);
Rf = 0.15 (5% EtOAc in hexanes);
m
max(neat)/cmꢁ1 3357w (OH),
[
a
]
D the insolubility of this amphiphilic compound at rt prevented
2919s, 2848s, 1459m, 1130w, 1057m, 1036m, 1013m, 975w,
725s; dH (300 MHz, CDCl3) 0.86 (3H, t, J 6.8, CH3CH2), 1.15–1.60
(38H, stack, alkyl chain), 2.12 (4H, app. t, J 7.1, CH2C„CCH2),
6.63 (2H, app. q, J 5.5, CH2OH), resonance for OH not visible; dC
(75 MHz, CDCl3) 14.1 (CH3, CH3CH2), 18.8 (CH2), 22.7 (CH2), 25.7
(CH2), 28.9 (CH2), 29.2 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2),
29.7 (CH2), 32.0 (CH2), 32.8 (CH2), 63.1 (CH2, CH2OH), 80.2 (quat.
C, C„C), 80.3 (quat. C, C„C), some overlap in the alkyl chain reso-
nances; m/z (TOF ES+) 471.3 ([M+Ag]+, 100%); HRMS m/z (TOF ES+)
471.2745 ([M+Ag]+. C25H48OAg requires 471.2756).
us from obtaining reliable optical rotation data; Rf = 0.1 (20% MeOH
in CHCl3); dH (300 MHz, CDCl3) 0.79 (3H, t, J 6.7, CH2CH3), 1.08–
1.32 (23H, stack, alkyl chain), 1.38–1.50 (1H, m), 1.55–1.69 (2H,
m), 3.14–3.21 (1H, m), 3.25–3.65 (9H, stack), 3.68–3.75 (1H, m),
exchangeable protons were exchanged with deuterium from
CD3OD for clarity prior to running the 1H NMR spectrum; dC
(75 MHz, CDCl3) 13.9 (CH3, CH2CH3), 22.5 (CH2), 25.6 (CH2), 29.2
(CH2), 29.6 (CH2), 31.8 (CH2), 33.5 (CH2), 52.9 (CH), 63.4 (CH2),
70.1 (CH), 71.0 (CH2), 71.6 (CH), 72.5 (CH2), 73.2 (CH), 73.9 (CH),
some overlap in alkyl chain resonances; m/z (TOF ES+) 422.3
([M+H]+, 100%); HRMS m/z (TOF ES+) 422.3489 ([M+H]+.
C22H48NO6, requires 422.3482).
4.10. 1-Bromo-pentacosane 20
Pd/C (29 mg, 27 lmol) was added to a solution of alkyne 18
4.8. 1-O-[
octadecantriol 3
L
-Threitol]-2-hexacosanoylamino-1,3,4-
D
-ribo-
(1.00 mg, 2.70 mmol) in Et2O (10 mL). The reaction vessel was
evacuated and replaced with a H2 atmosphere, and then the mix-
ture was stirred overnight. The slurry was then filtered, washing
the residue with hot THF (2 ꢂ 5 mL). The solvent was evaporated
under reduced pressure to afford alcohol 19 as an oil (943 mg,
Hexacosanoic acid (37.6 mg, 94.9 lmol) was placed in (COCl)2
(1.0 mL) and stirred at 70 °C for 2 h after which time, the solution
was cooled to rt, and the (COCl)2 was removed under a stream of
dry argon. The residual volatiles were removed under reduced
pressure. The resulting crude acyl chloride was dissolved in dry
THF (0.5 mL) and added with vigorous stirring to a solution of
95%): Rf = 0.15 (5% EtOAc in hexanes);
m
max(neat)/cmꢁ1 3278br
(OH), 2917s, 2849s, 1473m, 1452m, 1062m, 731m, 719m; dH
(300 MHz, CDCl3) 0.87 (3H, t, J 6.5, CH3CH2), 1.20–1.37 (44H, stack,
alkyl chain), 1.50–1.60 (2H, m, CH2CH2OH), 3.63 (2H, t, J 6.4,
CH2OH), resonance for OH not visible; dC (75 MHz, CDCl3) 14.1
(CH3, CH2CH3), 22.7 (CH2), 25.7 (CH2), 29.38 (CH2), 29.45 (CH2),
29.7 (CH2), 31.9 (CH2), 32.8 (CH2), 63.1 (CH2, CH2OH), some overlap
in alkyl chain resonances. Satisfactory mass spectral analysis could
not be obtained on alcohol 19. Alcohol 19 was used without further
purification in the next step: PPh3 (531 mg, 2.02 mmol) and CBr4
(492 mg, 1.48 mmol) were added sequentially to a solution of alco-
hol 19 (500 mg, 1.35 mmol) in CH2Cl2 (10 mL) at 0 °C. After 1 h,
hexanes (10 mL) were added slowly, and the resulting precipitate
was filtered. The filtrate was diluted with CH2Cl2 (20 mL) and
washed with H2O (10 mL). The phases were separated, and the or-
ganic phase was dried (Na2SO4). The volatiles were evaporated un-
der reduced pressure to afford a residue, which was purified by
column chromatography (eluent: 2% Et2O in hexanes) to afford al-
kyl bromide 20 as a white solid (378 mg, 65%): Rf = 0.4 (hexanes);
dH (300 MHz, CDCl3) 0.88 (3H, t, J 6.6, CH2CH3), 1.20–1.31 (43H,
stack, alkyl chain), 1.39–1.48 (1H, m), 1.79–1.87 (2H, m), 3.40
(2H, t, J 6.9, CH2Br); dC (75 MHz, CDCl3) 14.1 (CH3, CH2CH3), 22.7
(CH2), 28.2 (CH2), 28.8 (CH2), 29.4 (CH2), 29.5 (CH2), 29.7 (CH2),
31.9 (CH2), 32.8 (CH2), 34.0 (CH2), some overlap in alkyl chain res-
onances. Satisfactory mass spectral analysis could not be obtained
on bromide 20.
amine 4 (20 mg, 47.4 lmol) in THF / NaOAc(aq) (8 M) (1:1,
0.8 mL). Vigorous stirring was maintained for 2 h, after which time
the mixture was left to stand and the phases were separated. The
aqueous phase was extracted with THF (2 ꢂ 1.0 mL), and the com-
bined organic phases were evaporated under reduced pressure.
Purification of the residue by column chromatography (gradient
from CHCl3 to 15% MeOH in CHCl3) afforded threitol ceramide 3
as a white solid (22.5 mg, 60%); Rf = 0.3 (8% MeOH in CHCl3); [a]
the insolubility of this amphiphilic compound at rt prevented us
D
from obtaining reliable optical rotation data; mp 107–109 °C;
m
max(neat disk)/cmꢁ1 3308br m (OH, NH), 2915s, 2849s, 2098w,
1634m (C@O), 1540m, 1471m, 1108m, 1070m, 1026m, 718m; dH
(500 MHz, THF-d8, 45 °C) 0.89 (6H, app t, J 6.7, 2 ꢂ CH2CH3),
1.25–1.63 (72H, stack, alkyl chain), 2.12 (2H, t, J 7.7, C(O)CH2),
3.40–3.47 (2H, stack, C(30)H, C(40)H), 3.47–3.57 (5H, stack, C(1)H2,
C(4)H2, C(2)H or C(3)H), 3.59–3.63 (1H, m, C(10)HaHb) 3.67–3.72
(2H, stack, C(10)HaHb, C(3)H or C(2)H), 4.14–4.18 (1H, m, C(20)H),
6.93–6.96 (1H, m, NH); dC (125 MHz, THF-d8, 45 °C) 14.3 (CH3,
2 ꢂ CH2CH3), [23.5 (CH2), 26.6 (CH2), 26.8 (CH2), 29.3 (CH2), 30.3
(CH2), 30.5 (CH2), 30.59 (CH2), 30.64 (CH2), 30.7 (CH2), 30.8
(CH2), 32.8 (CH2), 34.1 (CH2) alkyl chain resonances, some overlap],
36.9 (CH2, C(O)CH2), 51.8 (CH, C(20)), 64.6 (CH2, C(4)), 71.3 (CH,

Y. R. Garcia Diaz et al. / Tetrahedron: Asymmetry 20 (2009) 747–753
753
4.11. [14C]Hexacosan-1-nitrile 21
Acknowledgements
Alkyl bromide 20 (13 mg, 30
lmol) was added to a solution of
G.S.B. acknowledges support in the form of a Personal Research
Chair from Mr. James Bardrick, Royal Society Wolfson Research Mer-
it Award, as a former Lister Institute-Jenner Research Fellow, the
Medical Research Council and The Wellcome Trust. We also thank
the University of Birmingham for PhD funding (to Y.R.G.D. and J.W.).
Na14CN (1 mg, 20 mol, 53 mCi mmolꢁ1 (1.961 GBq mmolꢁ1)) in
l
DMSO (1 mL). The mixture was heated at 85 °C for 72 h, and then
cooled to rt. Et2O (1 mL) was added to the mixture followed by
H2O (1 mL). The phases were separated, and the aqueous phase
was extracted with Et2O (3 ꢂ 1 mL). The combined organic phases
were dried with MgSO4, and the volatiles were removed under re-
duced pressure. The residue was purified by column chromatogra-
phy (eluent: 5% EtOAc in hexanes) to provide nitrile 21 (7 mg,
95%): Characterisation data for non-radiolabelled nitrile,33 which
was prepared as described above: Rf = 0.35 (5% EtOAc in hexanes);
References
1. Dutronc, Y.; Porcelli, S. A. Tissue Antigens 2002, 60, 337–353.
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Today 1998, 19, 362–368.
3. Porcelli, S. A. Adv. Immunol. 1995, 59, 1–98.
4. Moseley, W. S.; Watson, M. L.; Kingsmore, S. F.; Seldin, M. F. Immunogenetics
1989, 30, 378–382.
m
max(film)/cmꢁ1 2914s (br), 2848s, 2241w (CN), 1633w, 1471s,
1424w, 1377w, 730w, 716s; dH (300 MHz, CDCl3) 0.86 (3H, t, J
7.0, CH2CH3), 1.90–1.32 (42H, stack, alkyl chain), 1.35–1.49 (2H,
m), 1.60–1.72 (2H, m), 2.31 (2H, t, J 6.7, CH2CN); dC (75 MHz, CDCl3)
14.1 (CH3, CH2CH3), 17.1 (CH2), 22.7 (CH2), 25.4 (CH2), 28.7 (CH2),
28.8 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.7 (CH2), 30.0
(CH2), 31.9 (CH2), some overlap in the alkyl chain resonances; ni-
trile resonance not observed; m/z (TOF ES+) 400.3 ([M+Na]+,
100%); HRMS m/z (TOF ES+) 400.3932 ([M+Na]+. C26H51NNa re-
quires 400.3919).
5. Bradbury, A.; Belt, K. T.; Neri, T. M.; Milstein, C.; Calabi, F. EMBO J. 1988, 7,
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4.12. [14C]Hexacosanoic acid [14C]-5
A solution of NaOH (220 mg, 5.5 mmol) in EtOH (2.4 mL)/H2O
(0.3 mL) was prepared. [14C]Nitrile 21 (7 mg, 20
lmol) was placed
in 1 mL of this solution, and the mixture heated at 70 °C for 6 d,
after which time TLC indicated that the alkyl nitrile had been con-
sumed. The mixture was diluted with H2O (2 mL), and the pH re-
duced to 3 with concd hydrochloric acid. The solution was
extracted with Et2O (3 ꢂ 2 mL). The phases were separated, and
the organic fraction was dried (MgSO4). The volatiles were evapo-
rated under reduced pressure to afford the fatty acid [14C]-5 as a
white solid (7.0 mg, 95%): Characterisation data for non-radiola-
belled acid,33 which was prepared as described above: Rf = 0.2
(20% EtOAc in hexanes); dH (300 MHz, CDCl3) 0.88 (3H, t, J 6.7,
CH2CH3), 1.25–1.35 (44H, stack, alkyl chain), 1.60–1.69 (2H, m),
2.35 (2H, t, J 6.9, CH2CO2H), carboxylic acid resonance not visible;
spectroscopic data in agreement with those reported in the litera-
ture.36 m/z (TOF ESꢁ) 395.3 ([MꢁH]ꢁ, 100%); HRMS m/z (TOF ESꢁ)
395.3908 ([MꢁH]ꢁ C26H51O2 requires 395.3889).
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1626–1629.
20. Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413, 531–534.
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4.13. [14C]-1-O-[
octadecantriol [14C]-3
L-Threitol]-2-hexacosanoylamino-1,3,4-D-ribo-
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in (COCl)2 (0.5 mL) was stirred at 70 °C for 2 h after which time,
the solution was cooled to rt, and the (COCl)2 was removed under
a stream of dry argon. The residual volatiles were removed under
reduced pressure. The resulting crude acyl chloride (20 lmol,
assuming 100% conversion) was dissolved in dry THF (0.5 mL)
and added with vigorous stirring to a solution of amine 4
(16.8 mg, 40 lmol) in THF/NaOAc(aq) (8 M) (1:1, 0.8 mL). Vigorous
stirring was maintained for 2 h, after which time TLC showed that
the fatty acid had been consumed. The mixture was let to stand,
and the phases were separated. The aqueous phase was extracted
with THF (2 ꢂ 1.0 mL), and the combined organic phases were
evaporated under reduced pressure. Purification of the residue by
column chromatography (gradient from CHCl3 to 15% MeOH in
CHCl3) afforded threitol ceramide [14C]-3 as a white solid33
(13.6 mg, 85%).
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radiolabelled products were then characterised using standard analytical
techniques. The progress of the reactions involving [14C]-labelled analogues
and assessment of purity of these radiolabelled products were then assessed by
comparison with characterised authentic samples using thin-layer
chromatography and autoradiography.
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