Synthesis of some second-generation substrate analogues of early intermediates in the biosynthetic pathway of glycosylphosphatidylinositol membrane anchors

Article abstract of DOI:10.1016/S0008-6215(99)00170-6

1-D-6-O-(2-Amino-2-deoxy-α-D-glucopyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (23) and the corresponding 2-O-hexadecyl-D-myo-inositol compound 24 have been prepared as substrate analogues of an early intermediate in the biosynthetic pathway of glycosylphosphatidylinositol (GPI) membrane anchors. 1-D-6-O-(2-Amino-2-deoxy-α-D-glucopyranosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate) has also been prepared as a substrate analogue. Biological evaluation of the analogues 23 and 24 revealed that they are neither substrates nor inhibitors of GPI biosynthetic enzymes in the human (HeLa) cell-free system but are potent inhibitors at different stages of GPI biosynthesis in the Trypanosoma brucei cell-free system. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00170-6

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Carbohydrate Research 321 (1999) 42–51
Synthesis of some second-generation substrate analogues
of early intermediates in the biosynthetic pathway of
glycosylphosphatidylinositol membrane anchorsꢀ
Arthur Crossman Jr. a, John S. Brimacombe a,*, Michael A.J. Ferguson b,
Terry K. Smith b
a Department of Chemistry, Uni6ersity of Dundee, Dundee DD1 4HN, UK
b Department of Biochemistry, Uni6ersity of Dundee, Dundee DD1 4HN, UK
Received 18 April 1999; accepted 26 June 1999
Abstract
1-
phosphate) (23) and the corresponding 2-O-hexadecyl-
analogues of an early intermediate in the biosynthetic pathway of glycosylphosphatidylinositol (GPI) membrane
anchors. 1- -6-O-(2-Amino-2-deoxy-a- -glucopyranosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate)
D
-6-O-(2-Amino-2-deoxy-a-
D
-glucopyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-
D
-myo-inositol compound 24 have been prepared as substrate
D
D
has also been prepared as a substrate analogue. Biological evaluation of the analogues 23 and 24 revealed that they
are neither substrates nor inhibitors of GPI biosynthetic enzymes in the human (HeLa) cell-free system but are potent
inhibitors at different stages of GPI biosynthesis in the Trypanosoma brucei cell-free system. © 1999 Elsevier Science
Ltd. All rights reserved.
Keywords: Glycosylphosphatidylinositol (GPI) membrane anchors; 1-
and -2-O-hexadecyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate); 1-
nosyl)-myo-inositol 1-(1,2-di-O-octyl-sn-glycerol 3-phosphate); Inhibition of GPI biosynthesis
D
-6-O-(2-Amino-2-deoxy-a-
D
-glucopyranosyl)-2-O-octyl-
-glucopyra-
D
-6-O-(2-Amino-2-deoxy-a-
D
tached to the plasma membrane by means of
glycosylphosphatidylinositol (GPI) anchors,
the principal function of which is to provide a
stable association of protein or oligosaccha-
ride with the lipid bilayer [3]. Studies in our
laboratories have focused largely on those en-
zymes involved in the early stages of GPI
biosynthesis in the belief that disruption of
GPI-anchor biosynthesis would seriously im-
pair the parasite’s ability to survive in the host
[4,5].
1. Introduction
Glycoconjugates on the cell surface of para-
sitic protozoa of the Trypanosomatidae (in-
cluding, for example, African and American
trypanosomes and Leishmania spp.) frequently
have a crucial role in determining parasite
infectivity [2]. Many glycoconjugates are at-
All GPI anchors that have been character-
ised to date (from protozoan, yeast, slime
mould, fish and mammalian sources) contain
ꢀ Parasite glycoconjugates, Part 10. For Part 9, see Ref. [1].
* Corresponding author. Tel.: +44-1382-223-181, ext.
4329; fax: +44-1382-345-517.
E-mail address: j.s.brimacombe@dundee.ac.uk (J.S. Brima-
combe)
an identical ethanolamine phosphate-6-a-
D-
Manp-(1“2)-a- -Manp-(1“6)-a- -Manp-
D
D
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00170-6

A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
43
(1“4)-a-
D
-GlcpNH2-(1“6)-
D
-myo-inositol
occurs before the first a-
D
-Manp residue is
backbone, suggesting that this sequence is
likely to be conserved in all GPI anchors [2].
To this conserved region may be added other
sugars in a species- and stage-specific manner.
The biosynthesis of GPI membrane anchors
in bloodstream forms of Trypanosoma brucei
[6,7] occurs in the endoplasmic reticulum and
involves the sequential glycosylation of phos-
attached to form, for example, a-
D
-GlcpNH2-
(1“6)-2-O-acyl-PI 2.
Methylation of 2-OH of the
D
-myo-inositol
residue, as in the synthetic analogue 3 [9],
prevents inositol acylation and allows the role
of inositol acylation in GPI biosynthesis to be
assessed. Studies with the methylated ana-
logue 3 showed [10] that it is recognised and
phatidylinositol (PI) as follows: a-
is transferred from UDP- -GlcpNAc to PI to
form a- -GlcpNAc-(1“6)-PI, which is then
N-deacetylated to form a- -GlcpNH2-(1“6)-
PI. An a- -Manp residue is then transferred
from dolichol phosphate -mannose to form
a- -Manp-(1“4)-a- -GlcpNH2-(1“6)-PI, to
which is added a fatty acyl group [e.g. hexade-
canoyl(palmitoyl)] at 2-OH of the -myo-inos-
itol residue to give the 2-O-acyl compound 1.
-Manp residues are trans-
D
-GlcpNAc
D-mannosylated by the GPI biosynthetic en-
D
zymes of T. brucei but not by those of human
(HeLa) cells, thereby providing the first direct
evidence that trypanosomal and mammalian
D
D
D
a-(1“4)-
ent substrate specificities and that inositol 2-
O-acylation of a- -GlcpNH2-(1“6)-PI (“2)
is a prerequisite for -mannosylation (“1) in
D-mannosyltransferases have differ-
D
D
D
D
D
D
the HeLa cell-free system. Furthermore, they
confirmed that inositol 2-O-acylation is re-
quired in trypanosomes for the efficient addi-
tion of ethanolamine phosphate [7], since
2-O-methylation prevented the addition of
ethanolamine phosphate to a later biosyn-
Two further a-
ferred in turn from dolichol phosphate
mannose to form a- -Manp-(1“2)-a-
D
D
-
-
D
D
Manp-(1“6)-a-
D
-Manp-(1“4)-a- -Glcp-
D
NH2-(1“6)-2-O-acyl-PI. Ethanolamine phos-
thetic intermediate, namely a-
a- -Manp-(1“6)-a- -Manp-(1“4)-a-
GlcpNH2-(1“6)-2-O-methyl-PI. It is also no-
-GlcpNAc-2-O-methyl-PI 4 is a
D
-Manp-(1“2)-
phate (from phosphatidyl ethanolamine) is
D
D
D
-
subsequently added at the terminal a- -Manp
D
residue and the fatty acyl group is then re-
moved from the -myo-inositol residue. The
table that a-
D
D
substrate for both trypanosomal and human
N-deacetylases, exhibiting turnover rates of
resulting structure undergoes a complex series
of fatty acid remodelling reactions before the
pre-assembled GPI precursor (known as gly-
colipid A) is transferred en bloc to newly
roughly 60% of that of a- -GlcpNAc-PI
[10,11].
D
synthesised protein. Some a- -galactosylation
D
of glycolipid A takes place in the endoplasmic
reticulum but mainly in the Golgi apparatus
during transport to the outer surface of the
cell membrane.
The GPI biosynthetic pathway in mam-
malian cells [8] appears to be broadly similar
to that outlined for the bloodstream form of
T. brucei. One notable difference is that acyla-
tion of 2-OH of the
D-myo-inositol residue
The finding [10] that the synthetic analogue
3 is recognised and -mannosylated by the
D
GPI biosynthetic pathway of T. brucei, but
not by that of human (HeLa) cells, suggests
that the discovery and development of in-
hibitors that are selective for parasite GPI
pathways are attainable goals. With this in
mind, we have prepared for biological testing
a number of analogues in which the inositol
2-O-methyl group of the compound 3 is re-

44
A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
placed by a medium or long alkyl chain, as in
the 2-O-octyl and 2-O-hexadecyl analogues 23
and 24, respectively. Following on from ear-
lier work [9], we also wished to test as a
with each of the alcohols 14 and 15 in the
presence of pivaloyl chloride [15,16] to give
the phosphonic diesters 17 and 18, respec-
tively, as mixtures of diastereoisomers
(Scheme 2). These were oxidised with iodine in
pyridine–water [16] to the phosphoric di-
esters, which were isolated and characterised
as the triethylammonium (TEA) salts 19 and
20. Finally, hydrogenolysis of the correspond-
ing sodium salts 21 and 22 over 20% Pd(OH)2/
substrate for the various
D
-mannosyltrans-
ferases of GPI anchor biosynthesis, the ether-
substituted analogue 31 in which octyl groups
are introduced at O-1 and O-2 of the glycerol
residue. The corresponding octadecyl com-
pound 32 [9] proved to be an excellent sub-
strate for the GPI -mannosyltransferases and
D
C gave a- -GlcpNH2-(1“6)-2-O-octyl-PI 23
D
it was of interest to discover whether shorter
alkyl chains would suffice. Our reasons for
wanting ether-linked substituents on the glyc-
erol residue have been recorded earlier [9].
and the 2-O-hexadecyl derivative 24, respec-
tively (Scheme 3).
In pursuing a similar approach to the 1,2-
di-O-octyl-sn-glycerol-PI analogue 31, the
pseudodisaccharide 25 [12] is required to react
with the hydrogenphosphonate 27, which is
readily prepared by a standard procedure [17]
from the enantiopure alcohol 26 [18]. The
reaction of compounds 25 and 27 in the pres-
ence of pivaloyl chloride [15,16] gave a mix-
ture of diastereoisomeric phosphonic esters
28, which, as before, was transformed first
into the TEA phosphate derivative 29 for
2. Results and discussion
Alkylation of 2-OH of the otherwise pro-
tected -myo-inositol 5 [12] with octyl or hexa-
D
decyl bromide in N,N-dimethylformamide in
the presence of sodium hydride produced the
2-O-octyl and 2-O-hexadecyl derivatives 6
and 7, respectively. These derivatives gave the
deallylated compounds 8 and 9 following
isomerisation of the O-allyl group with potas-
sium tert-butoxide in dimethyl sulfoxide and
mild acid hydrolysis of the resulting propenyl
derivatives (Scheme 1).
The 2-O-octyl derivative 8 gave a ꢀ2:1
mixture of the a- and b-linked pseudodisac-
charides 11 on coupling with the glycosyl
fluoride 10 [9,12] in dry diethyl ether in the
presence of zirconocene dichloride and silver
perchlorate [13]. Treatment of the coupled
products with trifluoroacetic acid (TFA) in
CH2Cl2 then afforded a mixture of the com-
pounds 13 (J1%,2% 7.9 Hz) and 14 (J1%,2% 3.6 Hz),
which was resolved by a combination of crys-
tallisation (of 13) and radial-band chromatog-
raphy. Similar coupling of the glycosyl
fluoride 10 [9,12] and the 2-O-hexadecyl
derivative 9 gave a ꢀ3:1 mixture of the a-
and b-linked pseudodisaccharides 12, which,
following hydrolysis with TFA and radial-
band chromatography, furnished the required
a-linked compound 15 (J1%,2% 3.6 Hz).
In following the serviceable hydrogenphos-
phonate route to phosphonic diesters [9,12],
the hydrogenphosphonate 16 [9,14] reacted
Scheme 1.

A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
45
Scheme 2.
characterisation purposes and thereafter into
the sodium salt 30. Hydrogenolysis of the
sodium salt 30 over 20% Pd(OH)2/C furnished
the 1,2-di-O-octyl-sn-glycerol-PI 31 (Scheme
4).
Scheme 4.
Details of the results of enzymic studies
with the above analogues are reported else-
where [11,19]. In brief, neither trypanosomal
nor HeLa a-(1“4)-
can act on a- -GlcpNH2-2-O-hexadecyl-PI
24, which turns out to be an inhibitor of
trypanosomal a-(1“4)- -mannosyltrans-
ferase but not of the corresponding HeLa
-GlcpNH2-2-O-octyl-PI 23 is nei-
D-mannosyltransferases
D
D
enzyme. a-
D
ther a substrate for, nor an inhibitor of, any
of the early GPI biosynthetic enzymes in the
HeLa cell-free system but is a potent inhibitor
of inositol 2-O-acylation of
D
-mannosylated
species in the T. brucei cell-free system. The
importance of inositol 2-O-acylation in the
trypanosomal GPI biosynthetic pathway [7],
coupled with the selective inhibition of this
activity with the 2-O-octyl analogue 23, sug-
gest trypanosomal inositol acyltransferase as a
potential target for the development of try-
panosome-specific therapeutic agents.
Scheme 3.

46
A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
The 1,2-di-O-octyl-sn-glycerol-PI analogue
0.80 (t, 3 H, J 7.1, CH2Me), ꢀ1.20 (10 H,
[CH2]5), 1.47 (m, 2 H, OCH2CH2), 3.13 (dd, 1
H, J2,3 2.3, J3,4 9.8, H-3), 3.20 (dd, 1 H, J1,2
2.3, J1,6 9.8, H-1), 3.25 (t, 1 H, J4,5=J5,6 9.8,
H-5), 3.64 (t, 2 H, J 6.5, OCH2), 3.70 (4 H,
ArOCH3 and H-2), 3.75 (t, 1 H, H-4), 3.88 (t,
1 H, H-6), 4.20–4.35 (m, 2 H, CH2CHꢀCH2),
4.45–4.85 (8 H, 4×CH2Ar), 5.00–5.20 (m, 2
31 resembled the corresponding octadecyl
analogue 32 [9] in being an excellent substrate
for both mammalian and trypanosomal
mannosyltransferases.
D-
3. Experimental
H, CH2CHꢀCH2), 5.85–5.94 (m,
1
H,
CH2CHꢀCH2), 6.75–7.40 (19 H, C6H4 and
3×Ph); Anal. Calcd for C46H58O7: C, 76.4; H,
8.1. Found: C, 76.1; H, 8.2.
Melting points were determined on a Re-
ichert hot-plate and are uncorrected. Optical
rotations were measured with a Perkin–Elmer
1 - - 6 - O - Allyl - 3,4,5 - tri - O - benzyl - 2 - O-
D
1
141 polarimeter at ambient temperature. H
hexadecyl-1-O-(4-methoxybenzyl)-myo-inositol
(7).—To a stirred solution of the alcohol 5
[12] (1.0 g, 1.6 mmol) in DMF (30 mL) at 0 °C
were added sodium hydride (0.16 g, 6.7 mmol)
and hexadecyl bromide (734 mL, 2.4 mmol).
The reaction mixture was stirred for 2 h at
room temperature (rt) before MeOH was
added to quench any excess of sodium hy-
dride. The resulting solution was extracted
with diethyl ether (3×25 mL) and the com-
bined extracts were washed successively with
water (25 mL) and brine (25 mL), dried
(Na2SO4), and concentrated under reduced
pressure. RBC (1:2 diethyl ether–cyclohexane)
of the residue gave the hexadecyl derivative 7
(1.32 g, 96.5%), mp 42–43 °C (from hexane);
[h]D −7° (c 1.0, CHCl3); lH 0.80 (t, 3 H, J
7.0, CH2Me), ꢀ1.20 (26 H, [CH2]13), 1.48 (m,
2 H, OCH2CH2), 3.13 (dd, 1 H, J2,3 2.2, J3,4
9.8, H-3), 3.18 (dd, 1 H, J1,2 2.2, J1,6 9.8, H-1),
3.28 (t, 1 H, J4,5 =J5,6 9.8, H-5), 3.62 (m, 2 H,
OCH2), 3.69 (4 H, ArOCH3 and H-2), 3.75 (t,
1 H, H-4), 3.86 (t, 1 H, H-6), 4.15–4.35 (m, 2
H, CH2CHꢀCH2), 4.45–4.85 (8 H, 4×
CH2Ar), 5.00–5.25 (m, 2 H, CH2CHꢀCH2),
5.85–5.94 (m, 1 H, CH2CHꢀCH2), 6.75–7.30
(19 H, C6H4 and 3×Ph). Anal. Calcd for
C54H74O7: C, 77.7; H, 8.9. Found: C, 77.4; H,
8.9.
NMR and COSY spectra were recorded on a
Bruker AM 200 MHz or AC 500 MHz spec-
trometer using deuteriochloroform as the sol-
vent and tetramethylsilane as the internal
reference and 31P spectra were performed at 81
MHz using aq 85% H3PO4 as the external
reference, unless otherwise indicated. Chemi-
cal shifts (l) and J values are given in ppm
and Hz, respectively. ES mass spectra were
recorded with a VG Quattro instrument. TLC
was performed on Silica Gel 60F254 (E.
Merck) with A, a 1:1 diethyl ether–hexane; B,
9:1 CHCl3–MeOH; C, 19:1 CHCl3–MeOH;
D, 10:10:3 CHCl3–MeOH–water; and E,
2:1:1 butanol–EtOH–water as developers and
detection by UV light or by charring with
3:17:1 H2SO4–water–EtOH as appropriate.
Radial-band chromatography (RBC) was per-
formed using a Chromatotron (model 7924T,
TC Research, UK) with Adsorbosil Plus-P
(6–15 mm) (Alltech) as the adsorbent.
1- -6-O-Allyl-3,4,5-tri-O-benzyl-1-O-(4-
D
methoxybenzyl)-2-O-octyl-myo-inositol (6).—
To a stirred solution of the alcohol 5 [12] (514
mg, 0.84 mmol) in DMF (15 mL) at 0 °C were
added sodium hydride (78 mg, 3.25 mmol)
and octyl bromide (212 mL, 1.23 mmol). The
cooling bath was removed and the solution
was stirred for 24 h, whereafter MeOH was
added to decompose any excess of sodium
hydride. The resulting solution was extracted
with diethyl ether (25 mL) and the organic
extract was washed successively with water
and brine, dried (Na2SO4), and concentrated
under reduced pressure to an oil which soli-
dified on standing. Recrystallisation from hex-
ane gave the octyl derivative 6 (0.6 g, 98%),
mp 32–33 °C; [h]D −7° (c 1.2, CHCl3); lH
1- -3,4,5-Tri-O-benzyl-1-O-(4-methoxyben-
D
zyl)-2-O-octyl-myo-inositol (8).—A solution
of the allyl derivative 6 (491 mg, 0.68 mmol)
in anhyd Me2SO (15 mL) containing potas-
sium tert-butoxide (662 mg, 5.9 mmol) was
heated and stirred at 60 °C for 1 h, cooled and
then poured into ice-water (75 mL). The re-
sulting aqueous solution was extracted with
EtOAc (3×25 mL) and the combined organic

A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
47
C6H4 and 3×Ph). Anal. Calcd for C51H70O7:
extracts were washed successively with water
and brine, dried (Na2SO4), and concentrated
under reduced pressure. The resulting
propenyl derivative in 1 M hydrochloric acid–
acetone (20 mL, 1:9) was boiled under reflux
for 10 min, whereafter the solvents were re-
moved under reduced pressure. A solution of
the residue in diethyl ether was percolated
through a short column of silica gel and the
eluent was concentrated under reduced pres-
sure to give the deallylated compound 8 (0.37
g, 80%), mp 62–63 °C (from hexane), [h]D
−14° (c 1.1, CHCl3); lH 0.85 (t, 3 H, J 7.1,
CH2Me), ꢀ1.25 (10 H, [CH2]5), 1.55 (m, 2 H,
OCH2CH2), 2.45 (s, 1 H, OH), 3.10 (d, 1 H,
J2,3 2.2, J3,4 9.8, H-3), 3.31 (dd, 1 H, J1,2 2.2,
J1,6 9.8, H-1), 3.34 (t, 1 H, J4,5=J5,6 9.8, H-5),
3.70 (m, 2 H, OCH2), 3.80 (s, 3 H, ArOCH3),
3.84 (t, 1 H, H-2), 3.95 (t, 1 H, H-6), 4.05 (t,
1 H, H-4), 4.45–4.90 (8 H, 4×CH2Ar), 6.80–
7.40 (19 H, C6H4 and 3×Ph). Anal. Calcd for
C43H54O7: C, 75.6; H, 8.0. Found: C, 75.6; H,
8.3.
C, 77.0; H, 8.9. Found: C, 76.9; H, 9.0.
1-
D
-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-
- glucopyranosyl) - 3,4,5 - tri - O-
deoxy - h,i -
D
benzyl-1-O-(4-methoxybenzyl)-2-O-octyl-myo-
inositol (11).—The glycosyl fluoride 10 [12]
(140 mg, 0.29 mmol) and the acceptor 8 (143
mg, 0.21 mmol) were dried overnight under
vacuum in a desiccator containing P2O5. They
were then dissolved in anhyd diethyl ether (10
mL) and zirconocene dichloride (338 mg, 1.16
,
mmol) and 4 A molecular sieves (1.0 g) were
added to the solution. After 15 min at rt, the
mixture was cooled to 0 °C and pre-dried
silver perchlorate (236 mg, 1.05 mmol) sus-
pended in anhyd diethyl ether (10 mL) was
added dropwise, followed by 1,1,3,3-te-
tramethylurea (30 mL, 251 mmol). The mixture
was stirred vigorously at 0 °C under argon
overnight. It was then percolated through a
short column of silica gel (elution with diethyl
ether) and the eluent was concentrated under
reduced pressure. Purification of the residue
by RBC (1:6 diethyl ether–hexane) gave the
pseudodisaccharide 11 (119 mg, 51%) as a
mixture of the a, b anomers in the ratio of
1- -3,4,5-Tri-O-benzyl-2-O-hexadecyl-1-O-
D
(4-methoxybenzyl)-myo-inositol (9).—A solu-
tion of the allyl derivative 7 (800 mg, 0.96
mmol) in anhyd Me2SO (10 mL) containing
potassium tert-butoxide (931 mg, 8.3 mmol)
was heated and stirred at 60 °C for 1 h,
cooled, and poured into ice-water. The result-
ing solution was extracted with EtOAc (3×25
mL) and the combined organic extracts were
washed successively with water and brine,
dried (Na2SO4), and concentrated under re-
duced pressure. The resulting propenyl deriva-
tive was boiled under reflux in 1 M
hydrochloric acid–acetone (20 mL; 1:9) for 10
min, after which the solvents were removed
under reduced pressure. Purification of the
residue by RBC (elution with 1:3 diethyl
ether–cyclohexane) gave the deallylated com-
pound 9 (0.72 g, 94%), mp 44–46 °C (from
hexane); [h]D −11° (c 1.4, CHCl3); lH 0.80 (t,
3 H, J 7.1, CH2Me), ꢀ1.20 (26 H, [CH2]13),
1.50 (m, 2 H, OCH2CH2), 2.50 (s, 1 H, OH),
3.03 (dd, 1 H, J2,3 2.0, J3,4 9.8, H-3), 3.24 (dd,
1 H, J1,2 2.1, J1,6 9.8, H-1), 3.27 (t, 1 H,
J4,5 =J5,6 9.8, H-5), 3.60 and 3.65 (m, 2 H,
OCH2), 3.68 (s, 3 H, ArOCH3), 3.78 (t, 1 H,
H-2), 3.90 (t, 1 H, H-6), 4.00 (t, 1 H, H-4),
4.40–4.80 (8 H, 4×CH2Ar), 6.75–7.30 (19 H,
1
ꢀ2:1 (determined by H NMR spectroscopy);
[h]D +17.5° (c 1.2, CHCl3); lH 0.80 (t, 3 H, J
7.1, CH2Me), ꢀ1.20 (10 H, [CH2]5), 1.50 (m,
2 H, OCH2CH2), 3.09 (dd, 1 H, J5%,6%a 2.2,
J6%a,6%b 11.0, H-6%a), 3.14 (dd, 1 H, J1%,2% 3.7, J2%,3%
10.5, H-2% a anomer), 3.16 (dd, 1 H, J5%,6%b 2.0,
H-6%b), 3.27 (dd, 1 H, J1,2 2.2, J1,6 9.8, H-1),
3.31 (t, 1 H, J4,5 =J5,6 9.5, H-5), 3.34 (dd, 1 H,
J2,3 2.2, J3,4 9.5, H-3), 3.60 (3 H, OCH2 and
H-4%), 3.74 (s, 3 H, ArOCH3), 3.78 (t, 1 H,
H-2), 3.88 (t, 1 H, J2%,3%=J3%,4% 10.5, H-3%), 3.96
(2 H, H-5%, 6), 4.09 (t, 1 H, H-4), 4.32 (d, 1 H,
J1%,2% 8.5, H-1% b anomer), 4.40–4.95 (14 H,
7×CH2Ar), 5.65 (d, 1 H, 1%-H a anomer),
6.80–7.40 (34 H, C6H4 and 6×Ph); ESMS
(+): m/z 1157 [M+NH4]+.
1-
D
-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-
-glucopyranosyl)-3,4,5-tri-O-benzyl-
deoxy-h,i-
D
2-O-hexadecyl-1-O-(4-methoxybenzyl)-myo-
inositol (12).—After drying overnight over
P2O5 in a vacuum desiccator, the glycosyl
donor 10 [12] (280 mg, 0.59 mmol) and the
acceptor 9 (340 mg, 0.43 mmol) were dissolved
in anhyd diethyl ether (15 mL) containing
zirconocene dichloride (691 mg, 2.36 mmol)

48
A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
,
HO-1), 3.17 (dd, 1 H, J5%,6%b 2.3, H-6%b), 3.27 (t,
1 H, J4,5=J5,6 9.4, H-5), 3.34 (dd, 1 H, J2,3
2.3, J3,4 9.8, H-3), 3.40 (dd, 1 H, J1%,2% 3.6, J2%,3%
10, H-2%), 3.51 (m, 1 H, H-1), 3.56 and 3.86
(2×m, 2 H, OCH2), 3.65 (t, 1 H, J3%,4%=J4%,5%
9.3, H-4%), 3.75 (t, 1 H, J1,2 =J2,3 2.3, H-2),
3.80–3.88 (3 H, H-3%, 5%, 6), 3.93 (t, 1 H, H-4),
4.05–4.95 (12 H, 6×CH2Ph), 5.45 (d, 1 H,
H-1%), 6.90–7.50 (30 H, 6×Ph); ESMS(+):
m/z 1037 [M+NH4]+.
and 4 A molecular sieves (1 g). After stirring
of the mixture at rt for 15 min under argon, it
was cooled to 0 °C and pre-dried silver
perchlorate (484 mg, 2.15 mmol) and 1,1,3,3-
tetramethylurea (62 mL, 520 mmol) were
added. Stirring of the mixture under argon at
0 °C was continued overnight, whereafter it
was percolated through a short column of
silica gel (elution with diethyl ether) and the
eluent was concentrated under reduced pres-
sure. RBC (1:4 diethyl ether–cyclohexane) of
the residue gave a mixture of the a- and
b-linked compounds 12 (377 mg, 70%) in the
1-
oxy-h-
D
-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-de-
-glucopyranosyl)-3,4,5-tri-O-benzyl-2-
D
O-hexadecyl-myo-inositol (15).—To a stirred
solution of the methoxybenzyl compound 12
(280 mg, 0.22 mmol) in CH2Cl2 (10 mL) at rt
was added TFA (241 mL, 3.1 mmol). After 1
h, the solution was neutralised with Et3N and
washed successively with water and brine,
dried MgSO4, and evaporated under reduced
pressure. RBC (1:1 diethyl ether–hexane) of
the residue gave the a-coupled compound 15
(169 mg, 67%) as an oil; Rf 0.48 (solvent A);
[h]D +21° (c 1.2, CHCl3); lH 0.80 (t, 3 H, J
7.1, CH2Me), ꢀ1.20 (26 H, [CH2]13), 1.52 (m,
2 H, OCH2CH2), 3.02 (dd, 1 H, J5%,6%a 2.0,
J6%a,6%b 11.0, H-6%a), 3.07 (d, 1 H, J1,OH 6.9,
HO-1), 3.17 (dd, 1 H, J5%,6%b 2.1, H-6%b), 3.27 (t,
1 H, J4,5=J5,6 9.5, H-5), 3.33 (dd, 1 H, J2,3
2.2, J3,4 9.5, H-3), 3.40 (dd, 1 H, J1%,2% 3.6, J2%,3%
10.3, H-2%), 3.50 (m, 1 H, H-1), 3.55 and 3.82
(2×m, 2 H, OCH2), 3.65 (t, 1 H, J3%,4%=J4%,5%
9.4, H-4%), 3.75 (t, 1 H, J1,2 =J2,3 2.2, H-2),
3.85 (3 H, H-3%, 5%, 6), 3.93 (t, 1 H, H-4),
4.05–5.00 (12 H, 6×CH2Ph), 5.43 (d, 1 H,
1%-H), 6.90–7.40 (30 H, 6×Ph); ESMS(−):
m/z 1130 [MꢁH]−.
1
ratio of ꢀ3:1 (determined by H NMR spec-
troscopy); [h]D +18.4° (c 1.3, CHCl3); lH 0.80
(t, 3 H, J 7.1, CH2Me), ꢀ1.20 (26 H,
[CH2]13), 1.47 (m, 2 H, OCH2CH2), 3.07 (dd, 1
H, J5%,6%a 2.1, J6%a,6%b 11.0, H-6%a), 3.10–3.15 (2
H, H-2%, 6%b), 3.25 (dd, 1 H, J1,2 2.2, J1,6 9.8,
H-1), 3.29–3.35 (2 H, H-3, 5), 3.55–3.61 (3 H,
OCH2, H-4%), 3.69 (s, 3 H, ArOCH3), 3.76 (t, 1
H, J2,3 2.2, H-2), 3.87 (t, 1 H, J2%,3%=J3%,4% 10.2,
H-3%), 3.94 (2 H, H-5%, 6), 4.07 (t, 1 H, J3,4=
J4,5 9.5, H-4), 4.32 (d, 1 H, J1%,2% 8.2, H-1% b
anomer), 4.30–5.00 (m, 14 H, 7×CH2Ar),
5.64 (d, 1 H, J1%,2% 3.7, H-1% a anomer), 6.70–
7.30 (34 H, C6H4 and 6×Ph); ESMS(+): m/z
1274 [M+Na]+.
1-
D
-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-de-
-glucopyranosyl)-3,4,5-tri-O-benzyl-2-
oxy-h-
D
O-octyl-myo-inositol (14).—A solution of the
methoxybenzyl derivative 11 (110 mg, 96.5
mmol) in CH2Cl2 (10 mL) containing TFA
(104 mL, 1.35 mmol) was set aside at rt for 1
h, whereafter it was neutralised with Et3N,
washed successively with water and brine,
dried (MgSO4), and concentrated under re-
duced pressure. Trituration of the residue with
hexane deposited the crystalline b anomer 13
(20 mg, 20%), mp 137–139 °C; [a]D –34° (c
0.3, CHCl3); lH 4.57 (d, 1 H, J1%,2% 7.9, H-1%);
ESMS(+): m/z 1037 [M+NH4]+. Anal.
Calcd for C62H73O10N3: C, 73.0; H, 7.2; N,
4.1. Found: C, 73.0; H, 7.6; N, 4.1. RBC (1:2
diethyl ether–hexane) of the mother liquor
furnished the a-linked compound 14 (45 mg,
46%) as an opaque oil; Rf 0.40 (solvent A);
[h]D +18° (c 1.1, CHCl3); lH 0.81 (t, 3 H, J
7.1, CH2Me), ꢀ1.25 (10 H, [CH2]5), 1.52 (m,
2 H, OCH2CH2), 3.02 (dd, 1 H, J5%,6%a 2.0,
J6%a,6%b 11, H-6%a), 3.08 (d, 1 H, J1,OH 6.9,
Triethylammonium 1-
tri-O-benzyl-2-deoxy-h-
5-tri-O-benzyl-2-O-octyl-myo-inositol 1-(1,2-
di-O-hexadecanoyl-sn-glycerol 3-phosphate)
D-6-O-(2-azido-3,4,6-
D-glucopyranosyl)-3,4,
(19).—Compound 14 (45 mg, 0.044 mmol)
and 1,2-di-O-hexadecanoyl-sn-glycerol 3-hy-
drogenphosphonate TEA salt 16 [9] (65 mg,
0.089 mmol) were each dried overnight over
P2O5 and dried further by evaporation of an-
hyd pyridine (3×2 mL) therefrom. To a solu-
tion of the mixture in dry pyridine (10 mL)
was added pivaloyl chloride (68 mL,
0.55 mmol), and the resulting solution was
stirred under argon for 1 h; TLC (9:1

A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
49
toluene–EtOAc) then indicated that a mixture
of diastereoisomeric hydrogenphosphonates 17
had formed. Oxidation of the hydrogenphos-
phonates was accomplished by the addition of
a freshly prepared solution of iodine (46 mg,
0.18 mmol) in 19:1 pyridine–water (10 mL).
After 45 min, CHCl3 (25 mL) was added and
the resulting solution was washed successively
with 5% aq NaHSO3 (20 mL) and 1 M aq
CHCl3–MeOH) of the residue furnished the
TEA phosphate salt 20 (150 mg, 91%); [h]D
+30° (c 1.3, CHCl3); Rf 0.48 (solvent C); lH
0.80 (t, 9 H, J 7.1, 3×CH2Me), ꢀ1.20 (87 H,
3×CH2Me, [CH2]13, 2×[CH2]13), 1.50 (m, 6
H, OCH2CH2, 2×COCH2CH2), 2.20 (m, 4 H,
2×COCH2), 2.95 (m, 6 H, 3×CH2Me), 3.10
(dd, 1 H, J1%,2% 3.6, J2%,3% 10.3, H-2%), 3.30 (dd, 1
H, J5%,6%a 2.2, J6%a,6%b 11, H-6%a), 3.32–3.42 (3 H,
H-3, 5, 6%b), 3.64 (t, 1 H, J3%,4%=J4%,5% 9.7, H-4%),
3.74 (t, 2 H, J 6.6, OCH2), 3.90–4.00 (2 H,
H-3%, 4), 4.04–4.25 (5 H, H-1, 5%, 6, CH2 -1 or
-3 glycerol), 4.36 (3 H, H-2, CH2 -1 or -3
glycerol), 4.45–4.90 (12 H, 6×CH2Ph), 5.20
(m, 1 H, H-2 glycerol), 5.72 (d, 1 H, H-1%),
6.90–7.30 (30 H, 6×Ph); lP −1.6 (with het-
eronuclear decoupling); ESMS(−): m/z
1761.3 [M–NEt3–H]−.
triethylammonium
hydrogen
carbonate
(TEAB) buffer (3×15 mL), dried (MgSO4),
and concentrated under reduced pressure.
RBC (elution first with CHCl3 and then with
19:1 CHCl3 –MeOH) of the residue gave the
TEA salt 19 (54 mg, 70%); [h]D +42° (c 1.3,
CHCl3); Rf 0.45 (solvent B); lH 0.81 (t, 9 H, J
6.9, 3×CH2Me), ꢀ1.23 (67 H, 3×CH2Me,
[CH2]5, 2×[CH2]12), 1.55 (6 H, OCH2CH2,
2×COCH2CH2), 2.22 (m, 4 H, 2×COCH2),
3.00 (m, 6 H, 3×CH2Me), 3.12 (dd, 1 H, J1%,2%
3.6, J2%,3% 10.2, H-2%), 3.25–3.45 (4 H, H-3, 5,
6%a,b), 3.64 (t, 1 H, J3%,4%=J4%,5% 9.7, H-4%), 3.75
(t, 2 H, J 6.5, OCH2), 3.95–4.00 (2 H, H-3%, 4),
4.05–4.25 (5 H, H-1, 5%, 6, CH2 -1 or -3
glycerol), 4.34 (3 H, H-2, CH2 -1 or -3 glyc-
erol), 4.45–4.90 (12 H, 6×CH2Ph), 5.20 (m, 1
H, 2-H glycerol), 5.70 (d, 1 H, 1%-H), 6.90–7.40
(30 H, 6×Ph); lP −0.98 (with heteronuclear
decoupling); ESMS(−): m/z 1649 [M–NEt3–
H]−.
Sodium 1-
copyranosyl)-2-O-octyl-myo-inositol 1-(1,2-di-
O-hexadecanoyl-sn-glycerol 3-phosphate)
D
-6-O-(2-amino-2-deoxy-h-
D-glu-
(23).—A solution of the sodium salt 21 (33
mg, 0.02 mmol; prepared from the correspond-
ing TEA salt 19 as described below) in 4:4:1
THF–propan-1-ol–water (9 mL) containing
20% Pd(OH)2/C was shaken under a slight
overpressure of hydrogen at rt for 24 h. It was
then percolated through a short column con-
taining Chelex 100 on a bed of Celite (elution
with 1:1 propan-1-ol–water) and the eluent
was concentrated under reduced pressure to
give the inositol derivative 23 (20 mg, 92%);
[h]D +24° (c 1, 10:10:3 CHCl3–MeOH–H2O);
Rf 0.68 (solvent D) and 0.61 (solvent E); lH
(10:10:3 CDCl3–CD3OD–D2O) 0.60 (m, 9 H,
3×CH2Me), ꢀ1.00 (58 H, [CH2]5, 2×
[CH2]12), 1.25 (6 H, OCH2CH2, 2×
COCH2CH2), 2.05 (m, 4 H, 2×COCH2), 2.85
(dd, 1 H, J1%,2% 3.4, J2%,3% 9.0, H-2%), 3.00 (m, 1 H,
H-5), 3.15 (2 H, H-3, 4%), 3.35 (t, 1 H, J3,4 =J4,5
10.0, H-4), 3.42 (m, 2 H, OCH2), 3.50 (4 H,
Triethylammonium 1-
tri-O-benzyl-2-deoxy-h-
5-tri-O-benzyl-2-O-hexadecyl-myo-inositol 1-
(1,2-di-O-hexadecanoyl-sn-glycerol 3-phos-
D
-6-O-(2-azido-3,4,6-
D
-glucopyranosyl)-3,4,
phate) (20).—After drying overnight over
P2O5, the TEA salt 16 [9] (129 mg, 0.176
mmol) and the a-compound 15 (100 mg, 0.088
mmol) were dissolved in anhyd pyridine (10
mL) under argon and pivaloyl chloride (135
mL, 1.1 mmol) was added to the solution. After
1 h at rt, TLC (9:1 toluene–EtOAc) indicated
the presence of two diastereoisomeric hydro-
genphosphonates 18, which were then oxidised
using a freshly prepared solution of iodine (89
mg, 0.35 mmol) in 19:1 pyridine–water (10
mL). CHCl3 (20 mL) was added after 45 min
and the organic solution was washed succes-
sively with 5% NaHSO3 (20 mL) and 1 M aq
TEAB buffer (3×15 mL), dried (MgSO4), and
concentrated under reduced pressure. RBC
(elution first with CHCl3 and then with 19:1
H-2, 3%, 6%a,b), 3.60 (t, 1 H, J1,6 =J5,6 9.5, H-6),
3.70 (m, 2 H, CH2 -1 or -3 glycerol), 3.79 (m,
1 H, H-5%), 3.85 (3 H, H-1, CH2 -1 or -3
glycerol), 4.90 (m, 1 H, H-2 glycerol), 5.20 (d,
1 H, H-1%); lP 4.70 (with heteronuclear decou-
pling); ESMS(−): m/z 1082.2 [M–Na]−.
Sodium 1-
glucopyranosyl)-2-O-hexadecyl-myo-inositol 1-
(1,2-di-O-hexadecanoyl-sn-glycerol 3-phos-
phate) (24).—A solution of the TEA salt 20
D
-6-O-(2-amino-2-deoxy-h- -
D

50
A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
purification; lP 5.33 (with heteronuclear de-
coupling), JPH 640; ESMS(-): m/z 379 [M–
NEt3–H]−.
(54 mg, 0.029 mmol) in 1:1 THF–MeOH (10
mL) was stirred in the presence of Amberlite
DP-1 (Na+) resin for 3 h, filtered, and concen-
trated under reduced pressure to afford quan-
titatively the sodium salt 22. A solution of the
sodium salt 22 (40 mg, 0.022 mmol) in 4:4:1
THF–propan-1-ol–water (9 mL) containing
20% Pd(OH)2/C (50 mg), was shaken under a
slight overpressure of hydrogen at rt, for 24 h.
It was then percolated through a short column
containing Chelex 100 on a bed of Celite
(elution with 1:1 propan-1-ol–water) and the
eluent was concentrated under reduced pres-
sure to give the inositol derivative 24 (20 mg,
74%); [h]D +6° (c 1, 10:10:3 CHCl3–MeOH–
H2O); Rf 0.76 (solvent D) and 0.58 (solvent
E); lH (10:10:3 CDCl3–CD3OD–D2O) 0.60 (t,
9 H, J 7.0, 3×CH2Me), ꢀ0.90 (78 H, 3×
[CH2]13), 1.30 (6 H, OCH2CH2, 2×
COCH2CH2), 2.03 (m, 4 H, 2×COCH2), 2.87
(dd, 1 H, J1%,2% 3.6, J2%,3% 10, H-2%), 3.00 (m, 1 H,
H-5), 3.15 (m, 1 H, H-4%), 3.34 (2 H, H-3, 4),
3.43 (3 H, OCH2, H-6%a), 3.53 (3 H, H-2, 3%,
6%b), 3.61 (t, 1 H, J1,6=J5,6 9.3, H-6), 3.69 (m,
2 H, CH2 -1 or -3 glycerol), 3.80 (m, 1 H,
H-5%), 3.90 (3 H, H-1, CH2 -1 or -3 glycerol),
4.95 (m, 1 H, H-2 glycerol), 5.23 (d, 1 H,
H-1%); lP 0.95 (with heteronuclear decoupling):
ESMS(−): m/z 1194.6 [M–Na]−.
Triethylammonium 1,2-di-O-octyl-sn-glyc-
erol 3-hydrogenphosphonate (27).—The alco-
hol 26 [18] (150 mg, 0.475 mmol) was dried by
evaporation of pyridine therefrom and was
afterwards dissolved in dry 10:1 THF–
pyridine (11 mL). This solution was added
dropwise over 30 min to a stirred solution of
salicyl chlorophosphite (134 mg, 0.66 mmol)
in dry THF (5 mL) under argon at rt. After 1
h, TLC (19:1 CHCl3–MeOH) indicated com-
plete conversion of the starting material into a
product of lower mobility (Rf 0.26). The reac-
tion mixture was quenched with 1 M TEAB
buffer solution (15 mL) and the resulting
aqueous solution was stirred for 30 min.
CHCl3 (25 mL) was then added and the or-
ganic layer was separated and washed with 1
Triethylammonium 1-
tri-O-benzyl-2-deoxy-h-
D-6-O-(2-azido-3,4,6-
D-glucopyranosyl)-2,3,
4,5-tetra-O-benzyl-myo-inositol 1-(1,2-di-O-
octyl-sn-glycerol 3-phosphate) (29).—The
compounds 25 [12] (127 mg, 0.13 mmol) and
27 (170 mg, 0.35 mmol) were dried over P2O5
overnight, whereafter anhydrous pyridine (5
mL) was evaporated from them. They were
then dissolved in dry pyridine (10 mL), pival-
oyl chloride (269 mL, 2.19 mmol) was added
and the resulting solution was stirred under
argon at rt for 1 h. A freshly prepared solu-
tion of iodine (177 mg, 0.7 mmol) in 19:1
pyridine–water (10 mL) was then added and
stirring of the reaction mixture was continued
for 45 min. After the addition of CHCl3 (25
mL), the organic solution was washed succes-
sively with 5% aq NaHSO3 (25 mL), water (25
mL), and 1 M aq TEAB buffer (3×15 mL),
dried (MgSO4), and concentrated under re-
duced pressure. RBC of the residue (elution
first with CHCl3 and then with 25:1 CHCl3–
MeOH) afforded the TEA phosphate deriva-
tive 29 (150 mg, 80%); [h]D +51° (c 1.1,
CHCl3); Rf 0.45 (solvent C); lH 0.86 (t, 6 H, J
7.0, 2×CH2Me), ꢀ1.24 (29 H, 3×CH2Me,
2×[CH2]5), 1.50 (m, 4 H, 2×OCH2CH2),
2.92 (m, 6 H, 3×CH2Me), 3.18 (dd, 1 H, J1%,2%
2.2 and J2%,3% 10.2, H-2%), 3.35 (m, 2 H,
OCH2CH2), 3.40 (m, 2 H, H-6%a,b), 3.46 (t, 1
H, J4,5 =J5,6 9.0, H-5), 3.52 (2 H, m,
OCH2CH2), 3.54 (m, 4 H, 2×CH2 glycerol),
3.62 (1 H, H-3), 3.71 (t, 1 H, J3%,4%=J4%,5% 9.3,
H-4%), 3.96–4.14 (4 H, H-3%, 4, 5%, H-2 glyc-
erol), 4.32 (1 H, H-6), 4.52 (1 H, H-1), 4.72 (1
H, H-2), 5.86 (d, 1 H, H-1%), 4.20–5.00 (14 H,
7×CH2Ph), 6.80–7.40 (35 H, 7×Ph); lP −
0.96
ESMS(−): m/z 1374.1 [M–NEt3–H]−.
Sodium 1- -6-O-(2-amino-2-deoxy-h-
glucopyranosyl)-myo-inositol 1-(1,2-di-O-
(with
heteronuclear
decoupling);
D
D-
octyl-sn-glycerol 3-phosphate) (31).—The
sodium salt 30 was obtained quantitatively by
stirring a solution of the TEA salt 29 (73 mg,
0.049 mmol) in 1:1 THF–MeOH with Amber-
lite DP-1 (Na+) resin for 3 h, followed by
filtration and concentration of the filtrate un-
der reduced pressure. A solution of the
sodium salt 30 (68.5 mg, 0.049 mmol) in 4:4:1
M
TEAB solution (3×10 mL), dried
(MgSO4), and concentrated under reduced
pressure. The crude hydrogenphosphonate
TEA salt 27 (215 mg, 94%) so obtained was
used in the next experiment without further

A. Crossman et al. / Carbohydrate Research 321 (1999) 42–51
51
[2] M.J. McConville, M.A.J. Ferguson, Biochem. J., 294
(1993) 305–324.
[3] (a) M.A.J. Ferguson, Curr. Opin. Struct. Biol., 1 (1991)
522–529. (b) S.J. Turco, A. Descoteaux, Annu. Re6.
Microbiol., 46 (1992) 65–94.
[4] K.G. Milne, R.A. Field, W.J. Masterson, S. Cottaz, J.S.
Brimacombe, M.A.J. Ferguson, J. Biol. Chem., 269
(1994) 16403–16408.
[5] T.K. Smith, S. Cottaz, J.S. Brimacombe, M.A.J. Fergu-
son, J. Biol. Chem., 271 (1996) 6476–6482.
[6] P.T. Englund, Annu. Re6. Biochem., 62 (1993) 121–
138.
THF–propan-1-ol–water (9 mL) containing
20% Pd(OH)2/C was shaken under a slight
overpressure of hydrogen at rt for 24 h. It was
then percolated through a short column con-
taining Chelex 100 on a bed of Celite (elution
with 10:10:3 CHCl3–MeOH–water) and the
eluent was concentrated under reduced pres-
sure to give the di-O-octyl derivative 31 (35
mg, 96%); [h]D +25° (c 1.1, 10:10:3 CHCl3–
MeOH–H2O); Rf 0.72 (solvent D) and 0.60
(solvent E); lH (10:10:3 CDCl3–CD3OD–
D2O) 0.55 (t, 6 H, J 7.0, 2×CH2Me), ꢀ0.94
(20 H, 2×[CH2]5), 1.24 (m, 4 H, 2×
OCH2CH2), 2.85 (dd, 1 H, J1%,2% 3.9, J2%,3% 10.6,
H-2%), 3.02 (dd, 1 H, J2,3 2.0, J3,4 9.8, H-3),
3.12 (t, 1 H, J3%,4%=J4%,5% 9.7, H-4%), 3.16 and
3.26 (m, 4 H, 2×OCH2), 3.32 (5 H, H-4,
2×CH2 glycerol), 3.40 (dd, 1 H, J5%,6%a 4.9,
J6%a,6%b 12, H-6%a), 3.50 (dd, 1 H, J5%,6%b 2.1,
H-6%b), 3.54 (dd, 1 H, J3%,4% 9.7, H-3%), 3.60 (2
H, H-2, H-2 glycerol), 3.77 (2 H, H-5%, 6), 3.84
(dd, 1 H, J1,2 2.0, J1,6 9.5, H-1), 5.21 (d, 1 H,
H-1%); lP 1.02 (with heteronuclear decoupling);
ESMS(−): m/z 718.4 [M–Na]−.
[7] M.L.S. Gu¨ther, M.A.J. Ferguson, EMBO J., 14 (1995)
3080–3093.
[8] (a) S. Hirose, G.M. Prince, D. Sevlever, L. Ravi, T.L.
Rosenberry, E. Ueda, M.E. Medof, J. Biol. Chem., 267
(1992) 16968–16974; (b) A. Puoti, A. Conzelmann, J.
Biol. Chem., 268 (1993) 7215–7224; (c); W.T. Doerrler, J.
Ye, J.R. Falck, M.A. Lehrman, J. Biol. Chem., 271
(1996) 27031–27038.
[9] A. Crossman, Jr., J.S. Brimacombe, M.A.J. Ferguson, J.
Chem. Soc., Perkin Trans. 1, (1997) 2769–2774.
[10] T.K. Smith, D.K. Sharma, A. Crossman, A. Dix, J.S.
Brimacombe, M.A.J. Ferguson, EMBO J., 16 (1997)
6667–6675.
[11] D.K. Sharma, T.K. Smith, C.T. Weller, A. Crossman,
J.S. Brimacombe, M.A.J. Ferguson, Glycobiology, 9
(1999) 415–422.
[12] S. Cottaz, J.S. Brimacombe, M.A.J. Ferguson, J. Chem.
Soc., Perkin Trans. 1, (1993) 2945–2951.
[13] T. Matsumoto, H. Maeta, K. Suzuki, G. Tsuchihashi,
Tetrahedron Lett., 29 (1988) 3567–3570.
[14] I. Lindh, J. Stawinski, J. Org. Chem., 54 (1989) 1338–
1342.
Acknowledgements
[15] T.M. Slaghek, A.A.M. Maas, J.P. Kamerling, J.F.G.
Vliegenthart, Carbohydr. Res., 211 (1991) 25–39.
[16] A.V. Nikolaev, I.A. Ivanova, V.N. Shibaev, N.K. Ko-
chetkov, Carbohydr. Res., 204 (1990) 65–78, and Refs
cited therein.
[17] J.E. Marugg, M. Tromp, E. Kuyl-Yeheskiely, G.A. van
der Marel, J.H. van Boom, Tetrahedron Lett., 27 (1986)
2661–2664.
This work was supported by a programme
grant from the Wellcome Trust. One of the
authors (A.C.) is also indebted to the MRC
for financial support.
References
[18] B.R. Ganong, C.R. Loomis, Y.A. Hannun, R.M. Bell,
Proc. Natl. Acad. Sci. USA, 83 (1986) 1184–1188.
[19] T.K. Smith, D.K. Sharma, A. Crossman, J.S. Brima-
combe, M.A.J. Ferguson, EMBO J., submitted for publi-
cation.
[1] Part 9. I.A. Ivanova, A.J. Ross, M.A.J. Ferguson, A.V.
Nikolaev, J. Chem. Soc., Perkin Trans. 1, (1999) 1743–
1753.
.