Practical syntheses of [13C]- and [14C]-labelled glucosphingolipids

Article abstract of DOI:10.1039/b002104k

Synthetic routes to [glucose-U-¹⁴C]-1-O-(β-D-glucopyranosyl)-N-stearoyl-D-erythro-sp hingosine 1b and to [glucose-¹³C₆]-1-O-(β-D-glucopyranosyl)sphingosine ([glucose-¹³C₆]glucopsychosine, 2b) are described. Whereas the protected ceramide precursor for lb was prepared using conventional methodology, two new strategies were developed in the course of the synthesis of 2b. Of these, one relies on keeping a protecting group in place at all times to avoid the handling difficulties associated with sphingosine 4, while the other generates a protected derivative (24) of sphingosine indirectly by means of a Mitsunobu inversion.

Full text of DOI:10.1039/b002104k

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Practical syntheses of [13C]- and [14C]-labelled glucosphingolipids
Gordon R. Duffin,† George J. Ellames, Sascha Hartmann, John M. Herbert* and
David I. Smith
1
Isotope Chemistry and Metabolite Synthesis Department, Sanofi-Synthélabo, Willowburn Ave.,
Alnwick, Northumberland, UK NE66 2JH
Received (in Cambridge, UK) 15th March 2000, Accepted 8th June 2000
Published on the Web 3rd July 2000
Synthetic routes to [glucose-U-14C]-1-O-(β--glucopyranosyl)-N-stearoyl-D-erythro-sphingosine 1b and to
[glucose-13C6]-1-O-(β--glucopyranosyl)sphingosine ([glucose-13C6]glucopsychosine, 2b) are described. Whereas
the protected ceramide precursor for 1b was prepared using conventional methodology, two new strategies were
developed in the course of the synthesis of 2b. Of these, one relies on keeping a protecting group in place at all
times to avoid the handling difficulties associated with sphingosine 4, while the other generates a protected
derivative (24) of sphingosine indirectly by means of a Mitsunobu inversion.
Deficiency in the activity of the enzyme glucocerebrosidase,
which cleaves glucosylceramide 1a, results in an accumulation
Results and discussion
Since only limited quantities of 4 are available commercially, its
of 1a in human spleen, characteristic of the inherited lipidosis
disorder, Gaucher’s disease.1 In contrast, 1-O-β--glucosyl-
sphingosine (glucopsychosine, 2a) is a potent inhibitor of the
same enzyme.2 In order to facilitate pharmacological studies
involvingmammalianglucocerebrosidase,suppliesof[14C]gluco-
sylceramide 1b were needed, while a multiple [13C]-labelled
form, such as 2b, of glucopsychosine was required for use as an
internal standard in mass spectrometric assays.
synthesis was necessary. Of the various available routes to 4,7
that described by Garner et al.8 was chosen, and initial results
were satisfactory. This route involves addition of pentadec-1-
ynyllithium to the aldehyde 6 to give the acetylenic alcohol 7 in
a moderate diastereomeric excess. The enantiomeric excess of
this material was typically between 92 and 95%, as determined
by 19F NMR spectroscopy of the ester with (R)-MTPA.‡ Dis-
solving metal reduction of 7 (lithium–ethylamine) and acid
hydrolysis provided 4. In the course of resyntheses, however,
several side-products were observed of which the urea 8§ and
the reductive cleavage product 9¶ were identified. The over-
reduction product 10 was produced in small quantities, but
proved to be extremely difficult to separate from 4. A modified
route, which we found to be more reliable, involved partial
deprotection of 7 to give the Boc-protected species 11.9 Red-Al
With U-14C and 13C6-labelled forms of glucose (3a and 3b
respectively) available commercially, glucosylation of protected
forms of -erythro-sphingosine 4 and of the corresponding
stearamide (ceramide, 5) would provide a convenient approach
to 2b and 1b respectively, with the labelled moiety being intro-
duced at a late stage of the synthesis in both cases. Such an
approach has been used to prepare 2a3 and lower homologues
of 1a4,5 but, to date, [14C]glucosylceramide has been prepared
only in very small quantities by glucocerebrosidase-mediated
coupling of 4-methylumbelliferyl-β--glucose (as a glucose
donor) with [14C]-labelled 5,6 an approach which would not
have provided the quantities required.
‡ The 19F NMR spectrum (CDCl3) of the (S)-MTPA ester typically
contains two signals (attributed to two rotational isomers) arising from
the desired diastereomer at δ Ϫ71.48 and Ϫ71.60, the corresponding
signals arising from the minor isomer being observed at δ Ϫ71.76
and Ϫ72.27.
§ Data for 8: mp 120–121 ЊC; δH(CDCl3) 0.90 (3H, t), 1.15 (3H, t), 1.2–
1.45 (20H, m), 1.52 (2H, m), 2.22 (2H, t), 3.21 (2H, q), 3.55 (4H, br s),
3.71 (1H, dd), 3.78 (1H, dd), 3.86 (1H, dd), 4.54 (1H, d); m/z (FABϩ)
369 ([MH]ϩ, 100%), 351 ([MH Ϫ H2O]ϩ); HRMS 369.3112 (Calc. for
C21H41N2O3: m/z 369.3117).
¶ Data for 9: mp 44–47 ЊC; δH(CDCl3) 0.85 (3H, t), 1.1–1.4 (34H, m),
1.47 (2H, m), 2.81 (1H, m, NCHMe2), 3.26 (1H, m), 3.80 (2H, m), 3.87
(1H, m), 4.16 (3H, br s); m/z (CIϩ) 344 (100%), 102, 60. HRMS
344.3526 (MHϩ) (Calc. for C21H46NO2: m/z, 344.3529). This type of
process has been reported previously under other reductive conditions,
e.g. ref. 27.
† Current address: Development Centre, ChiRex, Cramlington,
Northumberland, UK NE23 7QG.
DOI: 10.1039/b002104k
J. Chem. Soc., Perkin Trans. 1, 2000, 2237–2242
This journal is © The Royal Society of Chemistry 2000
2237

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Table 1 Acylation conditions for the conversion of sphingosine 4 into
3-O-benzoyl-N-stearoyl--erythro-sphingosine 14
Acylation conditions
Yield (%)
Stearic acid, HBTUa–HOBt, Pri2NEt, DCM, room temp.
Stearic acid, TPTUb–HOBt, Pri2NEt, DCM, room temp.
Stearic acid, BOP, Et3N, THF, room temp.
4-Nitrophenyl stearate, DCM (ref. 11)
Stearic acid N-hydroxysuccinimide ester (ref. 12)
Stearoyl chloride, aq. NaOH, DCM, room temp.
Stearoyl chloride, pyridinec
47
28
70
53
27
0
26
46
Stearic anhydride, pyridine
a HBTU refers to O-(benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluron-
ium hexafluorophosphate. b TPTU refers to O-(1,2-dihydro-2-oxo-1-
pyridyl)-N,N,NЈ,NЈ-tetramethyluronium tetrafluoroborate. c Using this
method, even with a single molar equivalent of stearoyl chloride, diacyl
products were isolated also, and starting material was recovered.
reduction9 of this intermediate proceeded smoothly to give
Boc--erythro-sphingosine 12 in 90% yield; exposure of 12 to
hydrogen chloride in methanol resulted in acid hydrolysis
to provide 4 (89%).10
With both the protected sphingosine 14 and the glycosylating
Several methods were compared for the acylation of 4 to
form ceramide 5 (Table 1), of which the best yielding proved to
be a coupling using benzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate (BOP). The major draw-
back of this method proved to be that HMPA formed in the
reaction could not be removed completely from the product,
although this did not interfere with subsequent steps. Protec-
tion of 5 was carried out in the manner described by Schmidt
and Kläger4 for the corresponding palmitamide. Thus, pro-
tection of the primary alcohol as the trityl ether, followed by
benzoylation of the secondary alcohol, gave the fully protected
material 13, which was detritylated with acid to provide the
known intermediate 14.13 A protection sequence14 whereby the
agent 18a in hand, it was a relatively straightforward matter to
assemble 1b as outlined in Scheme 1. Lewis acid-promoted
Scheme 1 Assembly of fragments 14 and 18, and deprotection to give
1: (i) 18a or 18c, BF3ؒOEt2, DCM, 4 Å molecular sieves; (ii) MeONa,
MeOH.
primary alcohol is protected as a tert-butyldiphenylsilyl ether
rather than a trityl ether was examined, but was abandoned
since removal of the silyl ether with tetrabutylammonium fluor-
ide in THF resulted in a significant degree of acyl migration to
give the primary benzoate 15. A comparable process operates
during the acid-promoted detritylation step, but the isomeris-
ation is very much slower and only very small quantities of 15
were formed even after extended reaction times.||
coupling of 14 with 18a18 afforded the fully protected species
19a in 53% radiochemical yield. This was accompanied by a
small quantity of the acetate 20, presumably formed by nucleo-
philic attack of 14 upon the 2-O-acetate of the sugar, via the
cyclic tautomer of intermediate 21. The yield of this undesired
material was significantly lower when boron trifluoride–diethyl
ether was used to promote the coupling step than when the
reaction was carried out in the presence of trimethylsilyl tri-
flate.19 In addition, the product distribution appeared to be
dependent, at least in part, upon the temperature at which the
Lewis acid was added; formation of 20 was minimised by
maintaining the temperature below 0 ЊC during addition of
the Lewis acid. Final deprotection of 19a, using an excess of
sodium methoxide in methanol, gave the desired product, 1b,
in 77% radiochemical yield. The same coupling process was
carried out using unlabelled glucosylimidate 18c to provide
1a.
Protected stearoylsphingosine 14 was coupled with labelled
glucose by activation of the protected sugar as a trichloro-
acetimidate.15 Thus, [U-14C]glucose 3a was acetylated to give
pentaacetate 16a in 93% radiochemical yield, and this was
treated with benzylamine to deprotect the anomeric position
selectively.16 The resulting tetraacetate, 17a, was isolated in
70% radiochemical yield; unlabelled material 17c prepared in
1
the same manner was found from H NMR analysis to have
an anomeric ratio of 3:1 (α:β). 17a was converted into the
corresponding trichloroacetimide 18a, which appeared to be
the α-anomer exclusively (1H NMR), in 78% radiochemical
yield by treatment with trichloroacetonitrile in the presence of
caesium carbonate.17
Detailed 1H NMR studies were carried out on 1a. Definitive
assignments of all the resonances, other than those contained in
1
the methylene envelope, in the H NMR spectrum of 1a were
1
made with the assistance of the H/1H COSY spectrum. The
|| Data for 15: δH(CDCl3) 0.85 (6H, t), 1.1–1.35 (48H, m), 1.5–1.65 (4H,
m), 1.99 (2H, m), 2.17 (2H, t), 2.85 (1H, d), 4.25 (1H, m), 4.40 (2H, m),
4.55 (1H, m), 5.50 (1H, dd), 5.76 (1H, dt), 5.97 (1H, d), 7.43 (2H, m),
7.57 (1H, m), 8.01 (2H, d); m/z (CIϩ) 670 (MHϩ), 652 (670 Ϫ H2O), 530
(652 Ϫ PhCOOH).
stereochemistry at the anomeric centre could be assigned as
β from the coupling constant (7.6 Hz) from 1-H to 2-H of the
pyranose; further evidence for this assignment was provided by
NÖE enhancements in the signals due to 3-H and 5-H of the
2238
J. Chem. Soc., Perkin Trans. 1, 2000, 2237–2242

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pyranose, observed upon irradiation at the resonance frequency
of the anomeric proton. Since these can only occur if all three
protons are on the same face of the ring, the material
must be the β-anomer. The absence of any signals due to other
diastereomers also constitutes evidence that the route used to
prepare 4 had not resulted in isomerisation. The observed shifts
in the 13C NMR spectrum were also in good agreement with
those reported previously for 1a.20
With preparations of 1a and 1b complete, a similar approach
was chosen for that of the primary amine 2b. Nevertheless,
since the handling and solubility characteristics of 4 are poor, a
route was developed to the protected sphingosine 22 starting
from N-Boc-sphingosine 12, with the intention of maintaining
at least one protecting group in place at all times. Hence, 12 was
converted into fully protected derivative 23 in an analogous
sequence to that used already. Treatment with hydrogen chlor-
ide in methanol gave the known21 3-O-benzoyl--erythro-
sphingosine 24, which was acylated to give 22, identical to
material obtained using the published route.3 Hydrolysis of the
trityl group from 23 was very much faster than subsequent
hydrolysis of the tert-butyl carbamate, and so the alcohol 25
could be isolated by use of a smaller excess of hydrogen chlor-
ide or by hydrolysis in trifluoroacetic acid. Once again, despite
the use of extended reaction times, acyl migration was not
observed during deprotection.
Scheme 2 Alternative routes to 24: (i) (E)-C13H27CH᎐CHLi, THF;
᎐
(ii) Red-Al, toluene–Et2O, 0–5 ЊC to room temp.; (iii) PhCOCl, Py–
DMAP; (iv) Ref. 23; (v) PriOOCN᎐NCOOPri, PPh3, PhCOOH, THF;
᎐
(vi) HCl, MeOH.
More direct routes to the monoprotected species 24 are
outlined in Scheme 2. The fully protected intermediate 26 is
available from three different routes: addition of (E)-pentadec-1-
enyllithium22 to the complex formed by addition of diisobutyl-
aluminium hydride and triisobutylaluminium to the protected
serine 27 gave the protected threo-sphingosine 28.23 This was
successfully converted into the diastereomeric benzoate 26 by a
Mitsunobu reaction, as previously reported24 for the erythro-to-
threo conversion. Alternatively, reduction of 7 by Red-Al in the
same manner as that already described gave the allylic alcohol
29 in modest yield; the same intermediate is also available by
addition of (E)-pentadec-1-enyllithium to the Garner aldehyde
6 but, in this case, the yield was poor at best and this does
not appear to be a viable method. Thus, Mitsunobu inversion
(with concomitant esterification) of 28 and subsequent
hydrolysis of the acetonide provides a viable route to 26 and
thereby to 24.
that of the unlabelled form 2a, due to 13C–1H coupling in the
glucose moiety. Nevertheless, the signal arising from the ano-
meric proton is sufficiently separated from other signals to
enable confirmation that the material was the β-glycoside on
the basis of the observed J1,2-value of 7.8 Hz. The value of 1JCH
(117.7 Hz) is surprisingly low, but is still consistent with an
axial, rather than an equatorial, proton.25 The 13C NMR spec-
trum (acquired for the labelled portion of the molecule only)
also contained peaks corresponding to a single diastereomer
only.
Final assembly of [glucose-13C6]-1-O-(β--glucospyranoyl)-
-sphingosine 2b followed essentially the same sequence as that
described already for 1b. The glycosylating agent 18b was pre-
pared from [13C6]glucose via 16b and 17b, although in this case
the α- and β-anomeric forms were isolated. Since couplings to
13C complicate the signals observed due to pyranose protons in
Experimental
Mps were measured using a Buchi 510 capillary apparatus and
are uncorrected. IR spectra were recorded using a Nicolet 510
FTIR Instrument. Optical rotation measurements were made
using an Optical Activity digital polarimeter; [α]D-values are
given in units of 10Ϫ1 deg cm2 gϪ1, and concentrations (c) in
g/100 ml. NMR spectra were recorded using a JEOL GSX-270
spectrometer. Mass spectra were recorded using a VG Autospec
magnetic sector instrument at the University of York. [U-14C]-
-Glucose was obtained from Amersham International, Little
Chalfont, Buckinghamshire, UK, and [13C6]--glucose was
obtained from Cambridge Isotope Laboratories, Andover, MA,
USA.
1
the H NMR spectra of 16b–18b, making measurement of the
coupling constant J1,2 impractical, the stereochemistry at the
anomeric centre of these intermediates was assigned on the
1
basis of the coupling constant, JCH, for the anomeric proton.
In the case of 18b, for example, the (major) α-anomer has a
1JCH-value of 180 Hz, in line with that reported previously.25
Coupling of the α-anomer of 18b with 22 gave the protected
glucosylsphingosine 30, accompanied by the acetate 31. Depro-
tection of 30 with sodium methoxide in methanol3 gave 2b. The
NMR spectrum of this material is rather more complex than
J. Chem. Soc., Perkin Trans. 1, 2000, 2237–2242
2239

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N-Stearoyl-D-erythro-sphingosine 5
(1H, d, J 9.6 Hz, NH), 7.38 (2H, dd, J 7.6, 7.2 Hz), 7.52 (1H,
dd, J 7.6, 7.6 Hz), 7.96 (1H, d, J 7.2 Hz); m/z (NaI-FAB)
692.5594 (MNaϩ. C43H75NNaO4 requires m/z, 692.5594), 652
(692 Ϫ NaOH), 548 (670 Ϫ PhCOONa, 100%).
To a solution of octadecanoic acid (3.53 g, 12.43 mmol) in
dichloromethane (DCM) (45 ml) and triethylamine (1.75 ml)
was added a solution of BOP (5.57 g, 12.6 mmol) in DCM (25
ml), and the mixture was stirred at room temperature for 1 h.
After this time DCM (40 ml) and a solution of sphingosine 4
(3.12 g, 10.36 mmol) in THF (80 ml) were added and the
reaction mixture was stirred overnight at room temperature.
Solvents were removed under reduced pressure; the residue was
dissolved in chloroform, washed with aq. NaHCO3, and the
organic phase dried over MgSO4. After evaporation, the residue
was chromatographed on silica gel in methanol–chloroform
(3:100) to afford 5 (4.15 g, 70%), mp 85–87 ЊC (lit.,9 88–89 ЊC);
[α]D23 Ϫ2.0 (c 1.0 in EtOH; lit.,9 Ϫ2.4, c 1.1 in CHCl3); δH(CDCl3)
0.97 (6H, t), 1.1–1.4 (50H, m), 1.62 (2H, m), 2.04 (2H, m,
(ii) A solution of 4-nitrophenyl stearate26 (0.223 g, 0.55
mmol) and 24 (0.202 g, 0.50 mmol) (see below) in pyridine
(2 ml) was heated under nitrogen at 60 ЊC for 6 h, then stirred
overnight at room temperature. Pyridine was evaporated and
the residue was re-dissolved in ethyl acetate, washed twice with
1 M aq. sodium hydroxide and once each with water and brine.
The organic phase was dried (MgSO4) and evaporated to give
14 (0.197 g, 59%).
[glucose-U-14C]-1,2,3,4,6-Penta-O-acetyl-ꢀ-D-glucopyranose
16a
A solution of [U-14C]-glucose (20 mCi @ 302 mCi mmolϪ1
,
CH CH᎐), 2.21 (2H, t, J 8.2 Hz, CH CONH), 2.71 (2H,
᎐
2
2
m), 3.69 (1H, m), 3.8–4.0 (2H, m), 4.28 [1H, m, CH(OH)CH᎐],
5.52 [1H, ddt, J 15.4, 6.4, 1.0 Hz, CH(OH)CH᎐], 5.77 (1H,
᎐
0.066 mmol) in pyridine (10 ml) and acetic anhydride (6.5 ml)
under nitrogen was stirred at room temperature for 24 h.
Volatile materials were removed under reduced pressure and the
residue was partitioned between ethyl acetate and aq. NaHCO3.
The organic phase was washed with brine before being dried
over MgSO4. Solvent was removed under reduced pressure and
the residue was chromatographed on silica gel and eluted with
ethyl acetate–hexane (2:3) to afford 16a (18.75 mCi, 93%
radiochemical yield).
᎐
dtd, J 15.4, 6.7, 1.1 Hz, CH CH᎐), 6.22 (1H, d, J 6.8 Hz,
᎐
2
NH).
3-O-Benzoyl-N-stearoyl-1-O-trityl-D-erythro-sphingosine 13
A suspension of 5 (6.52 g, 11.5 mmol), triethylamine (12.8 ml),
DMAP (122 mg) and trityl chloride (4.80 g, 17.2 mmol) in
DCM (150 ml) was heated at reflux for 60 h. Volatile materials
were evaporated, the residue was re-dissolved in ethyl acetate,
and the mixture was washed successively with 1 M hydrochloric
acid, aq. NaHCO3 and brine. The organic phase was dried
(MgSO4) and evaporated, and the residue was chromato-
graphed on silica gel in ethyl acetate–hexane (3:7) to afford
N-stearoyl-1-O-trityl--erythro-sphingosine (7.20 g, 77%) as an
oil, δH(CDCl3) 0.88 (6H, t), 1.15–1.4 (m), 1.64 (2H, m), 1.91
(2H, m), 2.20 (2H, t, J 8.2 Hz), 3.28 (1H, dd, J 9.6, 4.0 Hz),
3.35–3.4 (2H, m), 4.04 (1H, m), 4.17 (1H, m), 5.24 (1H, dd,
J 15.4, 6.2 Hz), 5.62 (1H, dt, J 15.4, 6.6 Hz), 6.06 (1H, d, J 7.5
Hz, NH), 7.2–7.35 (9H, m), 7.35–7.45 (6H, m).
To a solution of this intermediate in pyridine (100 ml) under
nitrogen were added DMAP (163 mg) and benzoyl chloride
(1.74 ml, 15 mmol) and the mixture was stirred for 20 h. Solvent
was removed under reduced pressure and the residue was par-
titioned between aq. NaHCO3 and ethyl acetate. The organic
phase was washed with brine, dried (MgSO4), and evaporated,
and the residue chromatographed on silica gel in ethyl acetate–
hexane (15:85 to 1:1) to give 13 (5.61 g, 69%) as an oil,
δH(CDCl3) 0.87 (3H, t), 1.1–1.35 (50H, m), 1.54 (2H, m), 1.99
(2H, m), 2.08 (2H, t), 3.17 [1H, dd, J 7.4, 3.9 Hz, CH(HЈ)OH],
3.43 [1H, dd, J 9.7, 3.9 Hz, CH(HЈ)OH], 4.47 [1H, m, CH-
Similarly prepared, from [13C6]glucose (1.0 g, 5.4 mmol), was
16b (2:1 ratio of α- to β-anomer; 2.141 g, 100%), δH(CDCl3)
2.00 (9H, s), 2.00 (3H, s), 2.07 (3H, s), 3.5–6.0 (6H, m), 5.41
1
(0.33H, dm, JCH 147 Hz, H-1 of β-anomer), 6.32 (0.66H, dm,
1
1JCH 177 Hz, H-1 of α-anomer); δC(CDCl3) 61.3 (d, J 45 Hz),
67.6 (br dd, 1J 53, 44 Hz), 69–71 (2C, m), 72.6 (dd, 1J 45, 42 Hz),
1
1
89.0 (br d, J 46 Hz, C-1 of α-anomer), 91.6 (ddd, J 48 Hz,
2J 5.5, 5.5 Hz, C-1 of β-anomer).
[glucose-U-14C]-2,3,4,6-Tetra-O-acetyl-D-glucopyranose 17a
Compound 16a (18.75 mCi, 0.62 mmol) was diluted to 50 mCi
mmolϪ1 by addition of unlabelled 1,2,3,4,6-penta-O-acetyl-α--
glucopyranose (16c; 121 mg) in THF (3 ml) under nitrogen, and
this material (18.75 mCi, 155 mg, 0.371 mmol) was treated with
benzylamine (55 ml, 0.49 mmol) and stirred at room temper-
ature for 24 h. Solvent was removed under reduced pressure and
the residue was chromatographed on silica gel and eluted with
ethyl acetate–hexane (2:3) to afford the desired [glucose-U-14C]-
17a (13.2 mCi, 70% radiochemical yield).
Similarly prepared, from 16b (2.124 g, 5.4 mmol), was 17b
(1.942 g, 100%), δH(CDCl3) 2.03 (6H, s), 2.06 (6H, s), 3.4–5.6
1
(6H, m), 5.45 (1H, dm, JCH 174 Hz, 1-H); δC(CDCl3) 61.9 (d,
1J 41 Hz), 66–74 (m, 4C), 90.1 (d, 1J 44 Hz, C-1 of α-anomer),
95.5 (dt, 1J 46 Hz, 2J 5.5, 5.5 Hz, C-1 of β-anomer); m/z (NH3-
CIϩ) 372 (MNH4ϩ, 100%), 337; HRMS 372.1602 (Calc. for
C813C6H20O10: M, 372.1602).
(NHCOR)], 5.43 [1H, dd, J 15.3, 7.3 Hz, CH(OCOPh)CH᎐],
᎐
5.6–5.75 [2H, m, NH, CH(OCOPh)], 5.86 (1H, dt, J 15.3, 7.9
Hz, CH CH᎐), 7.1–7.25 (9H, m), 7.3–7.4 (8H, m), 7.54 (1H, t,
᎐
2
J 7.5 Hz), 7.92 (2H, d, J 7.3 Hz); m/z (NaI-FAB) 934.6690
(MNaϩ. C62H89NNaO4 requires m/z, 934.6689), 796, 548, 264
(100%).
[glucose-U-14C]-2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl
trichloroacetimidate 18a
3-O-Benzoyl-N-stearoyl-D-erythro-sphingosine 14
To a solution of 17a (13.2 mCi, 0.264 mmol) in DCM (3 ml)
under nitrogen were added trichloroacetonitrile (48 ml, 0.61
mmol) and caesium carbonate (23 mg, 0.07 mmol) and the mix-
ture was stirred at room temperature for 5 h. Additional DCM
was added and the solution was washed with aq. NaHCO3,
dried (MgSO4) and evaporated. The residue was chromato-
graphed on silica gel in ethyl acetate–hexane (2:3) to afford 18a
(10.35 mCi, 78% radiochemical yield).
(i) A solution of 13 (5.60 g, 6.1 mmol) and toluene-p-sulfonic
acid monohydrate (1.3 g, 6.7 mmol) in DCM (100 ml) and
methanol (100 ml) was stirred under nitrogen for 3 h. Solvent
was evaporated and the residue was partitioned between aq.
NaHCO3 and chloroform. The organic phase was washed with
brine, dried over MgSO4, and evaporated to dryness. The resi-
due was chromatographed on silica gel and eluted with ethyl
acetate–hexane (1:1) to give 14 (3.70 g, 91%), mp 85.5–86 ЊC
(lit.,13 86–88 ЊC); [α]D20 ϩ16.0 (c 0.4 in EtOH; lit.,13 ϩ16.7, c 1.5 in
CHCl3); δH(CDCl3) 0.87 (6H, t), 1.1–1.3 (50H, m), 1.54 (2H,
m), 1.96 (2H, m), 2.14 (2H, m), 2.77 (2H, br s), 3.71 (2H, m,
CH2O), 4.24 (1H, m, CHN), 5.4–5.6 [2H, m, CH(OC-
Similarly prepared, from 17b (1.388 g, 3.9 mmol), was 18b.
α-Anomer (1.108 g, 57%): δH(CDCl3) 2.02 (9H, s), 2.06 (3H, s),
3.7–6.0 (6H, m), 6.52 (1H, dm, 1JCH 180 Hz, 1-H), 8.68 (1H, s).
β-Anomer (0.286 g, 15%): δH(CDCl3) 2.02 (9H, s), 2.06 (3H,
1
s), 3.7–6.0 (6H, m), 5.82 (1H, dm, JCH 162 Hz, 1-H), 8.70
OPh)CH᎐], 5.79 (1H, dt, J 15.0, 6.8 Hz, CH CH᎐), 6.18
(1H, s).
᎐
᎐
2
2240
J. Chem. Soc., Perkin Trans. 1, 2000, 2237–2242

View Article Online
[glucose-U-14C]-3-O-Benzoyl-1-O-(2,3,4,6-tetra-O-acetyl-ꢁ-D-
glucopyranosyl)-N-stearoyl-D-erythro-sphingosine 19a
This intermediate was dissolved in pyridine (10 ml) and
DMAP (0.888 g) was added, followed by benzoyl chloride
(0.551 ml, 4.8 mmol). The suspension was stirred for 2 h,
pyridine was removed under reduced pressure, and the residue
was chromatographed on silica gel in hexane–ethyl acetate
(from 85:15 to 1:1) to give 23 (2.775 g, 98%), δH(CDCl3) 0.88
(3H, t), 1.1–1.5 (22H, m), 1.43 (9H, s), 1.97 (2H, m), 3.20 [1H,
dd, J 10.9, 4.1 Hz, CH(HЈ)OTr], 3.32 [1H, dd, J 10.2, 4.1 Hz,
CH(HЈ)OTr], 4.13 (1H, m), 4.77 (1H, br d, J 10.2 Hz), 5.41 (1H,
dd, J 15.3, 7.5 Hz), 5.65 (1H, m), 5.85 (1H, dt, J 15.3, 7.9 Hz),
7.1–7.5 (17H, m), 7.52 (1H, dd, J 8.2, 4.0 Hz), 7.89 (2H, d, J 7.5
Hz); m/z (NaI-FAB) 768.4601 (MNaϩ. C49H63NNaO5 requires
m/z, 768.4604), 664, 526, 326, 243 (100%).
A suspension of 14 (169 mg, 0.252 mmol) and dried, powdered
4 Å molecular sieves (643 mg) in DCM (6 ml) was stirred under
nitrogen for 90 min before the addition of 18a (10.3 mCi, 0.207
mmol) in DCM (5 ml). The mixture was cooled to Ϫ10 ЊC,
boron trifluoride–diethyl ether (35 ml, 0.288 mmol) was added,
and the mixture was allowed to warm to room temperature and
was stirred for 10 h. After this time the molecular sieves were
removed by filtration and washed with further DCM. The
filtrate was washed with aq. NaHCO3 and dried over MgSO4
before evaporation under reduced pressure. The residue was
chromatographed on silica gel in acetone–toluene (4:96) to give
19a (5.46 mCi, 53% radiochemical yield).
Similarly prepared, from 18c,17 was 19b (55%), mp 74–74.5 ЊC,
[α]D22 ϩ11.25 (c 0.4 in EtOH); δH(CDCl3) 0.86 (6H, t), 1.15–1.35
(50H, m), 1.95 (3H, s), 1.99 (6H, s), 2.00 (3H, s), 2.14 (2H, t),
3.6–3.7 (2H, m), 3.9–4.3 (4H, m), 4.45 (1H, d, J 7.8 Hz, H-6),
4.85–5.0 (1H, m), 5.04 (1H, dd, J 9.2, 4.3 Hz), 5.15 (1H, d, J 9.5
Hz), 5.4–5.6 (2H, m), 5.75–5.9 (2H, m), 7.41 (2H, dd, J 8.1, 7.6
Hz), 7.54 (1H, dd, J 7.6, 7.6 Hz), 7.99 (2H, dd, J 8.1, 1.1 Hz);
m/z (NaI-FAB) 1022.6551 (MNaϩ. C57H93NNaO13 requires
m/z, 1022.6545), 878, 548, 331, 264, 169 (100%). This was
accompanied by acetylated aglycone 20 (10%), mp 88–89 ЊC;
δH(CDCl3) 0.87 (6H, t), 1.1–1.4 (50H, m), 1.57 (2H, m), 2.00
(2H, m), 2.01 (3H, s), 2.17 (2H, t, J 8.5 Hz), 4.16 [1H, dd, J 11.6,
5.4 Hz, CH(HЈ)OH], 4.34 [1H, dd, J 11.6, 7.5 Hz, CH(HЈ)OH],
4.58 (1H, m, CHN), 5.4–5.6 (2H, m), 5.77 (1H, d, J 9.6 Hz,
tert-Butyl 4-[(1R,2E)-1-hydroxyhexadec-2-enyl]-2,2-dimethyl-
oxazolidine-3-carboxylate 29
Red-Al in toluene (3.2 M; 3.5 ml, 11 mmol) was added under
nitrogen to an ice-cooled solution of 7 (0.969 g, 2.2 mmol) in
diethyl ether (20 ml). The mixture was warmed to room tem-
perature and stirred for 22 h. Methanol (1 ml) was added, fol-
lowed by an excess of aq. ammonium chloride, and the mixture
was filtered through Celite. The phases were separated, the
aqueous phase was re-extracted with ethyl acetate, and the
combined organic phases were dried (MgSO4), solvent was
removed under reduced pressure, and the residue was chrom-
atographed on silica in 9:1 hexane–ethyl acetate to give 29
(0.337 g, 35%), δH(CDCl3) 0.88 (3H, t), 1.1–1.65 (37H, m), 1.99
(2H, m), 3.52 (2H, m), 3.61 (1H, m), 4.06 (1H, m), 5.34 (1H, m),
5.53 (1H, m); m/z (CIϩ) 440.3736 (MHϩ. C26H50NO4 requires
m/z, 440.3740), 366, 340 (100%), 100.
NH), 5.88 [1H, dt, J 15.0, 7.5 Hz, CH(OH)CH᎐], 7.45 (2H, dd,
᎐
J 7.6, 7.2 Hz), 7.56 (1H, dd, J 7.6, 7.6 Hz), 8.01 (1H, d, J 7.2
Hz); m/z (NaI-FAB) 734.5699 (100%, MNaϩ. C45H77NNaO5
requires m/z, 734.5699), 612, 264, 176.
tert-Butyl 4-[(1R,2E)-1-benzoyloxyhexadec-2-enyl]-2,2-
dimethyloxazolidine-3-carboxylate 26
[glucose-U-14C]-1-O-(ꢁ-D-Glucopyranosyl)-N-stearoyl-D-
erythro-sphingosine 1b
(i) A solution of benzoyl chloride (116 ml, 1 mmol), DMAP
(0.184 g) and 29 (0.320 g, 0.73 mmol) in pyridine (5 ml) was
stirred for 2 h, concentrated to dryness, and the residue chrom-
atographed on silica gel in 1:9 ethyl acetate–hexane to give
26 (0.210 g, 53%) as a colourless oil, δH(CDCl3) 0.87 (3H, t),
1.1–1.4 (22H, m), 1.45 (9H, s), 1.46 (3H, s), 1.50 (3H, s), 2.03
(2H, m), 4.0–4.3 (3H, m), 5.4–5.65 (1H, m), 5.7–5.9 (2H, m),
7.45 (2H, m), 7.54 (1H, m), 8.04 (1H, m), 8.09 (1H, d); m/z
(NaI-FAB) 566.3814 (100%, MNaϩ. C33H53NNaO5 requires
m/z, 566.3821).
(ii) A solution of diisopropyl azodicarboxylate (20 ml, 0.1
mmol) and triphenylphosphine (26 mg, 0.1 mmol) in THF
(2 ml) was stirred for 20 min, following which the S-alcohol 2823
(20 mg, 46 µmol) as a solution in THF (1 ml) was added
followed, immediately, by benzoic acid (31 mg). The mixture
was stirred for 36 h, then purified by preparative TLC on silica
gel in 1:3 ethyl acetate–hexane to give 26 (18 mg, 72%).
A suspension of 19a (5.46 mCi, 0.11 mmol) and sodium meth-
oxide (30.8 mg, 0.57 mmol) in methanol (5 ml) was stirred for
24 h. Additional methanol (25 ml) and chloroform (5 ml) were
added followed by Dowex 50W-X8 cation-exchange resin (532
mg). The mixture was stirred for 10 min, the exchange resin was
removed by filtration, and solvent was removed under reduced
pressure. The residue was chromatographed on silica gel in
methanol–chloroform (1:4) to afford 1b (4.2 mCi, 77% radio-
chemical yield).
Similarly prepared, from 19b, was 1a (95%) as a white solid,
mp 194–195 ЊC; δH(CDCl3–CD3OD, 1:1) 0.89 (6H, t), 1.2–1.45
(50H, m), 1.60 (2H, m, COCH CH ), 2.03 (2H, dt, ᎐CHCH ),
᎐
2
2
2
2.17 (2H, t, COCH2), 3.20 (1H, dd, 3Ј-H), ≈3.28 (2H, partly
obscured by solvent, CH2OH), 3.35 (1H, dd, J 10.1, 2.8 Hz,
4-H), 3.64 (1H, m, 6-H), 3.68 (1H, m, NCH), 3.86 (1H, dd,
6-H), 3.98 (1H, ddd, 6-HЈ), 4.08 [1H, m, ᎐CHCH(OH)], 4.11
᎐
(1H, m, 5-H), 4.26 (1H, d, J 7.6 Hz, 1-H), 5.46 [1H, br dd,
3-O-Benzoyl-D-erythro-sphingosine 24
J 15.4, 7.3 Hz, ᎐CHCH(OH)], 5.69 (1H, dt, J 15.4, 6.5 Hz,
᎐
᎐CHCH ); m/z 750 (MHϩ), 710 (750 Ϫ NaOH), 548, 264;
᎐
2
(i) Acetyl chloride (0.995 ml, 14 mmol) was added to methanol
(25 ml) and, after 5 min, the solution was added to the fully
protected sphingosine 23 (2.638 g, 3.5 mmol) to give a suspen-
sion, which was stirred for 90 min. The mixture was concen-
trated to dryness, the residue was partitioned between ethyl
acetate and aq. NaHCO3, and the organic extract was washed
with brine, dried over (MgSO4), and solvent removed under
vacuum. The residue was purified by chromatography on silica
gel in 99:1 ethyl acetate–acetic acid followed by 96:2:2
propan-2-ol–methanol–acetic acid to give 24 (809 mg, 57%) as a
white solid, mp 96–97 ЊC; [α]D22 ϩ3.2 (c 3.1 in EtOH); δH(CDCl3)
0.92 (3H, t), 1.1–1.5 (22H, m), 2.13 (2H, dt, J 8.2, 7.2 Hz), 3.51
(1H, dd, J 13.0, 5.5 Hz, CHN), 3.76 [1H, m, CH(HЈ)OH], 3.90
[1H, m, CH(HЈ)OH], 5.59 (1H, dd, J 15.6, 7.5 Hz), 5.71 [1H,
dd, J 7.2, 6.2 Hz, CH(OCOPh)], 6.00 (1H, dt, J 15.6, 7.1 Hz),
7.51 (2H, dd, J 8.2, 7.5 Hz), 7.66 (1H, dd, J 7.5, 7.5 Hz), 8.09
HRMS 750.5864 (Calc. for C42H81NNaO8: m/z, 750.5860).
3-O-Benzoyl-N-tert-butoxycarbonyl-1-O-tritylsphingosine 23
A solution of N-Boc-sphingosine 12 (2.002 g, 5 mmol), DMAP
(107 mg), pyridine (2 ml) and trityl chloride (1.422 g, 5.1 mmol)
in DCM (40 ml) was stirred for 24 h, then heated to 75 ЊC for
2.5 h. Solvent was removed and the residue was chromato-
graphed on silica gel in hexane–ethyl acetate (95:5, then 9:1) to
give N-tert-butoxycarbonyl-1-O-tritylsphingosine (2.452 g,
77%), δH(CDCl3) 0.89 (3H, t), 1.1–1.4 (22H, m), 1.48 (9H, s),
1.93 (2H, m), 3.24 (1H, m), 3.40 (1H, m), 3.70 (1H, m), 4.25
(1H, m), 5.15 (1H, dd, J 15.3, 6.7 Hz), 5.24 (1H, m), 5.64 (1H,
dt, J. 15.3, 6.6 Hz), 7.15–7.35 (9H, m), 7.35–7.5 (6H, m); m/z
(NaI-FABϩ) 664 (MNaϩ), 484, 243 (Ph3Cϩ); HRMS 664.4340
(Calc. for C42H59NNaO4: m/z, 664.4342).
J. Chem. Soc., Perkin Trans. 1, 2000, 2237–2242
2241

View Article Online
(2H, d, J 8.2 Hz); δH for HCl salt (CD3OD) 0.90 (3H, t), 1.1–1.5
(22H, m), 2.11 (2H, m), 3.58 (1H, m), 3.79 [1H, dd, J 10.8, 3.4
Hz, CH(HЈ)OH], 3.94 [1H, dd, J 10.8, 2.7 Hz, CH(HЈ)OH],
CH CH᎐), 2.9–3.2 (2.5H, m), 3.3–3.65 (4H, m, part obscured
᎐
2
by solvent), 3.8–4.05 (2.5H, m), 4.11 [1H, br d, J 6.5 Hz,
1
CH(OH)CH᎐], 4.36 (1H, dd, J 117.7 Hz, J 7.8 Hz, 1-H),
᎐
CH
5.56 [1H, dd, J 15.6, 7.1 Hz, CH(OCOPh)CH᎐], 5.74 [1H, dd,
5.48 [1H, dd, J 15.6, 7.5 Hz, CH(OH)CH᎐], 5.80 (1H, dt, J 15.6,
᎐
᎐
J 7.1, 3.0 Hz, CH(OCOPh)], 6.03 (1H, dt, J 15.6, 6.8 Hz,
6.5 Hz, ᎐CHCH ); δ (CD OD) 62.6 (d, J 170 Hz), 71.5
᎐
2 C 3
᎐CHCH ), 7.51 (2H, dd, J 7.1, 7.5 Hz), 7.64 (1H, dd, J 7.1, 7.1
(dd, J 162, 153 Hz), 74.9 (dd, J 184, 158 Hz), 77.9 (2C, 2 dd),
᎐
2
Hz), 8.07 (2H, d, J 7.5 Hz); m/z (FAB) 404.3166 (MHϩ.
C25H42NO3 requires m/z, 404.3165), 281 (100%).
104.25 (d, J 188 Hz); m/z (NaI-FABϩ) 490.3455 (MNaϩ.
C1813C6H47NO7Na requires m/z, 490.3452), 473, 323, 282;
᎐
(ii) A solution of 26 (0.210 g, 0.37 mmol) in trifluoroacetic
acid (4.2 ml) was stored for 3 h. Volatile materials were removed
under reduced pressure and the residue was partitioned
between ethyl acetate and aq. NaHCO3. The organic phase was
dried (MgSO4) and evaporated. Preparative TLC of the residue
on silica gel in 99:1 ethyl acetate–acetic acid gave 25 (84 mg,
43%); δH(CDCl3) 0.88 (3H, t), 1.1–1.5 (31H, m), 2.35 (2H, m),
3.55–4.10 (4H, m), 5.03 [1H, m, CH(OCOPh)], 5.64 (1H, dd,
J 15.3, 7.3 Hz), 5.84 (1H, dt, J 15.3, 7.1 Hz), 7.44 (2H, m), 7.57
(1H, m), 8.06 (2H, m); m/z (NaI-FAB) 526.3507 (MNaϩ.
C30H49NNaO5 requires m/z, 526.3508), 326 (100%), 154, and 24
(60 mg, 39%).
(CI) 468, 450, 282 (468 Ϫ C H C᎐CH), 264.
᎐
13 27
Acknowledgements
We thank Dr S. J. Byard for NMR spectra, Dr T. Dransfield
(University of York) for mass spectra, and Mr T. W. Mathers
for assistance with the preparation of precursors.
References
1 T. Kolter and K. Sandhoff, Angew. Chem., Int. Ed., 1999, 38, 1532;
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3-O-Benzoyl-N-trifluoroacetyl-D-erythro-sphingosine 22
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9 P. Herold, Helv. Chim. Acta, 1988, 71, 354.
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1988, 1103.
11 M. Morita, K. Motok, K. Akimoto, T. Natori, T. Sakai, E. Sawa,
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A solution of 4-nitrophenyl trifluoroacetate (1.014 g, 4.1 mmol)
and 24 (664 mg, 1.65 mmol) in pyridine (10 ml) under nitrogen
was stirred for 2 h at 55 ЊC. Volatile material was removed under
reduced pressure, and the residue was partitioned between ethyl
acetate and 0.3 M aq. KHSO4. The organic phase was washed
twice with 2 M aq. NaOH, three times with water, and once
with brine, then dried (MgSO4). Evaporation under reduced
pressure, and column chromatography of the residue on silica
gel in ethyl acetate–hexane (1:9), gave 22 (444 mg, 54%), mp
80–81 ЊC (lit.,2 80.5–81.5 ЊC); [α]D21 ϩ11.2 (c 1.0 in EtOH) (lit.,2
[α]D ϩ11.1, c 1.0, CHCl3); δH(CDCl3) 0.88 (3H, t), 1.15–1.4
(22H, m), 2.05 (2H, dt, CH CH᎐), 2.69 (1H, br s, OH), 3.69
᎐
2
[1H, dd, J 12.0, 3.4 Hz, CH(HЈ)OH], 3.80 [1H, dd, J 12.0,
2.8 Hz, CH(HЈ)OH], 4.26 (1H, m, CHN), 5.55 [1H, m, CH-
(OCOPh)], 5.60 [1H, dd, J 14.4, 7.2 Hz, ᎐CHCH(OH)], 5.90
᎐
(1H, dt, J 14.4, 6.7 Hz, ᎐CHCH ), 6.95 (1H, d, J 8.95 Hz, NH),
᎐
2
7.47 (2H, dd, J 8.5, 7.5 Hz), 7.61 (1H, dd, J 7.5, 7.5 Hz), 8.04
(1H, d, J 8.5 Hz); m/z (NaI-FAB) 522.2809 (MNaϩ. C27H40F3-
NNaO4 requires m/z, 522.2807), 173. Further elution returned
24 (100 mg, 15% recovery).
15 K. Toshima and K. Tatsuta, Chem. Rev., 1993, 93, 1503; R. R.
Schmidt, Angew. Chem., Int. Ed. Engl., 1986, 25, 212.
16 M. M. Sim, H. Kondo and C.-H. Wong, J. Am. Chem. Soc., 1993,
115, 2260.
17 F. J. Urban, B. S. Moore and R. Breitenbach, Tetrahedron Lett.,
1990, 31, 4421.
[glucose-U-13C]-3-O-Benzoyl-1-O-(2,3,4,6-tetra-O-acetyl-ꢁ-D-
glucopyranosyl)-N-trifluoroacetyl-D-erythro-sphingosine 30
18 P. Zimmerman, R. Bommer, T. Bär and R. R. Schmidt,
J. Carbohydr. Chem., 1988, 7, 435; N. P. Singh and R. R. Schmidt,
J. Carbohydr. Chem., 1989, 8, 199 and references therein; R. R.
Schmidt and W. Kinzy, Adv. Carbohydr. Chem. Biochem., 1994, 50,
21; T. Murakami, H. Minamikawa and M. Hato, J. Chem. Soc.,
Perkin Trans. 1, 1992, 1875; T. Sakai, H. Ueno, T. Natori,
A. Uchimura, K. Motoki and Y. Koezuka, J. Med. Chem., 1998, 41,
651.
19 H. Minamikawa, T. Murakami and H. Masakatsu, Chem. Phys.
Lipids, 1994, 72, 111.
20 F. Sarmientos, G. Schwarzmann and K. Sandhoff, Eur. J. Biochem.,
1985, 146, 59.
21 D. Shapiro and H. M. Flowers, J. Am. Chem. Soc., 1961, 83, 3327.
22 M. A. Peterson and R. A. Polt, Synth. Commun., 1992, 22, 477.
23 R. Polt, M. A. Peterson and L. De Young, J. Org. Chem., 1992, 57,
5469.
24 S. Nimkar, D. Menaldino, A. H. Merrill and D. Liotta, Tetrahedron
Lett., 1988, 29, 3037.
25 I. Tvaroska and F. Taravel, Adv. Carbohydr. Chem. Biochem., 1995,
51, 15.
26 J. H. Bowie and B. J. Stapleton, Aust. J. Chem., 1975, 28, 1011.
27 K. Ito and M. Sekiya, Chem. Pharm. Bull., 1972, 20, 1792;
T. E. Saavedra, J. Org. Chem., 1985, 50, 2271.
As described for 19a, coupling of 18b (316 mg, 0.6 mmol) with
22 gave 30 (140 mg, 27%), mp 100–102 ЊC (lit.,2 103–105 ЊC);
[α]D22 Ϫ4.3 (c 0.8 in EtOH; lit.,2 Ϫ4.5, c 0.75 in CHCl3);
δH(CDCl3) 0.88 (3H, t), 1.05–1.3 (22H, m), 1.95 (3H, s), 1.98
(6H, s), 2.00 (3H, s), 2.08 (2H, m), 3.0–5.3 (11H, m), 5.34 (1H, t,
J 7.9 Hz), 5.45 (1H, dd, J 14.4, 6.8 Hz), 5.80 (1H, dt, J 14.4, 7.7
Hz), 7.30 (2H, m), 7.45 (1H, m), 7.88 (2H, d, J 7.9 Hz); m/z
(NH3-FABϩ) 853.4408 (MNH4ϩ, 100%. C3513C6H62F3N2O13
requires m/z, 853.4405), 733, 337. Also recovered was 31 (58
mg, 18%), δH(CDCl3) 0.87 (3H, t), 1.1–1.4 (22H, m), 2.05 (3H,
s), 2.06 (2H, m), 4.26 (1H, dd), 4.37 (1H, dd), 4.57 (1H, m), 5.47
(1H, t), 5.57 (1H, dd), 5.96 (1H, dd), 6.84 (1H, d), 7.46 (2H, t),
7.60 (1H, t), 8.01 (2H, d).
[glucose-13C6]-1-O-(ꢁ-D-Glucopyranosyl)-D-erythro-sphingosine
2b
Hydrolysis of 307 (0.240 g, 0.29 mmol) gave 2b (105 mg, 78%),
δH(CD3OD) 0.90 (3H, t), 1.15–1.5 (2H, m), 2.11 (2H, dt,
2242
J. Chem. Soc., Perkin Trans. 1, 2000, 2237–2242