The synthesis of some deoxygenated analogues of early intermediates in the biosynthesis of glycosylphosphatidylinositol (GPI) membrane anchors

Article abstract of DOI:10.1016/j.carres.2004.02.026

Syntheses are described of 2-azido-4,6-di-O-benzyl-2,3-dideoxy-D-ribo- hexopyranosyl fluoride, 6-O-acetyl-2-azido-3-O-benzyl-2,4-dideoxy-D-xylo- hexopyranosyl fluoride and 2-azido-3,4-di-O-benzyl-2,6-dideoxy-D-glucopyranosyl fluoride. These glycosyl donor

Full text of DOI:10.1016/j.carres.2004.02.026

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1263–1277
The synthesis of some deoxygenated analogues of early
intermediates in the biosynthesis of glycosylphosphatidylinositol
(GPI) membrane anchorsq
Alexander P. Dix,a Charles N. Borissow,a Michael A. J. Fergusonb and
John S. Brimacombea,*
aSchool of Life Sciences (Chemistry), Carnelley Building, University of Dundee, Dundee DD1 4HN, UK
bSchool of Life Sciences (Biochemistry), Wellcome Trust Building, University of Dundee, Dundee DD1 5EH, UK
Received 20 October 2003; received in revised form 13 February 2004; accepted 26 February 2004
Available online 24 March 2004
Abstract—Syntheses are described of 2-azido-4,6-di-O-benzyl-2,3-dideoxy-
benzyl-2,4-dideoxy- -xylo-hexopyranosyl fluoride and 2-azido-3,4-di-O-benzyl-2,6-dideoxy-
cosyl donors were coupled with the acceptor 1 -2,3,4,5-tetra-O-benzyl-1-O-(4-methoxybenzyl)-myo-inositol and the a-coupled
products were transformed into a- -3dGlcpN-PI, a- -4dGlcpN-PI and a- -6dGlcpN-PI by way of the H-phosphonate route. Brief
mention is made of the biological evaluation of these deoxy-sugar analogues and their N-acetylated forms as candidate substrate/
D
-ribo-hexopyranosyl fluoride, 6-O-acetyl-2-azido-3-O-
D
D
-glucopyranosyl fluoride. These gly-
D
D
D
D
inhibitors of the N-deacetylase and a-(1fi4)-
system.
D-mannosyltransferase activities present in trypanosomal and HeLa (human) cell-free
Ó 2004 Published by Elsevier Ltd.
Keywords: Glycosylphosphatidylinositol (GPI) membrane anchors; Deoxy-sugar analogues of 1-
D
-6-O-(2-amino-2-deoxy-a-D-glucopyranosyl)-myo-
inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate); GPI biosynthesis in Trypanosoma brucei and HeLa (human) cell-free systems
1. Introduction
parasite Trypanosoma brucei.2 This work is predicated
on the belief that disruption of GPI-anchor biosynthesis
would seriously impair the parasite’s ability to survive in
a mammalian (human) host. It is equally important
to test these analogues against the related enzymes
present in a HeLa (human) system, whose GPI-anchor
biosynthesis must survive the assault on that of the
parasite.
Our efforts over the past decade or so to produce ana-
logues of 1-
D
-6-O-(2-acetamido-2-deoxy-a-D-glucopyr-
anosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol
3-phosphate) (1) and the N-deacetylated form 2 have
been summarised previously.1 The various analogues
described therein were used to probe the substrate
specificities or inhibition of two enzymes, a N-deacetyl-
The N-deacetylase activities of interest transform a-D-
ase and an a-(1fi4)-
D
-mannosyltransferase (MT-1), that
GlcpNAc-PI (1) into a-D-GlcpN-PI (2) in both T. brucei
exert their activities on substrates identical in all essen-
tial features to the synthetic glycosylphosphatidylinosi-
tols 1 and 2, respectively, in GPI membrane-anchor
biosynthesis in the bloodstream form of the protozoan
and HeLa cell-free systems.3 The N-deacetylated com-
pound 2 is then acted upon by MT-1, which in the
trypanosome transfers an a-
chol phosphate -mannose to form a-
a- -GlcpN-PI (3). This step is followed by the addition
of a fatty acyl group (most often hexadecanoyl) to 2-OH
of the -myo-inositol residue. This sequence is reversed
in the mammalian system wherein inositol 2-acylation
D
-Manp residue from doli-
D
D
-Manp-(1fi4)-
D
qParasite glycoconjugates, Part 14. For Part 13, see Ref. 1.
* Corresponding author. Tel.: +44-1382-779-214; fax: +44-1382-345-
517; e-mail: j.s.brimacombe@dundee.ac.uk
D
0008-6215/$ - see front matter Ó 2004 Published by Elsevier Ltd.
doi:10.1016/j.carres.2004.02.026

1264
A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
OH
1
in situ oxidation provides the related phosphoric diester,
which is transformed into the fully deprotected analogue
(e.g., 28) on hydrogenolysis. In keeping with previous
experience,1;6–9 we opted for the glycosyl fluorides 16, 37
and 56 as the glycosyl donors (see Figs. 1, 2, 4 and 7),
thereby ensuring that the requisite deoxy function was
already in place in the coupled product. The nonpar-
ticipating 2-azido group favours the formation of an
a-glycosidic linkage in the coupling reaction and is
reduced to a 2-amino group during the final deprotec-
tion step.
HO
OH
6
OR3
O
O
HO
1'
O
P
NHR1
O
O
OCOC15H31
OCOC15H31
R2O
O
OH
R1
R2
R3
1
2
3
4
5
Ac
H
H
H
H
H
Two essentially traditional approaches to the synthe-
sis of 2-azido-4,6-di-O-benzyl-2,3-dideoxy-
pyranosyl fluoride (16) are illustrated in Figures 1 and 2.
In the first approach, ring opening of the b- -manno-
D-ribo-hexo-
H α-
D
-Manp H
H
H
H
H
CH2C15H31
COC15H31
D
epoxide 9 with LiAlH4 in boiling Et2O furnished the
3-deoxy compound 10,12 which was straightforwardly
transformed into the axial tosylate 11 and thereafter into
the 2-azide 12, with inversion of configuration at the
reaction centre. Removal of the 4,6-O-benzylidene
group with acid (fi13) and a conventional benzylation
(fi14) was followed by glycoside hydrolysis (fi15) and
subsequent treatment with DAST (fi16).
OBn
O
P
BnO
HO
OBn
OBn
Et3NH
O
O
OCOC15H31
OCOC15H31
H
7
OMBn
6
MBn = p-methoxybenzyl
An alternative approach (Fig. 2) was based on
conjugate addition of hydrazoic acid to ethyl 2,3-dide-
-glycero-hex-2-enopyranoside (18), which is
-glucal
occurs before the first a-D
-Manp residue is attached.4
oxy-a-
D
Thus there is a fundamental difference between the try-
panosomal and mammalian biosynthetic pathways with
respect to the timing of inositol 2-acylation that might
be exploited in the development of parasite-specific
therapeutic agents. A synthetic analogue displaying
available in three steps from 3,4,6-tri-O-acetyl-
D
(17).13 This 1,4-addition yields the threo-adduct 19 as the
kinetically favoured product, which slowly equilibrates
with the thermodynamically more-stable erythro-adduct
20.14 Reduction of a mixture of the azido-ketones 19 and
20 with NaBH4 in MeOH gave ethyl 2-azido-2,3-
parasite-specific inhibition is a-D-GlcpN-2-O-hexadecyl-
PI (4), which inhibits trypanosomal MT-1 in a cell-free
dideoxy-a-D
-ribo-hexopyranoside (22)14 as one of the
system but is not a substrate for HeLa MT-1 despite
D-
products. Radial-band chromatography (RBC), with
sacrificial cuts, permitted the separation of the pure
2-azido-2,3-dideoxy compound 22 in a lowly 18% yield
from the other dominant product, which on the evidence
being a close analogue of the proven substrate a-
GlcpN-2-O-hexadecanoyl-PI (5).5
To date we have described the synthesis of substrate
analogues of the developing GPI anchors 1 and 2 having
of previous work14 was assumed to be the a-
D-lyxo
structural modifications to the phospholipid, a-
cosamine and
herein focuses on further modifications to the a-
D
-glu-
isomer 21. Because no physical data were recorded for
the targeted 2-azido-2,3-dideoxy compound 22, a sam-
ple of this compound in MeOH was hydrogenated over
20% Pd(OH)2 on carbon and the resulting amine was
N-acetylated in situ to give ethyl 2-acetamido-2,3-dide-
D
-myo-inositol components.6–9 Attention
D
-glu-
cosamine residue of compounds 1 and 2, which has been
deoxygenated at each of positions 3, 4 and 6 with the
intent of establishing which of these OH groups are in-
volved in essential hydrogen bonding in the active site of
the respective enzymes. Preliminary communications on
this work have appeared.10
oxy-a-D-ribo-hexopyranoside, also known as ethyl
N-acetyl-lividosaminide, whose physical data matched
those reported.14 Conventional benzylation of the diol
22 (fi23), followed by successive acidolysis (fi15) and
DAST treatment provided the glycosyl fluoride 16.
There is little to choose between the two routes to the
glycosyl fluoride 16 since, besides the penultimate gly-
coside hydrolysis (14fi15, 23fi15, see Experimental for
details), each of them has one other low-yielding step
that reduces the overall efficiency of the synthesis; for
2. Results and discussion
The synthesis of the targeted deoxy analogues followed
a familiar approach.1;6–9 In this, a suitable glycosyl
donor is coupled with the protected
D-myo-inositol
acceptor 6,6 whereafter at the next or a later stage the
1-OH group of the a-coupled product is exposed for
attachment of the H-phosphonate 7.8;11 Thereafter
the approach outlined in Figure 1, the b-
epoxide 9 was obtained in only 22% yield, along with the
isomeric b- -allo-epoxide (31%), in the second of the
D-manno-
D

A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
1265
OH
O
Ph
2 steps12
O
O
O
OMe
a
d
HO
HO
O
OMe
OH
9
8
O
O
Ph
Ph
RO
O
c
O
O
O
OMe
OMe
N3
10 R = H
12
b
11 R = Ts
OR
OBn
f
OMe
O
F
O
O
BnO
R =
RO
BnO
N3
N3
BnO
N3
13 R = H
14 R = Bn
15 R = α-OH
16 R = F
16
e
g
Figure 1. Synthesis of the 3-deoxyglycosyl fluoride 16: (a) LiAlH4, Et2O; (b) TsCl, DMAP, py, CH2Cl2; (c) NaN3, DMF, 18-crown-6; (d) 70%
AcOH; (e) BnBr, NaH, DMF; (f) 1 M HCl, 80% AcOH; (g) DAST, ClCH2CH2Cl.
dichloride and silver perchlorate15 to give a 3:1 mixture
OAc
OH
O
0
0
of the a-linked pseudodisaccharide 24 (J1 2 3.0 Hz) and
the b-anomer in 35% yield. Removal of the 1-O-meth-
oxybenzyl group from the pseudodisaccharide 24 and
coupling of the resulting alcohol 25 with the H-phos-
phonate 78;11 provided a mixture of two diastereoiso-
meric phosphonic diesters 26 that was oxidised in situ to
the phosphoric diester 27 with iodine in wet pyridine.16
The final transformation (27fi28) was accomplished by
hydrogenolysis over 20% Pd(OH)2 on carbon.
O
O
3 steps13
a
AcO
AcO
OEt
17
18
OH
OH
HO
O
N3
N3
O
O
19
OEt
OEt
OEt
b
d,e
Our approach to the 2-azido-2,4-dideoxyglycosyl
fluoride 37 (Fig. 4) was somewhat speculative insofar as
it required the radical-mediated removal of a thiocar-
bonyl group17 in the presence of a reducible azide.18 The
selective reduction of alkyl iodides brought about by
Bu3Sn radicals in the presence of an azido group is not
unknown;19 most notably, a recent review20 cites the
conversion of the disaccharide 20-iodide 29 (X ¼ I) into
the 20-deoxy compound 29 (X ¼ H) with Bu3SnH in
boiling benzene in the presence of 2,20-azobisisobuty-
ronitrile (AIBN) as the radical initiator. Whilst the
introduction of a 4-iodo group along the lines of a recent
synthesis21 of the 4-fluoro compound 30 suggested itself,
we decided first to test whether the related alcohol 32
could be deoxygenated by way of Bu3SnH treatment of
the 4-O-(thiocarbonylimidazolyl) derivative 33. The
overriding attraction of this route is that it is readily
16
21 (removed by RBC)
OH
OR
O
O
O
RO
N3
N3
20
OEt
22 R = H
c
23 R = Bn
Figure 2. Alternative synthesis of the 3-deoxyglycosyl fluoride 16: (a)
NaN3, AcOH, H2O; (b) NaBH4, MeOH; (c) BnBr, NaH, DMF; (d)
1 M HCl, 80% AcOH; (e) DAST, ClCH2CH2Cl.
two steps proceeding from methyl b-D-glucopyranoside
(8).12 In mitigation, both approaches are rapidly devel-
oped from readily available starting materials and,
despite the deficiencies already noted, yielded ample
quantities of the glycosyl fluoride 16.
developed from
dures.22
D-glucal (31) by established proce-
The route depicted in Figure 3 for the synthesis of
a-D-3dGlcpN-PI (28) was, with minor adjustments, also
Gratifyingly, radical-mediated deoxygenation of the
imidazolide 33 with Bu3SnH and AIBNin boiling benz-
ene occurred without significant reduction of the
2-azido group to give the 2-azido-2,4-dideoxy com-
pound 34 in a satisfactory 55% yield. The presence of the
followed for those of the corresponding 40- and 60-deoxy
analogues (see later). Thus the 3-deoxyglycosyl fluoride
16 was coupled with the
4:1 1,4-dioxane–toluene in the presence of zirconocene
D
-myo-inositol acceptor 66 in

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A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
OBn
OBn
BnO
OBn
OBn
BnO
OBn
OBn
a
c
f
6 + 16
O
O
O
O
BnO
BnO
BnO
BnO
O
P
OR
O
O
OCOC15H31
OCOC15H31
N3
N3
24 R = MBn (J1',2' = 3 Hz)
25 R = H
R
b
26 R = H
d, e
27 R = O NHEt3
OH
HO
OH
OH
O
O
HO
O
P
O
O
OCOC15H31
NH2
HO
O NHEt3 OCOC15H31
28
Figure 3. Synthesis of a-D-3dGlcpN-PI (28): (a) Cp2ZrCl2, AgClO4, tetramethylurea, 1,4-dioxane–toluene; (b) TFA, CH2Cl2; (c) 7, Me3CCOCl, py;
(d) I2, py, H2O; (e) TEAB buffer; (f) 20% Pd(OH)2/C.
OSiMe2But
O
O
O
OBn
OBn
O
N3
O
Me
F
N3
30
OBn
X
AcO
29 (X = I
H)
O
O
OAc
OAc
OH
OBn
OBn
O
O
O
O
O
5 steps22
c
b
e
BnO
BnO
HO
HO
N3
OR
N3
F
N3
N3
32 R = H
RO
37
31
34
35 R = Ac
36 R = H
d
a
C
N
N
33 R =
S
Figure 4. Synthesis of the 4-deoxyglycosyl fluoride 37: (a) 1,10-thiocarbonyldiimidazole, toluene; (b) Bu3SnH, AIBN, C6H6; (c) TFA, Ac2O;
(d) Me2NH, MeCN; (e) DAST, ClCH2CH2Cl.
4-deoxy group was indicated by distinctive signals for
two spin-coupled protons at d 2.17 and 1.79 (Jgem
15.1 Hz), well upfield from those of the other protons,
while the retention of the 2-azido group was signalled by
an IR absorption at 2100 cmÀ1. Thereafter, acetolysis
(fi35), selective removal of the anomeric O-acetyl group
(fi36), and DAST treatment provided the 4-deoxygly-
cosyl fluoride 37.
Coupling of the glycosyl fluoride 37 with the
D-myo-
inositol acceptor 66 in the customary manner1;6–9 fur-
0
0
nished the a-linked pseudodisaccharide 38 (J1 ;2 3.6 Hz)
in 35.5% yield following RBC (Fig. 5). In this particular
case, it was necessary to replace the 60-O-acetyl group
with an O-benzyl group (38fi39fi40) before completing
the remaining steps leading to a-D-4dGlcpN-PI
(41fi42fi43).

A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
1267
OBn
OBn
BnO
OBn
OBn
BnO
OBn
OBn
e, f, g
a
d
6 + 37
O
O
O
O
RO
BnO
OMBn
OH
N3
N3
OBn
41
OBn
38 R = Ac (J1',2' = 3.6 Hz)
b
c
39 R = H
40 R = Bn
OBn
OH
BnO
OBn
OBn
HO
OH
h
O
O
OH
O
O
BnO
HO
O
P
O
P
O
O
OCOC15H31
N3
O
O
OCOC15H31
NH2
O NHEt3 OCOC15H31
O NHEt3 OCOC15H31
OBn
OH
42
43
Figure 5. Synthesis of a-D-4dGlcpN-PI (43): (a) Cp2ZrCl2, AgClO4, tetramethylurea, Et2O; (b) MeONa, MeOH; (c) BnBr, NaH, DMF; (d) TFA,
CH2Cl2; (e) 7, Me3CCOCl, py; (f) I2, py, H2O; (g) TEAB buffer; (h) 20% Pd(OH)2/C.
As a safeguard against failure of the key deoxygen-
ation sequence (32fi33fi34), we had previously taken
the 4-O-methyl derivative of the alcohol 32 through an
identical series of reactions to those depicted in Figures
4 and 5 to give a-D-GlcpN4Me-PI (44) (no experimental
details are provided). Although less satisfactory in some
respects, this analogue might provide much the same
anoside (45) (Fig. 6). The 2-azide 46 (26%) was the
minor product, the major product being the keto sugar
47 (64%).27 The latter compound most likely results
from an initial b-elimination to give an enol benzoate,
which either simply loses the 3-O-benzoyl group during
heating in situ (cf. Ref. 24b) or undergoes attack by
azide ion at the ester carbonyl group to furnish the keto
sugar 47 by way of the enolate anion. In light of this
rationale, we reasoned that an SN2 displacement on the
butane-3,4-diacetal (BDA)-protected triflate 50 (Fig. 7)
was more likely to succeed because of the torsional
strain imposed on formation of the unsaturated linkage
at the ring-junction in the b-elimination reaction. This
might be regarded as torsional suppression of the elim-
ination process. Although the reactions outlined in
information as a-D-4dGlcpN-PI (43) in the enzymic
evaluations.
The route devised to a-
an SN2 displacement with azide ion on an a-
D-6dGlcpN-PI (60) required
D
-
rhamnopyranoside 2-sulfonate in the preparation of the
glycosyl fluoride 56. Such displacements are notoriously
difficult, sometimes impossible, to effect with charged
nucleophiles because of unfavourable dipolar interac-
tions in the transition state.23 Even with the weakly basic
azide ion, b-elimination leading to a 2,3-unsaturated
sugar is often dominant.24 An SN2 displacement on a
Figure 7 refer to those carried out in the D-series, the
practicability of the key displacement reaction (50fi51)
was established28 with the enantiomeric BDA-protected
b-
D
-rhamnopyranoside 2-sulfonate provides a more
a-
L-rhamnopyranoside 2-triflate (details not included).
promising option,25 since the unfavourable dipolar
interactions are minimised,23 but entails the more
The route to the latter compound from methyl a-L-
rhamnopyranoside is somewhat shorter since the
6-deoxy group is already in place.
involved preparation of a b-D-rhamnopyranoside. Since
the completion of this work, azide displacements have
been achieved26 on a-linked pseudodisaccharide 20-
triflates, using a hypervalent silicon azide as the nucleo-
phile, in yields comparable to that recorded below but
not without an accompanying b-elimination.
The BDA-protected a-D-rhamnopyranoside 2-triflate
50 (Fig. 7), readily obtained from the known29 alcohol
49, reacted with sodium azide in DMF at 75 °C to
produce the crystalline 2-azide 51 in 57% yield (over the
two steps) and a minor product (21%) tentatively
assigned as the 2,3-unsaturated sugar 52, based on the
presence of a single olefinic proton (H-2) as a doublet
We were confronted with this situation in attempting
an azide displacement on methyl 3-O-benzoyl-4,6-O-
1
benzylidene-2-O-trifluoromethylsulfonyl-a-
D
-mannopyr-
(J1;2 2.7 Hz) at d 5.05 in its H NMR spectrum. The

1268
A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
OH
HO
OH
OH
O
O
HO
MeO
O
P
O
O
OCOC15H31
NH2
O NHEt3 OCOC15H31
OH
44
O
O
Ph
O
Ph
Ph
TfO
O
O
O
NaN3
O
O
O
BzO
BzO
DMF
70 oC
N3
O
OMe
OMe
OMe
45
47 (64%)
46 (26%)
Figure 6. The products of an azide displacement on the a-
D
-mannopyranoside 2-triflate 45.
OH
OMe
RO
HO
O
Me
O
3 steps29
HO
HO
O
b
Me
Me
O
a
48
OMe
c
OMe
Me
OMe
49 R = H
50 R = Tf
OMe
Me
Me
O
O
O
e
BnO
BnO
RO
RO
O
Me
Me
O
O
N3
N3
X
N3
55 X = OH
56 X = F
OMe
OMe
OMe
f
53 R = H
51
d
54 R = Bn
OMe
Me
O
O
Me
Me
OMe
OMe
52 (removed by RBC)
Figure 7. Synthesis of the 6-deoxyglycosyl fluoride 56: (a) Tf2O, py, CH2Cl2; (b) NaN3, DMF, 75 °C; (c) TFA, H2O; (d) BnBr, NaH, DMF; (e) 1 M
HCl, 80% AcOH; (f) DAST, ClCH2CH2Cl.
unsaturated sugar 52 proved to be extremely unstable
and decomposed on standing or in CD3COCD3 or
CDCl3 solution, thereby precluding a more detailed
characterisation. Whilst elemental analyses and spec-
troscopic data served to identify the azido sugar 51, an
The remaining steps yielding the 6-deoxyglycosyl
fluoride 56 were conducted without incident and entailed
removal of the BDA protecting group (fi53), benzyl-
ation of the liberated diol (fi54), glycoside hydrolysis
(fi55) and treatment of the resulting hemiacetal with
X-ray crystal structure of its
L
-enantiomer confirmed
DAST (fi56). Progression to a-
equally incident-free, the a-coupled product 57 (J1 ;2
3.7 Hz) initiating a standard series of transforma-
D-6dGlcpN-PI (60) was
0
0
that no untoward changes had occurred during the
displacement reaction.28

A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
1269
tions (57fi58fi59fi60) to complete the synthesis
(Fig. 8).
N-acetylated derivatives of the foregoing analogues
inhibited both MT-1 activities in cell-free systems, with
a- -4dGlcpN-PI (43) exhibiting a more pronounced
effect.
D
(denoted, for example, as a-D-6dGlcpNAc-PI) required
for biological studies with the N-deacetylase activities
were prepared by existing procedures.30
3. Experimental
The details of biological tests with the various deoxy-
genated analogues are published elsewhere.31 These
3.1. General methods
showed that only a-D-3dGlcpNAc-PI was not accepted
as a substrate by the N-deacetylase activities in both
1H NMR and COSY spectra were routinely recorded on
either a Bruker AM 300 MHz or AC 500 MHz spec-
trometer using deuteriochloroform as the solvent and
tetramethylsilane as the internal standard, unless
otherwise indicated. 31P NMR spectra used 85% phos-
phoric acid in D2O as the external standard. Optical
rotations were measured using a Perkin–Elmer 141 or
343 polarimeter. Electrospray mass spectra (ESMS)
were recorded with a VG Quattro system (VG Biotech,
UK) and FAB mass spectra (FABS) with a VG 70-250
SE mass spectrometer using an Ion-tech xenon gun. IR
spectra were recorded on a Nicolet 205 FT-IR spec-
trometer either as liquid films or Nujol mulls. Melting
points were determined on a Reichert hot-plate appa-
ratus and are uncorrected. TLC was performed on
Kieselgel 60 F254 (Merck) with detection under UV light
or by charring with 3:17:1 H2SO4–water–ethanol.
Radial-band chromatography (RBC) was performed
using a Chromatotron (model 7924T, TC Research,
UK) with Adsorbosil Plus-P (6–15 lm) (Alltech) as the
adsorbent. Flash-column chromatography was per-
formed on silica gel 60 (230–400 mesh, E. Merck). Light
trypanosomal (T. brucei) and HeLa (human) cell-free
D-6dGlcpNAc-PI
systems, unlike a-
D
-4dGlcpNAc-PI, a-
and a- -GlcpNAc4Me-PI. This indicates that neither
D
the 40- nor 60-OH group is required for substrate rec-
ognition by the N-deacetylases, whereas the 30-OH
group is essential.
Only a-D-6dGlcpN-PI (60) was accepted as a sub-
strate by both the trypanosomal and HeLa MT-1
activities, requiring prior inositol 2-acylation in situ in
the latter instance. Since the 60-OH group is not required
for recognition by either the N-deacetylase or MT-1
activities, it raises the possibility of introducing fluo-
rescent or other probes at this position for further
in-depth studies of GPI-anchor biosynthesis. As already
noted, a-D-3dGlcpN-PI (28) was not recognised by
either of the MT-1 activities, once again demonstrating
the essentialness of the 30-OH group. Obviously neither
a-
D
-4dGlcpN-PI (43) nor a-D-Glcp4Me-PI (44) can act
as a substrate for the MT-1 activities because of the
removal or blocking of the 40-OH group required for
D
-mannosylation. On the other hand, these analogues
OBn
OBn
BnO
OBn
OBn
BnO
OBn
OBn
c, d, e
a
f
6 + 56
Me
O
O
Me
O
O
O
P
OR
O
O
OCOC15H31
N3
BnO
BnO
N3
O NHEt3 OCOC15H31
OBn
OBn
b
59
57 R = MBn (J1',2' = 3.7 Hz)
58 R = H
OH
HO
OH
OH
Me
HO
O
O
O
P
O
O
OCOC15H31
NH2
O NHEt3 OCOC15H31
OH
60
Figure 8. Synthesis of a-D-6dGlcpN-PI (60): see legend to Figure 3 for reagents (a)–(f).

1270
A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
petroleum refers to the fraction having a boiling point
60–80 °C. All anhydrous solvents were purchased from
Aldrich Chemical Company Ltd.
(0.42 g, 17.5 mmol). The mixture was allowed to warm
to room temperature and stirring was continued for
30 min. Benzyl bromide (1.56 mL, 13.12 mmol) was then
added dropwise and stirring of the mixture was contin-
ued overnight, whereupon MeOH was added to destroy
the excess of NaH. The resulting mixture was parti-
tioned between EtOAc (25 mL) and water (25 mL), and
the aqueous phase was separated and extracted with
EtOAc (4 · 25 mL). The organic extracts were combined,
washed with brine (25 mL), dried (MgSO4) and con-
centrated under reduced pressure. An ethereal solution
of the residue was passed down a short silica-gel column
(further elution with Et2O) and the eluent was concen-
trated under reduced pressure. RBC (first with hexane
and then with 16:1 hexane–EtOAc) of the residue gave
3.2. Methyl 4,6-O-benzylidene-3-deoxy-2-O-p-toluene-
sulfonyl-b- -arabino-hexopyranoside (11)
D
A solution of the alcohol 1012 (2.45 g, 9.2 mmol) in
CH2Cl2 (100 mL) containing p-toluenesulfonyl chloride
(2.62 g, 13.74 mmol), DMAP (0.11 g, 0.90 mmol) and
pyridine (3.71 mL. 46.3 mmol) was heated under reflux
overnight, whereafter it was cooled and poured into ice–
water (200 mL), and the aqueous solution was extracted
with CH2Cl2 (4 · 100 mL). The organic extracts were
combined, washed with 1 M HCl (3 · 50 mL), satd
NaHCO3 solution (2 · 50 mL) and water (50 mL), dried
(MgSO4) and concentrated under reduced pressure
the 4,6-dibenzylated compound 14 (1.1 g, 65%); mp 43–
25
44 °C (without recrystallisation); ½aŠ þ4 (c 1.0, CHCl3);
D
to give the tosylate 11 (2.26 g, 58%); mp 198–199 °C
1H NMR: d ꢀ7.10 (10H, 2 · C6H5), 4.36, 4.25 (2 · ABq,
4H, JAB 12.0 Hz, 2 · PhCH2), 3.97 (d, 1H, J1;2 8.0 Hz,
H-1), 3.33 (s, 3H, OCH3), 3.60–2.95 (5H, H-2, 4, 5,
6a,b), 2.19 (m, 1H, H-3eq), 1.17 (q, 1H,
Jgem ꢁ J2;3ax ꢁ J3ax;4 11.0 Hz, H-3ax); Anal. Calcd for
C21H25N3O4: C, 65.8; H, 6.6; N, 11.0. Found: C, 65.6;
H, 6.5; N, 10.9.
25
(from hexane–EtOAc); ½aŠ À42 (c 1.0, CHCl3); 1H
D
NMR: d ꢀ7.50 (9H, C6H4, C6H5), 5.55 (s, 1H, PhCH),
4.78 (br s, 1H, H-2), 4.44 (s, 1H, H-1), 3.35 (s, 3H,
OCH3), 2.43 (sþm, 4H, CH3Ar, H-3eq), 1.89 (t, 1H,
Jgem ꢁ J3ax;4 12.0, J2;3ax < 1 Hz, H-3ax); Anal. Calcd for
C21H24O7S: C, 60.0; H, 5.75; S, 7.6. Found: C, 60.1; H,
5.7; S, 7.5.
3.5. 2-Azido-4,6-di-O-benzyl-2,3-dideoxy-a- -ribo-hexo-
D
3.3. Methyl 2-azido-4,6-O-benzylidene-2,3-dideoxy-b-
ribo-hexopyranoside (12)
D
-
pyranose (15)
A mixture of the methyl b-glycoside 14 (100 mg,
0.26 mmol), 80% AcOH (3.2 mL) and 1 M HCl (1 mL)
was heated at 95 °C for 20 h, whereafter the reaction
mixture was diluted with CH2Cl2 (50 mL), washed with
water (50 mL) and satd NaHCO3 solution (100 mL),
dried (MgSO4) and concentrated under reduced pres-
sure. RBC (5:1fi1:1 light petroleum–Et2O) of the resi-
A mixture of the tosylate 11 (0.12 g, 0.29 mmol), sodium
azide (90 mg, 1.38 mmol) and 18-crown-6 (20 mg,
0.076 mmol) in DMF (5 mL) was stirred at 100 °C
overnight and then the solvent was removed under
reduced pressure (oil pump). A solution of the residue in
CH2Cl2 (50 mL) was washed with water (10 mL), dried
(MgSO4) and concentrated under reduced pressure to
give the azide 12 (70 mg, 84%); mp 112–114 °C (from
due and recrystallisation from hexane–Et2O gave the
25
a-hemiacetal 15 (30 mg, 31%); mp 96–100 °C; ½aŠ þ102
D
25
hexane); ½aŠ À51 (c 1.0, CHCl3); m 2120 cmÀ1 (N3); H
(c 0.96, CHCl3); 1H NMR: d 7.54–7.12 (10H, 2 · C6H5),
5.25 (t, 1H, J1;OH ¼ J1;2 3.2 Hz, H-1), 4.63–4.34 (4H,
2 · CH2Ph), 4.04 (m, 1H, H-5), 3.76–3.41 (4H, OH, H-4,
6a,b), 3.20 (m, 1H, H-2), 2.33, 2.05 (2 · m, 2H, H-3eq,
ax); Anal. Calcd for C20H23N3O4: C, 65.0; H, 6.3; N,
11.4. Found: C, 64.9; H, 6.3; N, 11.2.
1
D
NMR: d ꢀ7.40 (5H, C6H5), 5.53 (s, 1H, PhCH), 4.33 (d,
1 H, J1;2 7.8 Hz, H-1), 3.60 (s, 3H, OCH3), 4.35, 3.86–
3.35 (5H, H-2, 4, 5, 6a,b), 2.37 (dt, 1H, J2;3eq ¼ J3eq;4
4.8 Hz, H-3eq), 1.65 (q, 1H, Jgem ꢁ J2;3ax ꢁ J3ax;4 12.0 Hz,
H-3ax); Anal. Calcd for C14H17N3O4: C, 57.7; H, 5.9; N,
14.4. Found: C, 57.75; H, 5.9; N, 14.2.
Hydrolysis of the ethyl a-glycoside 23 (see later) by
an identical procedure also provided the a-hemiacetal
15 in 29% yield.
3.4. Methyl 2-azido-4,6-di-O-benzyl-2,3-dideoxy-b-
D-
ribo-hexopyranoside (14)
3.6. 2-Azido-4,6-di-O-benzyl-2,3-dideoxy-D-ribo-hexo-
pyranosyl fluoride (16)
A solution of the azide 12 (1.28 g, 4.39 mmol) in 70%
AcOH (13 mL) was heated at 80 °C for 30 min and then
concentrated under reduced pressure with co-evapora-
tion with toluene. The diol 13, obtained in virtually
quantitative yield, was taken on to the next stage with-
out further purification.
To a cooled ()30 °C) and stirred solution of the hemi-
acetal 15 (0.104 g, 0.28 mmol) in anhyd 1,2-dichloro-
ethane (10 mL) under argon was added DAST (0.15 mL,
1.14 mmol). The reaction mixture was allowed to attain
room temperature and was then partitioned between
water (40 mL) and CHCl3 (20 mL). The aqueous phase
To a cooled (0 °C) and stirred solution of the diol 13
(0.89 g, 4.38 mmol) in DMF (25 mL) was added NaH

A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
1271
was separated and further extracted with CHCl3
3.8. Ethyl 2-azido-4,6-di-O-benzyl-2,3-dideoxy-a-
hexopyranoside (23)
D
-ribo-
(25 mL), and the organic extracts were combined, wa-
shed with brine (45 mL), dried (MgSO4) and concen-
trated under reduced pressure. An ethereal solution of
the residue was percolated through a short silica-gel
column (further elution with Et2O) and the eluent was
concentrated under reduced pressure. RBC (first with
hexane and then with 8:1 hexane–Et2O) gave the gly-
cosyl fluoride 16 (81 mg, 77%) as a 1:2 mixture of the a-
and b-anomers; 1HNMR (a-anomer): d 7.39–7.15 (10H,
2 · C6H5), 5.59 (dd, 1H, J1;2 2.8, J1;F 53.6 Hz, H-1), 4.64–
4.49 (4 H, 2 · CH2Ph), 4.43 (m, 1H, H-5), 3.77, 3.71
(2 · m, 2H, >H-6a,b), 3.62 (m, 1H, H-4), 3.20 (m, 1H,
H-2), 2.41, 1.97 (2 · m, 2H, H-3eq, ax); b-anomer: d 5.10
(dd, 1H, J1;2 7.0, J1;F 52.7 Hz, H-1); ESMS(+): m=z 394.2
[MþNa]þ.
To a stirred and cooled (0 °C) solution of the diol 22
(0.65 g, 2.99 mmol) in DMF (18 mL) was added NaH
(0.29 g, 12.08 mmol). Stirring of the mixture was con-
tinued at rt for 30 min before benzyl bromide (1.06 mL,
8.91 mmol) was added dropwise. After stirring of the
reaction mixture for a further 3 h, the excess of NaH was
destroyed by the addition of cold MeOH. Water (20 mL)
was then added and the aqueous solution was extracted
with EtOAc (4 · 25 mL). The organic extracts were
combined, washed with brine (20 mL), dried (MgSO4)
and concentrated under reduced pressure. An ethereal
solution of the residue was percolated through a short
silica-gel column (further elution with Et2O) and the
eluent was concentrated under reduced pressure. RBC
(first with hexane and then with 32:1 hexane–EtOAc) of
the residue gave the dibenzylated compound 23 (0.76 g,
3.7. Ethyl 2-azido-2,3-dideoxy-a- -ribo-hexopyranoside
D
(22)
25
64%); ½aŠ þ71 (c 1.0, CHCl3); m 2100 cmÀ1 (N3); H
1
D
To a stirred solution of the enone 1813 (1.3 g, 7.55 mmol)
in glacial AcOH (10.4 mL) was added a solution of
sodium azide (1.3 g, 20 mmol) in water (6.5 mL). Stir-
ring of the reaction mixture was continued for 5 h
before it was diluted with ice–water (10 mL) and
extracted with CHCl3 (4 · 15 mL). The organic extracts
were combined, washed with satd NaHCO3 solution
(20 mL), dried (MgSO4) and concentrated under
reduced pressure to give a ꢀ1:2 mixture of the azido
ketones 19 and 20 (1.3 g, 80%); m 2100 (N3), 1720 cmÀ1
NMR: d ꢀ7.30 (10H, 2 · C6H5), 4.88 (d, 1H, J1;2 3.2 Hz,
H-1), ꢀ4.57 (2 · ABq, 4H, JAB 11.3 Hz, 2 · CH2Ph), 3.12
(m, 1H, J1;2 ¼ J2;3eq 3.2 Hz, H-2), 2.35, 2.07 (2 · m, 2H,
H-3eq, ax), 1.23 (t, 3H, J 7.0 Hz, CH2CH3); Anal. Calcd
for C22H27N3O4: C, 66.5; H, 6.85; N, 10.6. Found: C,
66.5; H, 6.9; N, 10.5.
3.9. 1
ribo-hexopyranosyl)-2,3,4,5-tetra-O-benzyl-1-O-(4-meth-
D
-6-O-(2-Azido-4,6-di-O-benzyl-2,3-dideoxy-a-D-
oxybenzyl)-myo-inositol (24)
1
(C@O); H NMR: d 5.00 (d, 1H, J1;2 3.0 Hz, H-1) and
4.95 (s, 1H, H-1) for the azido ketones 20 and 19,
respectively.
To a stirred solution of the D
-myo-inositol derivative 66
(92 mg, 0.14 mmol) and the glycosyl fluoride 16 (70 mg,
0.19 mmol) in anhyd 1,4-dioxane (8 mL) under argon
To a stirred and cooled (0 °C) solution of the foregoing
mixture of compounds 19 and 20 (1.26 g, 5.85 mmol) in
MeOH (40 mL) was added NaBH4 (1.26 g, 33.31 mmol)
over a period of 30 min, whereafter stirring of the mixture
was continued for 2 h. It was then neutralised with
Amberlite IR-120 (Hþ) ion-exchange resin, filtered
and concentrated under reduced pressure. RBC (99:1fi
ꢀ
were added powdered 4 A molecular sieves (0.5 g) and a
mixture of zirconocene dichloride (0.225 g, 0.77 mmol)
and pre-dried silver perchlorate (0.145 g, 0.7 mmol)
in toluene (2 mL). 1,1,3,3-Tetramethylurea (23 lL,
0.19 mmol) was added after 5 min and stirring of the
reaction mixture was continued in the dark for 22 h. It
was then filtered and concentrated under reduced pres-
sure. An ethereal solution of the residue was passed
through a short silica-gel column (further elution with
Et2O) and concentrated under reduced pressure. RBC
(first with hexane and then with 1:2 hexane–Et2O) yiel-
ded a 3:1 mixture (50 mg, 35%) of the a-pseudodisac-
96:4 CHCl3–MeOH) of the residue, with sacrificial
25
D
cuts, furnished the ribo-diol 22 (0.23 g, 18%); ½aŠ þ126
(c 1.0, CHCl3); m 2100 cmÀ1 (N3); H NMR: d 4.80 (d,
1
1H, H-1), 3.56–3.50 (6H, CH2CH3, H-4, 5, 6a,b), 3.10
(dt, 1H, J1;2 ¼ J2;3eq 3.5 Hz, H-2), 2.90 (br s, 1H, OH),
2.16 (dt, 1H, J2;3eq ¼ J3eq;4 3.5 Hz, H-3eq), 2.03 (q, 1H,
Jgem ꢁ J2;3ax ꢁ J3ax;4 11.0 Hz, H-3ax), 1.26 (t, 3H, J 7.0 Hz,
CH2CH3); Anal. Calcd for C8H15N3O4: C, 44.2; H,
7.0; N, 19.3. Found: C, 44.0; H, 7.2; N, 19.1. Also
obtained was a small amount of the pure lyxo-isomer 21
(0.12 g, 9%).
1
charide 24 and the b-anomer; H NMR (a-anomer): d
0
0
7.35–6.69 (34H, C6H4, 6 · C6H5), 5.63 (d, 1H, J1 ;2
3.0 Hz, H-10), 4.96–4.36 (14H, 7 · CH2Ar), 4.23 (t, 1H,
J3;4 ¼ J4;5 9.5 Hz, H-4), 4.05 (t, 1H, J1;6 ¼ J5;6 9.5 Hz,
H-6), 3.95 (m, 1H, H-2), 3.88 (m, 1H, H-50), 3.71 (s, 3H,
OCH3), 3.57 (m, 1H, H-40), 3.37 (m, 1H, H-3), 3.32 (t,
1H, J4;5 ¼ J5;6 9.5 Hz, H-5), 3.30 (m, 1H, H-1), 3.20, 3.09
(2 · m, 2H, H-60a,b), 2.95 (m, 1H, H-20), 2.24, 1.98
(2 · m, 2H, H-30eq, ax); ESMS(+): m=z 1033.5
[MþNa]þ.
The compound 22 was characterised by its transfor-
mation14 into ethyl 2-acetamido-2,3-dideoxy-a-
D-ribo-
hexopyranoside; mp 184–187 °C (from EtOAc), lit.14 mp
25
25
185–187 °C; ½aŠ þ158 (c 1.0, MeOH), lit.14 ½aŠ þ149 (c
D
D
1.05, MeOH).

1272
A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
3.10. 1
ribo-hexopyranosyl)-2,3,4,5-tetra-O-benzyl-myo-inositol
(25)
D
-6-O-(2-Azido-4,6-di-O-benzyl-2,3-dideoxy-a-
D
-
H-4, 50, 1- or 3-CH2 glycerol), 3.65 (m, 1H, H-40), 3.58
(dd, 1H, J2;3 2.4, J3;4 9.8 Hz, H-3), 3.49 (t, 1H, J4;5 ¼ J5;6
9.3 Hz, H-5), ꢀ3.35 (m, 2H, H-60a,b), 2.97 (7H, H-20,
3 · CH2CH3), 2.33 (m, 1H, H-30eq), 2.25 (m, 4H,
2 · COCH2CH2), 2.09 (m, 1H, H-30ax), 1.56 (m, 4H,
2 · COCH2CH2), 1.25 (48H, 2 · [CH2]12), 1.22 (t, 9H,
J 7.4 Hz, 3 · CH2CH3), 0.88 (t, 6H, J 6.8 Hz, 2 ·
CH2CH3); 31P NMR: d À4.57 (with heteronuclear de-
coupling); ESMS(þ): m=z 1544.4 [MÀEt3NHþNa]þ.
A 3:1 mixture of the a-pseudodisaccharide 24 and the
b-anomer (50 mg, 0.05 mmol) in anhyd CH2Cl2 (10 mL)
containing TFA (54 lL, 0.70 mmol) was set aside at rt
for 1 h, whereafter it was neutralised with Et3Nand
diluted with CH2Cl2 (40 mL). The organic solution was
washed with water (50 mL) and brine (50 mL), dried
(MgSO4) and concentrated under reduced pressure. An
ethereal solution of the residue was percolated through a
short silica-gel column (further elution with Et2O) and
the eluent was concentrated under reduced pressure.
Flash-column chromatography (2 fi 25% EtOAc in
toluene) of the residue gave the pure a-pseudodisac-
3.12. Triethylammonium 1
D
-6-O-(2-amino-2,3-dideoxy-
a- -ribo-hexopyranosyl)-myo-inositol 1-(1,2-di-O-hexa-
D
decanoyl-sn-glycerol 3-phosphate) (28)
A solution of the protected compound 27 (70 mg,
0.043 mmol) in 2:2:1 THF–propanol–water (5 mL) con-
taining 20% Pd(OH)2 on carbon (328 mg) was stirred
under 3 atm of hydrogen for 6 h. It was then percolated
through a short column of Chelex 100 on a bed of Celite
(further elution with 2:2:1 THF–propanol–water) and
25
1
charide 25 (28.5 mg, 65%); ½aŠ þ54 (c 1.0, CHCl3); H
NMR: d 7.37–6.99 (30H, 6 ·DC6H5), 5.53 (d, 1H, J1 ;2
0
0
3.4 Hz, H-10), 5.00–4.05 (12H, 6 · CH2Ph), 4.01 (t, 1H,
J1;6 ¼ J5;6 9.3 Hz, H-6), 3.92 (t, 1H, J3;4 ¼ J4;5 9.3 Hz, H-
4), 3.90 (t, 1H, J1;2 ¼ J2;3 2.1 Hz, H-2), 3.84 (m, 1H, H-
50), 3.57 (2H, H-1, 40), 3.42 (dd, 1H, H-3), 3.33 (t, 1H, H-
the eluent was concentrated under reduced pressure to
25
D
give a-
D
-3dGlcpN-PI (28) (16 mg, 35%); ½aŠ þ12 (c 1.0,
5), 3.29 (2 · m, 2H, H-20, 60a), 3.04 (dd, 1H, J5 ;6 b 2.1,
10:10:3 CHCl3–MeOH–water); 1H NMR (10:10:3
0
0
gem 10.7 Hz, H-60b), 2.67 (d, 1H, J1;OH 8.0 Hz, OH), 2.24,
CDCl3–CD3OD–D2O): d 5.42 (d, 1H, J1 ;2 3.6 Hz, H-10),
5.29–5.25 (m, 1H, 2-H glycerol), 4.43 (dd, 2H, 1- or
3-CH2 glycerol), 4.23–4.18 (m, 2H, 1- or 3-CH2 glyc-
erol), 4.16 (dd, 1H, J1;2 2.6, J1;6 9.6 Hz, H-1), 4.09 (t, 1H,
J1;2 ¼ J2;3 2.6 Hz, H-2), 4.04–4.00 (m, 1H, H-50), 3.97 (t,
0
0
J
1.93 (2 · m, 2H, H-30eq, ax); ESMS(+): m=z 914.0
[MþNa]þ.
3.11. Triethylammonium 1
D
-6-O-(2-azido-4,6-di-O-benz-
0
0
yl-2,3-dideoxy-a- -ribo-hexopyranosyl)-2,3,4,5-tetra-O-
D
1H, J1;6 ¼ J5;6 9.6 Hz, H-6), 3.62 (dd, 1H, J5 ;6 a 2.2 Jgem
12.0 Hz, H-60a), 3.72 (m, 1H, H-60b), 3.69 (t, 1H,
J3;4 ¼ J4;5 9.9 Hz, H-4), 3.65–3.57 (m, 1H, H-40), 3.47
(dd, 1H, H-3), 3.41–3.37 (m, 1H, H-20), 3.36 (dd, 1H,
H-5), 2.32 (t, 4H, J 7.2 Hz, 2 · COCH2CH2), 2.21–2.14,
ꢀ1.90 (2 · m, 2H, H-30eq, ax), 1.60 (m, 4H,
2 · COCH2CH2), 1.27 (48H, 2 · [CH2]12), 0.89 (t, 6H,
J 7.1 Hz, 2 · CH2CH3); 31P NMR (10:10:3 CDCl3–
CD3OD–D2O): d À0.98 (with heteronuclear decou-
pling); ESMS(À): m=z 954.3 [MÀEt3NÀH]À.
benzyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol
3-phosphate) (27)
A mixture of the compounds 78 (104 mg, 0.14 mmol), 25
(55 mg, 0.061 mmol) and pivaloyl chloride (81 lL,
0.66 mmol) in anhyd pyridine (7 mL) was stirred at rt for
2 h. The diastereoisomeric phosphonic diesters 26
thereby formed in solution were oxidised by the addition
of a freshly prepared solution of iodine (72 mg,
0.28 mmol) in 19:1 pyridine–water (9.6 mL). After
45 min, the reaction mixture was dispersed between
CHCl3 (30 mL) and 5% aq NaHSO3 (30 mL). The
aqueous phase was separated and extracted with CHCl3
(25 mL), and the organic extracts were combined,
washed with 1 M triethylammonium hydrogen carbonate
(TEAB) buffer solution (3 · 10 mL), dried (MgSO4) and
concentrated under reduced pressure. RBC (elution first
with CHCl3 and then with 15:1 CHCl3–MeOH) of the
residue provided the TEA salt, which in CHCl3 solution
(20 mL) was washed again with 1 M TEAB buffer solu-
tion (3 · 10 mL), dried (MgSO4) and concentrated to
give finally the TEA phosphate salt 27 (75.8 mg, 76%);
3.13. 1,6-Anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(thio-
carbonylimidazol-1-yl)-b-D-glucopyranose (33)
A solution of the monobenzylated derivative 3222 (0.2 g,
0.72 mmol) and 1,1-thiocarbonyldiimidazole (0.19 g,
1.07 mmol) in toluene (7 mL) was heated under reflux
for 2.5 h, whereafter it was decanted from any solids and
concentrated under reduced pressure. An ethereal solu-
tion of the residue was passed down a short silica-gel
column (further elution with Et2O) and the eluent was
concentrated under reduced pressure. RBC (first with
hexane and then with 9:1 hexane–EtOAc) of the residue
25
D
1
½aŠ þ48 (c 1.0, CHCl3); H NMR: d 7.46–6.98 (30H,
gave the thiocarbonyl derivative 33 (0.22 g, 79%); mp
25
D
6 · C6H5), 5.76 (d, 1H, J1 ;2 3.1 Hz, H-10), 5.25 (m, 1H,
H-2 glycerol), 5.03–4.24 (12H, 6 · CH2Ph), 4.75 (m, 1H,
H-2), 4.39 (t, 1H, J1;6 ¼ J5;6 9.3 Hz, H-6), 4.34 (m, 2H,
1- or 3-CH2 glycerol), 4.28 (m, 1H, H-1), 4.16–4.02 (4H,
103–104 °C; ½aŠ À23 (c 1.0, CHCl3); 1H NMR: d ꢀ7.30
0
0
(5H, C6H5), 8.39, 7.67, 7.06 (3H, imidazole), 5.60, 5.50
(2 · br s, 2H, H-1, 4), 4.74 (s, 2H, CH2Ph), 4.79, 4.39,
3.87 (3 · m, 3H, H-5, 6a,b), 3.75 (br s, 1H, H-3), 3.45 (br

A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
1273
s, 1H, H-2); Anal. Calcd for C17H17N5O4S: C, 52.7; H,
4.4; N, 18.1; S, 8.3. Found: C, 52.7; H, 4.4; N, 18.0; S,
7.7.
3.4 Hz, H-1), 4.67 (ABq, 2H, JAB 11.4 Hz, CH2Ph), 4.30–
3.95 (4H, H-3, 5, 6a,b), 3.37 (dd, 1H, J2;3 10 Hz, H-2),
2.09 (s, 3H, OAc), 2.15, 1.50 (2 · m, 2H, H-4eq, ax);
b-anomer: d 4.50 (d, 1H, J1;2 8.0 Hz, H-1); Anal. Calcd
for C15H19N3O5: C, 56.1; H, 6.0; N, 13.1. Found: C,
56.1; H, 6.1; N, 12.6.
3.14. 1,6-Anhydro-2-azido-3-O-benzyl-2,4-dideoxy-b-
xylo-hexopyranose (34)
D-
To a solution of the thiocarbonyl derivative 33 (0.84 g,
2.17 mmol) in refluxing benzene (60 mL) was added
dropwise over 1 h a solution of Bu3SnH (0.7 mL,
2.6 mmol) and AIBN(71 mg, 0.43 mmol) in benzene
(20 mL). The reaction mixture was heated under reflux
for a further 30 min and then concentrated under
reduced pressure. An ethereal solution of the residue
was passed down a short silica-gel column (further elu-
tion with Et2O) and the eluent was concentrated under
reduced pressure. RBC (first with hexane and then with
3.16. 6-O-Acetyl-2-azido-3-O-benzyl-2,4-dideoxy-D-xylo-
hexopyranosyl fluoride (37)
The hemiacetal 36 (0.103 g, 0.32 mmol) in anhyd 1,2-
dichloroethane (10 mL) under argon at À30 °C was
treated with DAST (0.17 mL, 1.29 mmol) as previously
described for the preparation of the glycosyl fluoride 16.
RBC (first with hexane and then with 2:1 hexane–Et2O)
25
D
gave the fluoride a-37 (0.039 g, 38%); ½aŠ þ55 (c 2.2,
25
D
CHCl3); followed by the fluoride b-37 (0.058 g, 56%); ½aŠ
4:1 hexane–EtOAc) of the residue provided the 4-deoxy
À28 (c 3.3, CHCl3); 1H NMR (a-anomer): d 5.65 (dd, 1H,
J1;2 2.4, J1;F 52.4 Hz, H-1); b-anomer: d ꢀ7.38 (5H,
C6H5), 4.98 (dd, 1H, J1;2 7.2, J1;F 52.1 Hz, H-1), 4.67 (2H,
CH2Ph), ꢀ4.18 (2H, H-6a,b), 3.75 (m, 1H, H-5), 3.47
(2H, H-2, 3), 2.23 (dt, 1H, J3;4eq ¼ J4eq;5 2.0, Jgem 12.5 Hz,
H-4eq), 2.10 (s, 3H, OAc), 1.58 (q, 1H, H-4ax). The a-
and b-glycosyl fluorides were combined for the next step.
25
compound 34 (0.31 g, 55%); ½aŠ þ47 (c 1.0, CHCl3); m
D
2100 cmÀ1 (N3); 1H NMR: d ꢀ7.30 (5H, C6H5), 5.41 (br
s, 1H, H-1), ꢀ4.48 (mþABq, 3H, JAB 12.0 Hz, H-5,
CH2Ph), 4.15 (d, 1H, H-6a), 3.65 (t, 1H, J5;6b ¼ Jgem
6.6 Hz, H-6b), 3.60 (bd, 1H, H-3), 3.27 (br s, 1H, H-2),
2.17, 1.79 (mþbd, 2H, J3;4ax ¼ J4ax;5 4.9, Jgem 15.1 Hz, H-
4ax, eq); Anal. Calcd for C13H15N3O3: C, 59.8; H, 5.8;
N, 16.1. Found: C, 59.7; H, 5.7; N, 16.0.
3.17. 1
oxy-a-
O-(4-methoxybenzyl)-myo-inositol (38)
D
-6-O-(6-O-Acetyl-2-azido-3-O-benzyl-2,4-dide-
-xylo-hexopyranosyl)-2,3,4,5-tetra-O-benzyl-1-
D
3.15. 6-O-Acetyl-2-azido-3-O-benzyl-2,4-dideoxy-
hexopyranose (36)
D
-xylo-
The coupling between the
D
-myo-inositol acceptor 66
A solution of the compound 34 (0.64 g, 2.45 mmol) in
acetic anhydride (10.8 mL) containing TFA (1.2 mL)
was set aside at rt overnight, whereafter it was dispersed
in satd NaHCO3 solution (100 mL). Nitromethane
(100 mL) was then added and the organic phase was
separated, dried (MgSO4) and concentrated under
reduced pressure. A solution of the residue in CHCl3
(50 mL) was washed with water (25 mL), dried (MgSO4)
and concentrated under reduced pressure. An ethereal
solution of the residue was passed down a short silica-
gel column (further elution with Et2O) and the eluent
was concentrated to give the diacetate 35 (0.66 g, 74%)
(0.129 g, 0.195 mmol) and an a,b-mixture of the glycosyl
fluoride 37 (0.087 g, 0.27 mmol) was conducted essen-
tially as described for the preparation of the pseudo-
disaccharide 24. RBC (10:1fi4:1 hexane–Et2O) yielded
the a-pseudodisaccharide 38 (0.065 g, 35%); mp 109–
25
D
110 °C (from Et2O–hexane); ½aŠ þ38 (c 6.5, CHCl3); as
well as a small proportion of the unrecovered b-anomer;
1H NMR: d 7.40–6.77 (29H, C6H4, 5 · C6H5), 5.72 (d,
1H, J1 ;2 3.6 Hz, H-10), 5.08–4.44 (12H, 6 · CH2Ar), 4.24
(t, 1H, J3;4 ¼ J4;5 9.8 Hz, H-4), 4.12–4.05 (2H, H-50,6),
4.01 (t, 1H, J1;2 ¼ J2;3 2.1 Hz, H-2), 3.84–3.77 (sþm, 4H,
0
0
OCH3, H-30), 3.69 (dd, 1H, J5 ;6 a 2.9, Jgem 11.9 Hz,
0
0
1
H-60a), 3.49 (dd, 1H, J5 ;6 b 4.3 Hz, H-60b), 3.44 (dd, 1H,
0
0
as a ꢀ3:1 mixture of the a- and b-anomers; H N MR
0
0
(a-anomer): d ꢀ7.35 (5H, C6H5), 6.25 (d, 1H, J1;2 3.6 Hz,
H-1), 4.65 (ABq, 2H, JAB 11.3 Hz, CH2Ph), ꢀ4.10 (3H,
H-5, 6a,b), 3.94 (m, 1H, H-3), 3.52 (dd, 1H, J2;3 10.0 Hz,
H-2), ꢀ2.20 (m, 1H, H-4eq), 2.14, 2.08 (2 · s, 6H,
2 · OAc), 1.55 (q, 1H, J3;4ax ꢁ J4ax;5 ꢁ Jgem 11.7 Hz, H-4-
ax); b-anomer: d 5.41 (d, 1 H, J1;2 8.3 Hz, H-1).
H-3), 3.42–3.34 (2H, H-1,5), 3.14 (dd, 1H, J2 ;3 10.1 Hz,
H-20), 1.94 (s, 3H, OAc), 1.54 (dt, 1H, J3 ;4 eq ¼ J4 eq;5 2.1,
Jgem 12.2 Hz, H-40eq), 1.33 (m, 1H, H-40ax); Anal. Calcd
for C57H61N3O11: C, 71.0; H, 6.4; N, 4.4. Found: C,
71.0; H, 6.4; N, 4.3.
0
0
0
0
A solution of the diacetate 35 (0.457 g, 1.26 mmol) in
acetonitrile (10 mL) containing 2 M Me2NH in THF
(4.25 mL, 8.50 mmol) was set aside at rt for 6 h and then
concentrated under reduced pressure, with co-evapora-
tion with acetonitrile, to furnish an a,b mixture of the
hemiacetal 36 (0.327 g, 81%); m 2100 cmÀ1 (N3); 1H
NMR (a-anomer): d ꢀ7.35 (5H, C6H5), 5.33 (d, 1H, J1;2
3.18. 1
xylo-hexopyranosyl)-2,3,4,5-tetra-O-benzyl-1-O-(4-meth-
oxybenzyl)-myo-inositol (40)
D
-6-O-(2-Azido-3,6-di-O-benzyl-2,4-dideoxy-a-D-
A solution of the acetylated compound 38 (0.145 g,
0.15 mmol) in 5:4 THF–MeOH (18 mL) containing
0.5 M sodium methoxide in MeOH (2.5 mL, 1.25 mmol)

1274
A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
25
D
was kept at rt for 1.5 h and then neutralised with
Amberlite IR-120 (Hþ) ion-exchange resin. Filtration
and concentration of the filtrate under reduced pressure
gave the alcohol 39 (0.135 g, 97%), which was used in the
next step without further purification.
80%); ½aŠ þ35 (c 1.0, CHCl3); 1H NMR: d ꢀ7.17 (30H,
6 · C6H5), 5.69 (br s, 1H, H-10), 5.19 (m, 1H, H-2
glycerol), 4.95–4.42 (12H, 6 · CH2Ph), 4.64 (1H, H-2),
4.35–4.19 (4H, H-1, 6, 1- or 3-CH2 glycerol), 4.15
(m, 1H, H-50), 4.04 (m, 2H, 1- or 3-CH2 glycerol), 3.99
0
0
0
0
A cooled (0 °C) mixture of the alcohol 39 (0.13 g,
0.14 mmol) and NaH (0.02 g, 0.83 mmol) in DMF
(5 mL) was stirred for 30 min, whereafter benzyl bro-
mide (76 lL, 0.64 mmol) was added dropwise. Stirring of
the reaction mixture for 2 h, followed by a conventional
work-up and RBC (first with hexane and then with 2:1
hexane–Et2O) gave the etherified derivative 40 (0.096 g,
(t, 1H, J3;4 ¼ J4;5 9.7 Hz, H-4), 3.77 (m, 1H, J2 ;3 ¼ J3 ;4 ax
10.0 Hz, H-30), 3.47 (m, 1H, H-3), 3.35 (t, 1H, J4;5 ¼ J5;6
9.7 Hz, H-5), 3.16 (m, 1H, Jgem 10.7 Hz, H-60a), 2.99
(m, 1H, H-20), 2.86 (7H, H-60b, 3 · CH2CH3), 2.18
(m, 4H, 2 · COCH2CH2), 1.59, 1.35 (2 · m, 2H, Jgem
11.9 Hz, H-40eq, ax), 1.48 (m, 4H, 2 · COCH2CH2),
ꢀ1.19 (48H, 2 · [CH2]12), ꢀ1.06 (9H, 3 · CH2CH3),
0.82 (m, 6H, 2 · CH2CH3); 31P N MR:d À3.59 (with
heteronuclear decoupling); ESMS(À): m=z 1520.1 [MÀ
Et3NÀH]À.
25
1
67%); ½aŠ þ44 (c 1.1, CHCl3); H NMR: d 7.42–6.79
D
(34H, C6H4, 6 · C6H5), 5.78 (d, 1H, J1 ;2 3.5 Hz, H-10),
5.35–4.41 (14H, 7 · CH2Ar), 4.28 (t, 1H, J3;4 ¼ J4;5
10.6 Hz, H-4), 4.12–4.04 (2H, H-50, 6), 4.00 (m, 1H, H-
0
0
2), 3.83 (m, 1H, J2 ;3 ¼ J3 4 ax 10.1, J3 4 eq 2.7 Hz, H-30),
0
0
0
0
0 0
3.21. Triethylammonium 1
D
-6-O-(2-amino-2,4-dideoxy-
a- -xylo-hexopyranosyl)-myo-inositol 1-(1,2-di-O-hexa-
3.51 (s, 3H, OCH3), 3.45 (dd, 1H, H-3), 3.42–3.34 (2H,
H-1, 5), 3.18 (dd, 1H, J5 6 a 3.0, Jgem 10.8 Hz, H-60a), 3.14
D
0
0
decanoyl-sn-glycerol 3-phosphate) (43)
(dd, 1H, H-20), 2.62 (dd, 1H, J5 ;6 b 3.9 Hz, H-60b), 1.65
0
0
(dt, 1H, J3 ;4 eq ¼ J4 eq;5 2.7, Jgem 12.0 Hz, H-40eq), 1.52
0
0
0
0
A solution of the benzylated compound 42 (38.7 mg,
0.018 mmol) in 1:1 THF–MeOH (6 mL) containing 20%
Pd(OH)2 on carbon (206 mg) was stirred under 3 atm of
hydrogen for 4 h. Work-up as described for the isomeric
(m, 1H, H-40ax); ESMS(þ): m=z 1034.2 [MþNa]þ.
3.19. 1
xylo-hexopyranosyl)-2,3,4,5-tetra-O-benzyl-myo-inositol
D
-6-O-(2-Azido-3,6-di-O-benzyl-2,4-dideoxy-a-D-
compound 28 gave a-D-4dGlcpN-PI (43) (20.1 mg,
25
D
(41)
80%); ½aŠ þ29 (c 0.18, 10:10:3 CHCl3–MeOH–water);
1H NMR (10:10:3 CDCl3–CD3OD–D2O): d 5.55 (d, 1H,
A solution of the methoxybenzyl compound 40 (0.096 g,
0.095 mmol) in anhyd CH2Cl2 (10 mL) containing TFA
(0.1 mL, 1.3 mmol) was set aside at rt for 1.5 h and then
neutralised with Et3N. Work-up as described for the
isomeric compound 25 and RBC (elution first with
J1 2 2.7 Hz, H-10), 5.28 (m, 1H, H-2 glycerol), 4.43 (m,
2H, 1- or 3-CH2 glycerol), 4.32 (m, 1H, H-50), 4.25–4.22
(m, 2H, 1- or 3-CH2 glycerol), 4.15 (dd, 1H, J1;2 2.7, J1;6
9.5 Hz, H-1), 4.09 (t, 1H, H-2), 4.08–4.00 (m, 1H, H-30),
3.93 (t, 1H, H-6), 3.69–3.65 (m, 1H, H-4), 3.64–3.58 (m,
2H, H-60a,b), 3.46 (dd, 1H, J2;3 2.7, J3;4 9.9 Hz, H-3),
3.35–3.31, 3.06 (2H, H-5,20), ꢀ2.83 (6H, 3 · CH2CH3),
2.37 (t, 4H, 2 · COCH2CH2), 2.06–1.98, 1.56 (2 · m, Jgem
11.1 Hz, H-40eq, ax), 1.61 (m, 4H, 2 · COCH2CH2),
1.40–1.20 (57H, 2 · [CH2]12, 3 · CH2CH3), 0.94–0.63
(6H, 2 · CH2CH3); 31P NMR (10:10:3 CDCl3–CD3OD–
0
0
hexane and then with 2:1 hexane–Et2O) gave the
25
demethoxybenzylated derivative 41 (0.071 g, 84%); ½aŠ
D
þ29 (c 0.85, CHCl3); 1H NMR: d 7.52–7.10 (30H,
6 · C6H5), 5.49 (d, 1H, J1 2 2.6 Hz, H-10), 5.03–4.16
(12H, 6 · CH2Ph), 4.08 (t, J3;4 ¼ J4;5 9.6 Hz, H-4), 4.05–
3.96 (3H, H-2, 50, 6), 3.89 (m, 1H, H-30), 3.60 (m, 1H, J1;6
9.6 Hz, H-1), 3.47–3.35 (3H, H-20, 3, 5), 3.11 (dd, 1H,
0
0
D2O):
d
1.49 (with heteronuclear decoupling);
J5 ;6 a 3.7, Jgem 10.1 Hz, H-60a), 2.96 (dd, 1H, J5 6 b 3.7 Hz,
H-60b), 1.89, 1.52 (2 · m, 2H, Jgem 12.0 Hz, H-40eq, ax);
ESMS(+): m=z 914.3 [MþNa]þ.
ESMS(À): m=z 954.5 [MÀEt3NÀH]À.
0
0
0 0
3.22. (20S,30S)-Methyl 2-azido-2,6-dideoxy-3,4-O-(20,30-
dimethoxybutane-20,30-diyl)-a-
D-glucopyranoside (51)
3.20. Triethylammonium 1
D
-6-O-(2-azido-3,6-di-O-benzyl-
2,4-dideoxy-a- -xylo-hexopyranosyl)-2,3,4,5-tetra-O-
benzyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol
3-phosphate) (42)
D
To a cooled ()40 °C) and stirred solution of the
BDA derivative 4929 (0.55 g, 1.88 mmol) and pyridine
(0.73 mL) in CH2Cl2 (40 mL) was added Tf2O (0.4 mL,
2.38 mmol). The temperature of the mixture was then
allowed to rise to 10 °C over 2 h, whereafter it was
poured into cold satd NaHCO3 solution (20 mL) and the
resulting dispersion was stirred for 10 min. The organic
layer was separated and the aqueous layer was extracted
with CH2Cl2 (3 · 20 mL). The organic extracts were
combined, washed with cold 2 M HCl (10 mL), cold
satd NaHCO3 solution (10 mL) and water (10 mL),
This phosphoric diester was obtained from the pseudo-
disaccharide derivative 41 (0.07 g, 0.078 mmol) and the
H-phosphonate 78 (0.115 g, 0.16 mmol) essentially as
described for the preparation of the isomeric diester 27.
RBC (elution first with CHCl3 and then with 20:1
CHCl3–MeOH) and washing as before with 1 M TEAB
buffer solution gave the TEA phosphate salt 42 (0.102 g,

A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
1275
dried (MgSO4) and concentrated under reduced
pressure to give the crude 2-triflate 50 (0.82 g), which
was used in the next experiment without further
purification.
C21H25N3O4: C, 65.8; H, 6.6; N, 11.0. Found: C, 65.5;
H, 6.5; N, 10.8.
3.24. 2-Azido-3,4-di-O-benzyl-2,6-dideoxy-D-glucopyra-
nose (55)
A mixture of the 2-triflate 50 (0.82 g) and sodium
azide (0.68 g, 10.46 mmol) in DMF (25 mL) was heated
at 75 °C overnight and then concentrated under reduced
pressure (oil pump). A solution of the residue in CH2Cl2
(50 mL) was washed with water (10 mL) and the aque-
ous washing was further extracted with CH2Cl2
(3 · 20 mL). The combined organic extracts were dried
(MgSO4) and concentrated under reduced pressure. An
ethereal solution of the residue was passed down a short
silica-gel column (further elution with Et2O) and the
eluent was concentrated under reduced pressure. RBC
A mixture of the glycoside 54 (0.2 g, 0.52 mmol), 80%
AcOH (3.2 mL) and 1 M HCl (1 mL) was heated at 90–
100 °C for 48 h, whereafter the resulting solution was
concentrated under reduced pressure. The residue was
dissolved in EtOAc and the solution was washed with
satd NaHCO3 solution (3 · 5 mL) and water (10 mL),
dried (MgSO4) and concentrated under reduced pressure
to give the hemiacetal 55 (80 mg, 42%); mp 81–86 °C
(from EtOAc–hexane); m 2100 cmÀ1 (N3); as a mixture of
anomers; 1H NMR: d 5.23 (br s, H-1 a-anomer), 4.55 (d,
J1;2 8.9 Hz, H-1 b-anomer); Anal. Calcd for
C20H23N3O4: C, 65.0; H, 6.3; N, 11.4. Found: C, 64.9;
H, 6.2; N, 11.2.
(19:1 toluene–EtOAc) gave the 2-azide 51 (0.34 g, 57%
25
over the two steps); mp 89 °C (from MeOH); ½aŠ þ298
D
(c 1.0, CHCl3); m 2100 cmÀ1 (N3); H NMR: d 4.66 (d,
1
1H, J1;2 3.5 Hz, H-1), 4.14 (t, 1H, J2;3 ¼ J3;4 10.0 Hz, H-
3), 3.78 (m, 1H, H-5), 3.33 (dd, 1H, H-2), 3.28 (t, 1H,
J3;4 ¼ J4;5 10.0 Hz, H-4), 3.35, 3.30, 3.21 (3 · s, 9H,
3 · OCH3), 1.29, 1.26 (2 · s, 6H, 20, 30-CH3), 1.20 (d, 3H,
J5;6 6.2 Hz, 5-CH3); Anal. Calcd for C13H23N3O6: C,
49.2; H, 7.3; N, 13.2. Found: C, 49.3; H, 7.6; N, 13.2.
Also isolated was the 2,3-unsaturated compound 52
3.25. 2-Azido-3,4-di-O-benzyl-2,6-dideoxy-D-glucopyr-
anosyl fluoride (56)
To a cooled ()30 °C) and stirred solution of the hemi-
acetal 55 (0.107 g, 0.29 mmol) in anhyd 1,2-dichloro-
ethane (10 mL) under argon was added DAST (0.16 mL,
1.21 mmol), whereafter work-up and RBC as described
for the isomeric compound 16 furnished the glycosyl
fluoride 56 (86.9 mg, 81%) as a 1:3 mixture of the a- and
(0.11 g, 21% over the two steps); 1H
N
MR
(CD3COCD3): d 5.05 (d, 1H, J1;2 2.7 Hz, H-2), 4.89 (d,
1H, H-1), 3.90 (m, 1H, H-5), 3.70 (d, 1H, J4;5 9.0 Hz, H-
4), 3.37, 3.29, 3.27 (3 · s, 9H, 3 · OCH3), 1.33, 1.26 (2 · s,
6H, 20,30-CH3), 1.23 (d, 3H, J5;6 5.2 Hz, 5-CH3).
1
b-anomers; H N MR:d 5.46 (dd, J1;2 2.6, J1;F 52.6 Hz,
H-1 a-anomer), 4.91 (dd, J1;2 7.3, J1;F 52.6 Hz, H-1
3.23. Methyl 2-azido-3,4-di-O-benzyl-2,6-dideoxy-a-D-
glucopyranoside (54)
b-anomer); FABMS (þ): m=z 394.0 [MþNa]þ.
A solution of the BDA-protected 2-azide 51 (1.31 g,
4.13 mmol) in 9:1 TFA–water (26 mL) was stirred
at rt for 2 min and was then concentrated under
reduced pressure to give the diol 53 (0.79 g), which
was used in the next experiment without further
purification.
3.26. 1
glucopyranosyl)-2,3,4,5-tetra-O-benzyl-1-O-(4-meth-
oxybenzyl)-myo-inositol (57)
D
-6-O-(2-Azido-3,4-di-O-benzyl-2,6-dideoxy-a-D-
The coupling between the
D
-myo-inositol acceptor 66
(100 mg, 0.15 mmol) and the glycosyl fluoride 56 (79 mg,
0.21 mmol) was conducted essentially as described for
the preparation of the pseudodisaccharide 24. RBC (first
To a stirred and cooled (0 °C) solution of the diol 53
(0.79 g, 3.9 mmol) in DMF (40 mL) was added NaH
(0.37 g, 15.4 mmol) and stirring of the mixture was
continued at rt for 30 min, whereafter benzyl bromide
(1.4 mL, 11.8 mmol) was added dropwise. Work-up as
described for the preparation of compound 14 and RBC
(first with hexane and then with 19:1 hexane–EtOAc)
gave the 3,4-dibenzylated compound 54 (1.2 g, 76% over
the two steps); mp 77–78 °C (from aqueous ethanol);
with hexane and then with 4:1 hexane–Et2O) gave the
25
D
a-pseudodisaccharide 57 (76.9 mg, 50%); ½aŠ þ37
(c 2.4, CHCl3); as well as a small proportion of the
unrecovered b-anomer; 1H NMR: d 7.40–6.81 (34H,
C6H4, 6 · C6H5), 5.67 (d, 1H, J1 ;2 3.7 Hz, H-10), 5.02–
4.46 (14H, 7 · CH2Ar), 4.27 (t, 1H, J3;4 ¼ J4;5 9.3 Hz,
H-4), 4.14 (t, 1H, J1;6 ¼ J5;6 9.3 Hz, H-6), 4.09 (m, 1H,
H-50), 4.01 (dd, 1H, J1;2 2.1, J2;3 3.6 Hz, H-2), 3.94 (t, 1H,
0
0
25
D
1
½aŠ þ63 (c 1.0, CHCl3); m 2100 cmÀ1 (N3); H NMR: d
J2 ;3 ¼ J3 ;4 9.6 Hz, H-30), 3.78 (s, 3H, OCH3), 3.47 (dd,
0
0
0
0
ꢀ7.30 (10H, 2 · C6H5), 4.72 (d, 1H, J1;2 3.3 Hz, H-1),
4.89–4.63 (sþABq, 4H, JAB 11.0 Hz, 2 · CH2Ph), 3.93 (t,
1H, J2;3 ¼ J3;4 9.0 Hz, H-3), 3.77 (m, 1H, H-5), 3.40
(ddþs, 4H, H-2, OCH3), 3.19 (t, 1H, J3;4 ¼ J4;5 9.0 Hz,
H-4), 1.27 (d, 3H, J5;6 6.2 Hz, 5-CH3); Anal. Calcd for
1H, H-3), 3.45 (t, 1H, H-5), 3.39 (dd, 1H, H-1), 3.20 (dd,
1H, H-20), 3.08 (t, 1H, J3 ;4 ¼ J4 ;5 9.6 Hz, H-40), 0.92 (d,
0
0
0
0
3H, J5 ;6 6.2 Hz, 50-CH3); FABMS(þ): m=z 1034.7
0
0
[MþNa]þ.

1276
A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
3.27. Triethylammonium 1
D
-6-O-(2-azido-3,4-di-O-
Acknowledgements
benzyl-2,6-dideoxy-a- -glucopyranosyl)-2,3,4,5-tetra-O-
D
benzyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol
3-phosphate) (59)
This work was supported by a programme grant from
the Wellcome Trust. Two of us (A.P.D. and C.N.B.) are
indebted to the BBSRC for financial support. We also
thank Lisa M. C. Connolly for some preliminary
experiments.
Demethoxybenzylation of the a-pseudodisaccharide
derivative 57 (69 mg, 0.068 mmol) essentially as
described for the preparation of the isomeric compound
25 gave, after RBC (elution first with hexane and then
with 2:1 hexane–Et2O), the alcohol 58 (57 mg, 94%);
References
25
D
½aŠ þ31 (c 0.4, CHCl3); ESMS(+): m=z 914.1 [MþNa]þ.
1. Part 13. Crossman, A., Jr.; Paterson, M. J.; Ferguson, M.
A. J.; Smith, T. K.; Brimacombe, J. S. Carbohydr. Res.
2002, 337, 2049–2059.
The phosphoric diester 59 was obtained from the
pseudodisaccharide derivative 58 (42 mg, 0.047 mmol)
and the H-phosphonate 78 (94 mg, 0.128 mmol) essen-
tially as described for the preparation of the isomeric
diester 27. RBC (elution first with CHCl3 and then with
15:1 CHCl3–MeOH) and washing as before with 1 M
2. Ferguson, M. A. J.; Brimacombe, J. S.; Brown, J. R.;
€
Crossman, A.; Dix, A. P.; Field, R. A.; Guther, M. L. S.;
Milne, K. G.; Sharma, D. K.; Smith, T. K. Biochim.
Biophys. Acta 1999, 1455, 327–340.
3. (a) Englund, P. T. Annu. Rev. Biochem. 1993, 62, 121–138;
TEAB buffer solution gave the TEA phosphate salt 59
€
(b) Guther, M. L. S.; Ferguson, M. A. J. EMBO J. 1995,
14, 3080–3093.
25
D
(63 mg, 82%); ½aŠ þ57 (c 0.7, CHCl3); 1H N MR:d
0
0
7.40–6.97 (30H, 6 · C6H5), 5.61 (d, 1H, J1 ;2 3.1 Hz, H-
10), 5.18 (m, 1H, H-2 glycerol), 4.94–4.46 (12H,
6 · CH2Ph), 4.58 (m, 1H, H-2), 4.32–4.19 (4H, H-1, 6, 1-
or 3-CH2 glycerol), 4.09–4.03 (3H, H-50, 1- or 3-CH2
glycerol), 4.02 (t, 1H, J3;4 ¼ J4;5 9.6 Hz, H-4), 3.90 (dd,
4. (a) Hirose, S.; Prince, G. M.; Sevlever, D.; Ravi, L.;
Rosenberry, T. L.; Ueda, E.; Medof, M. E. J. Biol. Chem.
1992, 267, 16968–16974; (b) Puoti, A.; Conzelmann, A.
J. Biol. Chem. 1993, 268, 7215–7224; (c) Doerrler, W. T.;
Ye, J.; Falk, J. R.; Lehrmann, M. A. J. Biol. Chem. 1996,
271, 27031–27038.
5. Smith, T. K.; Sharma, D. K.; Crossman, A.; Brimacombe,
J. S.; Ferguson, M. A. J. EMBO J. 1999, 18, 5922–5930.
6. Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J.
J. Chem. Soc., Perkin Trans. 1 1993, 2945–2951.
7. Cottaz, S.; Brimacombe, J. S.; Ferguson, M. A. J.
J. Chem. Soc., Perkin Trans. 1 1995, 1673–1678.
8. Crossman, A., Jr.; Brimacombe, J. S.; Ferguson, M. A. J.
J. Chem. Soc., Perkin Trans. 1 1997, 2769–2774.
9. Crossman, A., Jr.; Brimacombe, J. S.; Ferguson, M. A. J.;
Smith, T. K. Carbohydr. Res. 1999, 321, 42–51.
1H, J2 ;3 10.2, J3 ;4 9.2 Hz, H-30), 3.50 (m, 1H, H-3),
0
0
0
0
3.42–3.38 (m, 1H, H-5), 3.13 (dd, 1H, H-20), 3.02 (t, 1H,
J3 ;4 ¼ J4 ;5 9.2 Hz, H-40), 2.89 (6H, 3 · CH2CH3), 2.18
0
0
0
0
(4H, 2 · COCH2CH2), 1.46 (m, 4H, 2 · COCH2CH2),
0
0
ꢀ1.17 (57H, 2 · [CH2]12, 3 · CH2CH3), 0.95 (d, 3H, J5 ;6
6.0 Hz, 50-CH3), 0.80 (t, 6H, J 7.0 Hz, 2 · CH2CH3);
31P N MR:d À6.06 (with heteronuclear decoupling);
ESMS(À): m=z 1520.4 [MÀEt3NÀH]À.
10. (a) Dix, A. P.; Borissow, C. N.; Ferguson, M. A. J.;
Brimacombe, J. S. Tetrahedron Lett. 2001, 42, 117–119; (b)
Borissow, C. N.; Smith, T. K.; Ferguson, M. A. J.;
Brimacombe, J. S. Tetrahedron Lett. 2001, 42, 121–123.
3.28. Triethylammonium 1
-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-
sn-glycerol 3-phosphate) (60)
D
-6-O-(2-amino-2,6-dideoxy-a-
D
ꢁ
11. Lindh, I.; Stawinski, J. J. Org. Chem. 1989, 54, 1338–1342.
12. Probert, M. A.; Zhang, J.; Bundle, D. R. Carbohydr. Res.
1996, 296, 149–170.
13. Fraser-Reid, B.; McLean, A.; Usherwood, E. W.; Tunker,
M. Can. J. Chem. 1970, 48, 2877–2884.
14. Jegou, E.; Cleophax, J.; Leboul, J.; Gero, S. D. Carbohydr.
Res. 1975, 45, 323–326.
15. Matsumoto, T.; Maeta, H.; Suzuki, K.; Tsuchihashi, G.
Tetrahedron Lett. 1988, 29, 3567–3570.
16. Nikolaev, A. V.; Ivanova, I. A.; Shibaev, V. N.; Kochet-
kov, N. K. Carbohydr. Res. 1990, 204, 65–78.
17. Rasmussen, J. R.; Slinger, C. J.; Kordish, R. J.; Newman-
Evans, D. D. J. Org. Chem. 1981, 46, 4843–4846.
18. (a) Samano, M. C.; Robins, M. J. Tetrahedron Lett. 1991,
32, 6293–6296; (b) Hays, D. S.; Fu, G. C. J. Org. Chem.
1998, 63, 2796–2797.
19. Kim, S.; Joe, G. H.; Do, J. Y. J. Am. Chem. Soc. 1994,
116, 5521–5522.
20. Veeneman, G. H. In Carbohydrate Chemistry; Boons,
G.-J., Ed.; Blackie Academic and Professional: London,
1998; p 165.
A solution of the benzylated compound 59 (85 mg,
0.052 mmol) in 2:2:1 THF–propanol–water (5 mL) con-
taining 20% Pd(OH)2 on carbon (240 mg) was stirred
under 3 atm of hydrogen for 5 h. Work-up as described
for the isomeric compound 28 gave a-D-6dGlcpN-PI
25
D
(60) (53.4 mg, 96%); ½aŠ þ17 (c 0.4, 10:10:3 CHCl3–
MeOH–water); 1H NMR (10:10:3 CDCl3–CD3OD–
D2O): d 5.37 (d, 1H, J1 2 4.1 Hz, H-10), 5.15 (m, 1H, H-2
glycerol), 4.30, 4.11 (2 · m, 4H, 1- and 3-CH2 glycerol),
4.06–4.00 (2H, H-1, 50), 3.97 (t, 1H, J1;2 ¼ J2;3 2.5 Hz, H-
2), 3.82 (t, 1H, J1;6 ¼ J5;6 9.4 Hz, H-6), 3.68 (t, 1H,
0
0
J2 ;3 ¼ J3 ;4 9.8 Hz, H-30), 3.56, 3.36 (2 H, H-4, 3), 3.25
(t, 1H, J4;5 ¼ J5;6 9.4 Hz, H-5), 3.07 (7H, H-20,
0
0
0
0
3 · CH2CH3), 2.98 (t, 1H, J3 ;4 ¼ J4 ;5 9.8 Hz, H-40), 2.22
(4H, 2 · COCH2CH2), 1.49 (m, 4H, 2 · COCH2CH2),
1.35–1.09 (60H, 2 · [CH2]12, 3 · CH2CH3, 50-CH3), 0.79
(6H, 2 · CH2CH3); 31P NMR (10:10:3 CDCl3–CD3OD–
0
0
0
0
21. Paterson, M. J. PhD Thesis, University of Dundee, 2001.
22. (a) Tailler, D.; Jacquinet, J. C.; Noirot, A. M.; Beau,
J.-M. J. Chem. Soc., Perkin Trans. 1 1992, 3163–3164;
D2O):
d
1.45 (with heteronuclear decoupling);
ESMS(À): m=z 954.1 [MÀEt3NÀH]À.

A. P. Dix et al. / Carbohydrate Research 339 (2004) 1263–1277
1277
(b) Hori, H.; Nishida, Y.; Ohrui, H.; Meguro, H. J. Org.
Chem. 1989, 54, 1346–1353.
26. Fraser-Reid, B.; Anilkumar, G.; Nair, L. G.; Olsson, L.;
Garcia Martin, M.; Daniels, J. K. Israel J. Chem. 2000, 40,
255–262.
27. Kabir, A. K. M. S.; Brimacombe, J. S., unpublished
results.
28. Barnes, J. C.; Brimacombe, J. S.; Connolly, L. M. C.; Dix,
A. P. Acta Cryst. E 2002, 58, 227–228.
29. Ley, S. V.; Owen, D. R.; Wessen, K. E. J. Chem. Soc.,
Perkin Trans. 1 1997, 2805–2806.
30. Smith, T. K.; Cottaz, S.; Brimacombe, J. S.; Ferguson,
M. A. J. J. Biol. Chem. 1996, 271, 6476–6482.
31. Smith, T. K.; Crossman, A.; Paterson, M. J.; Borissow,
C. N.; Brimacombe, J. S.; Ferguson, M. A. J. J. Biol.
Chem. 2002, 277, 37147–37153.
23. Richardson, A. C. Carbohydr. Res. 1969, 10, 395–402.
24. (a) Binkley, E. R.; Binkley, R. W. In Preparative
Carbohydrate Chemistry; Hanessian, S., Ed.; Marcel
Decker: New York, 1997; p 95; (b) Karpiesiuk, W.;
Banaszek, A.; Zamojski, A. Carbohydr. Res. 1989, 186,
156–162.
25. (a) Haradahira, T.; Maeda, M.; Kai, Y.; Omae, H.;
Kojima, M. Chem. Pharm. Bull. 1985, 33, 165–172;
ꢁꢂ
(b) Pavliak, V.; Kovac, P. Carbohydr. Res. 1991, 210,
333–337; (c) Vox, J. N.; van Boom, J. H.; van Boeckel,
C. A. A.; Beetz, T. J. Carbohydr. Chem. 1984, 3, 117–
124.