Synthesis of cyclophospho-glucoses and glucitols

Article abstract of DOI:10.1016/j.tet.2007.07.027

The syntheses of cyclophosphodiesters of 5-C-(hydroxymethyl)-hexoses and hexitols and of 6-C-(hydroxymethyl)-hexoses are reported, along with that of 6-deoxy-gluco-heptose 7-phosphate. These compounds proved to be reasonable substrate mimics and show inhibitory activity against human d-myo-inositol 3-phosphate synthase.

Full text of DOI:10.1016/j.tet.2007.07.027

Tetrahedron 63 (2007) 10042–10053
Synthesis of cyclophospho-glucoses and glucitols
E. J. Amigues,a M. L. Greenberg,b S. Ju,b Y. Chenb and M. E. Migauda,
*
aSchool of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir Building,
Stranmillis Road, Belfast, BT9 5AG Northern Ireland, UK
bCollege of Liberal Arts and Sciences, Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA
Received 1 May 2007; revised 28 June 2007; accepted 12 July 2007
Available online 19 July 2007
Abstract—The syntheses of cyclophosphodiesters of 5-C-(hydroxymethyl)-hexoses and hexitols and of 6-C-(hydroxymethyl)-hexoses are
reported, along with that of 6-deoxy-gluco-heptose 7-phosphate. These compounds proved to be reasonable substrate mimics and show
inhibitory activity against human D-myo-inositol 3-phosphate synthase.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Ki¼2.3 mM) and 6-deoxy-D-glucitol 6-(E)-vinyl phospho-
nate 8 (yeast Ki¼0.67 mM), Scheme 2).9,10 Amongst other
important residues, a series of four lysine side chains were
identified as playing a major role in complex-stabilisation
through hydrogen-bond interactions with the sugar hy-
droxyls, and ionic interactions with the phosphate moiety
of G6P (1). These analyses also established that the yeast en-
zyme was capable of binding the phosphorylated and phos-
phonylated sugar derivatives 7 and 8, in the same binding
pocket but in almost opposite orientations.11,12 This behav-
iour is thought to be facilitated by the flexibility and charge
stabilisation brought on by the lysine side chains. Further-
more, the phosphonate 8 is the most potent inhibitor of
MIP synthase while its Z-isomer showed no inhibitory prop-
erties. This potency is achieved through a slowly-reversible
competitive binding and oxidation catalysed by the NAD-
bound MIP synthase. These observations led to the proposal
that the enolisation step could be intramolecularly catalysed
by the phosphate moiety itself (Scheme 2).10
In some prokaryotic and most eukaryotic organisms, major
cellular events are controlled by inositol-containing com-
pounds; these include events such as signal transduction
and secondary messenger signalling,1,2 stress response and
cell wall biogenesis.3–5 The de novo synthesis of all inositol-
containing compounds involves a rate-limiting step, which
is the conversion of D-glucose 6-phosphate (1, G6P) into
D-myo-inositol 3-phosphate (also named L-myo-inositol
1-phosphate) catalysed by MIP synthase (D-myo-inositol
3-phosphate synthase; EC 5.5.1.4). For this isomerisation
step, MIP synthase uses b-nicotinamide adenine dinucleo-
tide (NAD) as a prosthetic group.6
The commonly accepted mechanism (Scheme 1) involves
the reaction intermediate 5-keto-glucose 6-phosphate 4 gen-
erated by oxidation of the C-5-hydroxyl of the open-form of
G6P 1, i.e., 3, with the concomitant reduction of NAD to
NADH, followed by the enolisation and aldol condensation
of 5 to yield the inosose phosphate 6. This reaction interme-
diate is subsequently reduced by NADH to produce MIP and
regenerate NAD.7 Several mechanistic studies indicate that
MIP synthase binds the open form of G6P 1 and that this
binding nucleates the active enzyme folding.8
A major structural difference between the phosphonate 8 and
the glucitol phosphate 7 is the introduction of rigidity in the
phosphonate region due to the conjugated alkene system.
Therefore, we wished to explore whether improved potency
over that of phosphate 7 could be achieved by introducing
rigidity in the phosphate region and whether ‘productive’
binding could result. To this end, five cyclophosphate-
containing hexoses and hexitols (9–13; Scheme 3) have
been synthesised, as inhibition studies on MIP synthase
have also shown that the 2-deoxy-sugar and the glucitol
series of substrate analogues display dramatically enhanced
inhibition properties when compared to that of their sugar
parents. Unlike the cyclophosphodiesters of 5-C-(hydroxy-
methyl)-hexoses (9–12), the five-membered ring cyclic
The human MIP synthase (G6P 1, Km¼0.57 mM) shares
high sequence and structural homology with the yeast en-
zyme (G6P 1, Km¼1.18 mM) for which Geiger reported
the crystal structures of MIP synthase bound to two different
inhibitors (2-deoxy D-glucitol 6-phosphate
7 (yeast
* Corresponding author. Tel.: +44 2890 974339; fax: +44 2890 976524;
e-mail: m.migaud@qub.ac.uk
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.07.027

E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
10043
OPO23-
H
OPO23-
OPO23-
OPO23-
5
5
5
HO
HO
HO
HO
OH
O
HO
HO
O
O
O
4
OH
3
HO
HO
H
HO
2
NAD
OPO23-
5
HO
HO
O
O
HO
HO
OH
HO
HO
OH
5
Glucose-6-phosphate
NADH
G6P 1
OPO23-
HO
HO
OPO23-
5
HO
HO
OH
OH
OH
HO
O
HO
myo-inositol 1-phosphate
6
MIP
Scheme 1. MIP synthase mechanism.
O_
O_
OPO32-
_
O
HO
HO
_
H
P
H
P
O
O
O
HO
HO
OH
HO
HO
OH
HO
O
O
OH
OH
OH
7
8
5
NAD-bound MIP sy_nthase
O
NAD-bound
MIP synthase
_
O
O
P
O_
HR
H
P
_
HO
HO
O
O
HO
HO
O
O
OH
O
HO
O
4
Scheme 2. Known potent MIP synthase inhibitors and phosphate-catalysed enolisation of 5-ketoglucose 6-phosphate.
phosphate 13, when in the open form, possesses a secondary
C-5 hydroxyl group. This hydroxyl could potentially be oxi-
dised to a keto-sugar once bound to the NAD–MIP synthase
complex, hence this becomes a mimic of the hydrogen-
abstraction transition state occurring during the enolisation
step, if such step is indeed phosphate-catalysed.
4). The 1,3 diols, 22 and 23, were accessed via an aldol-
type condensation with formaldehyde of the aldehydic sugar
precursors, 20 and 21, respectively), followed by a spontane-
ous Canizzaro reduction, as first developed by Schaeffer13
and applied to the synthesis of 5-C-(hydroxyoxymethyl)-
D-xylohexopyranoside by Hiler.14
Direct Wittig olefination of the aldehydic glucosides 20 and
21 afforded the methylene intermediates 24 and 25, which
were subsequently dihydroxylated to yield the 6-C-
(hydroxymethyl)-hexoses 26 and 27. In order to establish
the chirality at the C-6 carbon of 26 and 27, derivatisation
of 26b with an acetonide group was carried out and offered
a mixture of diastereoisomers in a 15:85 ratio (established
by H NMR). Selective crystallisation in dichloromethane/
hexane afforded crystals of the major isomer and crystallo-
graphicanalysesconfirmedtheC-6carbonconfigurationasR.
OH
P
O
OH
P
O
O
O
O
O
O
P
O
O
O
OH
R
OH OH
O
OH
OH
HO
HO
HO
R
HO
OH
OH
OH
9
R=OH
10 R= H
13
11 R=OH
12 R= H
1
Scheme 3. Cyclophospho-glucoses and glucitols.
2. Results and discussion
We previously reported the use of benzyloxy-bis(diisopropyl-
amino)phosphine as phosphitylating agent of 1,2- and 1,3-
diols.15 However, the use of the more reactive and rarely
used benzyloxydichlorophosphine in the presence of an ex-
cess of triethylamine was necessary to achieve quantitative
phosphitylation of the 1,2- and 1,3-diol sugars, 26/27 and
22/23, respectively (Scheme 5). Each cyclic phosphite triester
intermediate, 28a, 28b and 29 was isolated as a mixture of
diastereoisomers ((R)-P and (S)-P) in a 1:1 ratio. All three
cyclic phosphite triesters proved to be too unstable to allow
for extensive chromatographic purification and separation of
The aldehydes 20 and 21, precursors to the 5-C-(hydroxy-
methyl)-hexoses 9–12, were obtained from D-glucose and
2-deoxy-D-glucose in 33% and 17% overall yield, respec-
tively. Glucose and 2-deoxy-glucose were selectively trityl-
ated to yield the partially protected sugars 14 and 15,
which were subsequently benzylated (16 and 17) and selec-
tively detritylated to yield the primary alcohols 18 and 19,
which were readily converted under Swern conditions to
the aldehydic synthetic intermediates 20 and 21 (Scheme

10044
E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
OH
O
HO
HO
OH
O
OBn
R
OBn
R
HO
BnO
O
OH
R
BnO
HO
OBn
OBn
OH
, R = OBn
27 , R = H
26
22 , R = OBn
23 , R = H
R= OH
R= H
a
g
e
O
OH
OTr
OTr
O
OBn
O
OBn
R
O
OBn
R
O
OBn
O
OH
b
d
f
c
BnO
R
BnO
BnO
BnO
R
HO
R
OBn
OBn
OBn
OBn
OH
, R = OBn α/β
19 , R = H
20 , R = OBn α/β
21 , R = H
18
24
, R = OBn α/β
16 R= OBn
17 R= H
14 R= OH
15 R= H
25 , R = H
Scheme 4. Divergent syntheses of the 1,2- and 1,3-diols. (a) TrCl, pyridine, 70 ꢀC, 14: 97%, 15: 58%; (b) BnBr, NaH, Bu4NI, DMF, 16: 82%, 17: 80%; (c) AlCl3,
DCM, Et2O, 18: 58%, 19: 73%; (d) DMSO, (COCl)2, Et3N, DCM, ꢁ78 ꢀC, quant.; (e) 1,4 dioxane, formaldehyde, NaOH, rt, 22a/b: 69%, 23b: 50%; (f)
Ph3PCH3Br, BuLi, THF, ꢁ78 ꢀC to rt, 24a/b: 44%, 25b: 50% and (g) OsO4, NMO, DCM, 26a/b: 100%, 27b: 98%.
O
P
O
HO
O
P
O
O
HO
HO
BnO
O
BnO
c, b
HO
BnO
O
OBn
R
O
OBn
R
O
OBn
R
a, b
O
OBn
R
BnO
BnO
BnO
OBn
OBn
OBn
OBn
71%
71%
46%
71% 31 , R = H
22 , R = OBn
28 / , R = OBn
29 , R = H
26 / , R = OBn
27 , R = H
30 / , R = OBn
/
23 , R = H
Scheme 5. Synthesis of the phosphate triesters. (a) (BnO)PCl2, Et3N, DCM; (b) t-BuOOH, DCM and (c) 2,4-DNP, (BnO)P(NiPr)2, DCM.
the individual diastereoisomers. Subsequent oxidation of the
diastereoisomeric mixture using anhydrous tert-butyl hydro-
peroxide afforded the corresponding cyclic phosphate
triester diastereoisomers in high yields. The diastereoiso-
meric mixtures, while cumbersome for characterisation pur-
poses, could be used as such for the subsequent steps since
both the phosphorus chirality and the anomeric stereochem-
istry were to be lost at the deprotection step.
NaH for the phosphorylation of the resulting primary alcohol
to yield the fully benzylated 6-deoxy-gluco-heptose 7-
phosphate 36.
The debenzylation of 28, 29, 30, 31 and 36 proved to be less
straightforward than anticipated. Pd-catalysed hydrogena-
tion in methanol in the presence of a molar equivalent of
inorganic base was required for the debenzylation of cyclic
phosphotriesters and quantitative conversion to the cyclic
phosphate diester sugars with no cyclophosphate ring open-
ing.15 The protected sugars 28, 29 and 36 required two reac-
tion cycles with filtration of the catalyst and new addition of
Pd/C and 0.5 equiv K2CO3 over the course of the reaction, to
yield quantitatively the cyclic phosphate diesters 9, 10 and
35, respectively. Deprotection of the cyclic phosphate
triester 30 to cyclic phosphate diester 13 was only achieved
when Birch reduction conditions were employed. Unfortu-
nately, when applied to 31, neither of these sets of conditions
afforded the fully deprotected cyclic phosphate diester with
It must be noted that attempts to prepare the cyclic phos-
phates from the glucitol parents of 22 and 26, i.e. glucitols
32 and 33 (Scheme 6) only yielded to the detection of phos-
phate monoesters, with less than 10% cyclised phosphite
triesters identified by 31P NMR.
Additionally, the 6-deoxy-gluco-heptose-7-phosphate 35
was synthesised from intermediate 24 (Scheme 7) using
9-BBN for the regioselective hydroxylation of the alkene
bond and tetrabenzylpyrophosphate in the presence of
O
P
O
HO
O
P
O
HO
HO
BnO
O
O
BnO
OBn OBn a, b
HO
BnO
OBn OBn
a, b
X
OBn OBn
OBn OBn
X
OBn
OBn
BnO
OBn
OBn
BnO
OBn
BnO
OBn
OBn
OBn
33
32
Scheme 6. Attempted syntheses of the cyclic phosphate triesters from the glucitols 32 and 33. (a) (BnO)PCl2, Et3N, DCM and (b) t-BuOOH, DCM.

E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
10045
PO(OBn)2
O
PO(OH)2
O
sugar phosphates (tetrose and pentose derivatives) are
known MIP synthase inhibitors. Finally, the kinetic studies
of the effect of the five-membered ring cyclic phosphate
13 have so far been inconclusive. Further enzyme kinetic
experiments and compound stability studies are needed to
establish the inhibitory potency of this glucose 6-phosphate
mimic.
O
OBn
OBn
c
a, b
O
OBn
O
OH
BnO
BnO
OBn
HO
OH
OBn
OBn
OH
Scheme 7. Synthesis of 6-deoxy-gluco-heptose-7-phosphate. (a) i. 9-BBN,
THF; ii. H2O2, NaOH, 97%; (b) NaH, TBPP, 42% and (c) H2, Pd/C, MeOH,
quant.
3. Conclusions
In conclusion, we have synthesised five novel C7-sugar cy-
clophosphates, analogues of glucose 6-phosphate in addition
to 6-deoxy-gluco-heptose 7-phosphate and evaluated these
compounds against human MIP synthase. While conforma-
tional strain was introduced into the molecular scaffold in
a hope to increase conformational likeness to a reaction
intermediate, no improvement in MIP synthase inhibition
was observed when compared to the known inhibitors.
sufficient level of purity to allow for isolation and full char-
aterisation.
Since it was not possible to access the cyclic phosphate
triesters glucitol-derivatives from the glucitol diol precur-
sors 32 and 33 (Scheme 6), the glucose and 2-deoxy glucose
cyclic phosphates 9 and 10 were treated with an excess of
sodium borohydride in water to access the glucitols 11 and
12, respectively (Scheme 8). Surprisingly, no ring opening
of the cyclic phosphate diesters was detected, while the
hemiacetal carbonyl was reduced quantitatively.
4. Experimental
4.1. General experimental details
O
O
HPLC runs were performed on a Gilson Anachem system
with UV detector operating at 254 nm using a C18-RP Su-
pelcosyl HPLC column. IR spectra were recorded on a Perkin
O
O
P
P
HO
HO
O
OH
R
OH OH
a
O
O
28/29
Elmer spectrum RI I FT-IR system; absorption in cmꢁ1
.
HO
HO
R
OH
OH
Mass spectra were obtained on an LCT premier Micro-
massÒ Technologies Waters (ASEP Belfast). Optical
rotations were determined with a Perkin Elmer 341 polari-
9 R= OH
10 R= H
11 R= OH
12 R= H
1
meter. The H, 13C and 31P NMR spectra were recorded
Scheme 8. Synthesis of the glucitol cyclic phosphates 11 and 12 from the
glucose parents. (a) NaBH4, H2O then Dowex H+.
in CDCl3, D2O, CD3OD or (CD3)2SO on a Bruker AC
1
300 MHz spectrometer. HMQC and H–1H COSY experi-
ments were performed on a Bruker AMX 500 spectrometer.
The spirocyclophosphates 9–12 and 6-deoxy-gluco-heptose
7-phosphate 35 were then tested for activity against MIP
synthase. When compounds 9–12 were tested against puri-
fied human MIP synthase,16 only inhibition was observed
at low millimolar concentration (Table 1), the 2-deoxy cyclic
phosphates 10 and 12 possess better affinity for the enzyme
than their glucose parent. Unlike 2-deoxy-glucitol 6-
phosphate 7, known to inhibit eukaryotic MIP synthase at
low micromolar concentrations,9,10 the spirophosphate de-
rived from 2-deoxyglucitol 12, had limited affinity for the
human enzyme with a 10-fold loss in potency. This could
indicate that the charge and the conformational flexibility
of the phosphate moiety are crucial for recognition, binding
and inhibition of the human enzyme. Surprinsingly, no inhi-
bition was detected for the homoglucose 7-phosphate 35. It
is to be noted that the increased chain length of the sugar
moiety resulted in complete loss of inhibition while shorter
1
TMS (0 ppm, H NMR), CDCl3 (77 ppm, 13C NMR) and
triethyl phosphate (0.2 ppm, 31P NMR) were used as internal
references. The chemical shifts (d) are reported in parts per
million and the coupling constants (J values) are recorded in
hertz. The hydrogen and carbon assignments were done
1
when 2D experiments (1H–1H COSY and H–13C HMQC
experiments) allowed it. The benzylic methylene hydrogens
are described as two doublets instead of AB systems for clar-
ity purposes.
4.1.1. 6-O-Trityl-D-glucopyranose 14.17 To a solution of
D-glucose (10 g, 55.5 mmol) in dry pyridine (120 mL) was
added triphenylmethyl chloride (17 g, 61.0 mol). The result-
ing suspension was then heated at 75 ꢀC for 18 h. The reac-
tion mixture was then allowed to cool down to room
temperature. Pyridine was removed under reduced pressure
to afford a yellow gum. This residue was dissolved in
DCM (150 mL) and washed with NH4Cl (100 mL),
NaHCO3 (100 mL) and brine (100 mL). The organic layer
was the dried, filtered and concentrated under reduced pres-
sure to afford the crude 6-O-trityl-D-glucose as a yellow oil
(40 g). Purification by flash chromatography (100/0/90/
10, v/v, CHCl3/EtOH) afforded the pure compound 15 (mix-
ture of anomers) as a white foam (22.0 g, 94%). nmax (liquid
film) 3392, 3018, 1597, 1490, 1047 cmꢁ1; 1H NMR (CDCl3)
d 7.45–7.10 (15H, m, Ar), 5.69–4.50 (4H, m), 3.88 (2H, m),
3.47–3.22 (5H, m); 13C NMR (CDCl3) d 143.7, 143.6, 128.7,
Table 1. Inhibition constant values of spirocyclophosphates 9–12 and
6-deoxy-gluco-heptose 7-phosphate 35 against human MIP synthase deter-
mined at a G6P concentration of 5 mM
IC50 (human MIP synthase;
KmG6P¼0.57 mM)
No inhibition at 5 mM
0.6 mM
0.85 mM
Calculated Ki; Ki¼IC50
/
(1+[G6P]/Km)
9
N/A
10
11
12
35
32 mM
46 mM
30 mM
N/A
0.56 mM
No inhibition at 5 mM

10046
E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
127.8, 127.1, 127.0, 96.3, 92.3, 87.0, 86.7, 74.5, 73.4, 72.0,
71.3, 70.3, 64.0, 63.5; HRMS (ES) Calcd for C25H25O6
(MꢁH+) 421.1651 found 421.1654.
a-anomer, fraction 2 (681 mg) mixture of anomers and frac-
tion 3 (327 mg) b-anomer (overall yield 58%). b-anomer:
nmax (liquid film) 3469, 2925, 1497, 1360, 1214, 1070 cmꢁ1
;
[a]2D0 ꢁ8.4 (c 0.028, CHCl3) (lit.18b [a]D20 ꢁ10 (c 1, CHCl3));
1H NMR (CDCl3) d 7.37–7.25 (20H, m, Ar), 4.95 (1H, d, J
10.5 Hz, CH2–Ph), 4.93 (1H, d, J 11.9 Hz, CH2–Ph), 4.92
(1H, d, J 11.0 Hz, CH2–Ph), 4.86 (1H, d, J 11.0 Hz, CH2–
Ph), 4.80 (1H, d, J 10.9 Hz, CH2–Ph), 4.73 (1H, d, J
10.9 Hz, CH2–Ph), 4.69 (1H, d, J 11.8 Hz, CH2–Ph), 4.63
(1H, d, J 11.0 Hz, CH2–Ph), 4.57 (1H, d, J1–2 7.8 Hz, H-
4.1.2. Benzyl 6-O-trityl-2,3,4-tri-O-benzyl-D-glucopyra-
noside 16.18 Sodium hydride (1.42 g of 60% suspension in
mineral oil, 35.55 mmol) was added portionwise to a solu-
tion of 6-O-trityl-D-glucopyranoside 14 (3.0 g, 7.11 mmol),
benzyl bromide (4.2 mL, 35.55 mmol) and tetrabutylammo-
nium iodide (264 mg, 0.71 mmol) in dry DMF (80 mL) at
0 ꢀC. The resulting suspension was allowed to warm up to
room temperature and stirred overnight. The reaction mix-
ture was then diluted with Et2O (100 mL) and NH4Cl(aq)
(100 mL) was added to the resulting solution. The organic
layer was washed with NH4Cl(aq) (50 mL), water (50 mL),
brine (50 mL), dried, filtered and concentrated under vac-
uum to afford the crude sugar as a waxy yellowish solid
(6.61 g). Purification by flash chromatography (100/0/
50/50, v/v, PE/EtOAc) afforded the pure compound 16 as
a waxy solid (4.52 g, 82%). nmax (liquid film) 3028, 2926,
1955, 1659, 1450, 1077, 756 cmꢁ1; [a]D20 +17.3 (c 0.03,
0
1), 3.87 (1H, dd, J5–6 2.7 Hz, J6–6 5.7 Hz, H-6), 3.73–3.65
(1H, m, H-60), 3.67 (1H, appt, J3–4,4–5 9.0 Hz, H-4), 3.57
(1H, appt, J2–3,3–4 9.3 Hz, H-3), 3.49 (1H, appt, J1–2,2–3
0
8.6 Hz, H-2), 3.36 (1H, ddd, J5–6 2.8 Hz, J6–6 4.6 Hz, J4–5
9.6 Hz, H-5); 13C NMR (CDCl3) d 138.5 (Ar), 138.3 (Ar),
138.0 (Ar), 137.3 (Ar), 128.5 (Ar), 128.3 (2, Ar), 128.1
(Ar), 128.0 (Ar), 127.9 (Ar), 127.8 (Ar), 127.7 (Ar), 127.6
(Ar), 102.8 (C-1), 84.5 (C-3), 82.3 (C-2), 77.6 (C-4), 75.7
(CH2–Ph), 75.1 (2, CH2–Ph), 75.0 (C-5), 71.7 (CH2–Ph),
62.1 (C-6); HRMS (ES) Calcd for C34H35O6 (MꢁH+)
539.2434 found 539.2440; a-anomer: nmax (liquid film)
3469, 2925, 1497, 1360, 1214, 1070 cmꢁ1; [a]D20 +36.4 (c
1
CHCl3); H NMR (CDCl3) d 7.54–7.16 (33H, m, Ar), 6.86
1
(2H, appd, J 6.6 Hz), 5.07 (0.6H, d, J 11.8 Hz, b, CH2–
Ph), 5.01 (0.6H, d, J 10.9 Hz, b, CH2–Ph), 4.97 (0.4H, d, J
10.7 Hz, a, CH2–Ph), 4.95 (0.4H, d, J1–2 3.7 Hz, a, H-1),
4.89 (0.6H, d, J 10.8 Hz, b, CH2–Ph), 4.82–4.68 (3.8H, m,
b/a, CH2–Ph), 4.63 (0.4H, d, J 12.2 Hz, a, CH2–Ph), 4.61
(0.6H, d, J 12.0 Hz, b, CH2–Ph), 4.55 (0.6H, d, J1–2
7.2 Hz, H-1), 4.36 (0.6H, d, J 10.4 Hz, b, CH2–Ph), 4.30
(0.4H, d, J4–5 10.4 Hz, a, CH2–Ph), 4.02 (0.4H, t, J3–4,4–5
9.3 Hz, a, H-4), 3.87 (0.4H, ddd, J5–6 1.5 Hz, J4–5 4.3 Hz,
0.018, CHCl3) (lit.18a [a]D20 63.4 (c 1.5, CHCl3)); H NMR
(CDCl3) d 7.40–7.24 (20H, m, Ar), 5.01 (1H, d, J 10.9 Hz,
CH2–Ph), 4.88 (1H, d, J 11.0 Hz, CH2–Ph), 4.84 (1H, d, J
10.9 Hz, CH2–Ph), 4.81 (1H, d, J1–2 3.6 Hz, H-1), 4.68
(2H, 2d, J 11.7 Hz, 12.9 Hz, CH2–Ph), 4.64 (1H, d, J
11.0 Hz, CH2–Ph), 4.55 (1H, d, J 11.9 Hz, CH2–Ph), 4.54
(1H, d, J 12.3 Hz, CH2–Ph), 4.07 (1H, appt J2–3,3–4 9.3 Hz,
H-3), 3.72–3.66 (3H, m, H-6, H-60, H-5), 3.54 (1H, appt,
J3–4,4–5 9.2 Hz, H-4), 3.50 (1H, dd, J1–2 3.4 Hz, J2–3
9.4 Hz, H-2); 13C NMR (CDCl3) d 138.8 (Ar), 138.1 (Ar),
137.1 (Ar), 128.4 (Ar), 128.3 (Ar), 128.0 (Ar), 127.9 (Ar),
127.7 (Ar), 127.5 (Ar), 95.6 (C-1), 81.9 (C-3), 80.0 (C-2),
77.4 (C-4), 75.7 (CH2–Ph), 75.0 (CH2–Ph), 73.0 (CH2–
Ph), 71.0 (C-5), 69.2 (CH2–Ph), 61.8 (C-6); HRMS (ES)
Calcd for C34H35O6 (MꢁH+) 539.2434 found 539.2441.
4.1.4. 6-O-Trityl-2-deoxy-D-glucopyranose 15.19 To a solu-
tion of 2-deoxy-D-glucose (1 g, 6.1 mmol) in dry pyridine
(20 mL) was added triphenylmethyl chloride (1.86 g,
6.7 mol). The resulting suspension was then heated at
75 ꢀC for 18 h. The reaction mixture was then allowed to
cool down to room temperature. Pyridine was removed un-
der reduced pressure to afford a yellow gum. This residue
was dissolved in DCM (50 mL) and washed with NH4Cl
(30 mL), NaHCO3 (30 mL) and brine (30 mL). The organic
layer was the dried, filtered and concentrated under reduced
pressure to afford the crude 6-O-trityl-2-deoxy-D-glucose as
a yellow oil (4.2 g). Purification by flash chromatography
(100/0/90/10, v/v, CHCl3/EtOH) afforded the pure 15
(mixture of anomers) as a white foam (1.45 g, 58%). nmax
(liquid film) 3391, 3017, 1597, 1448, 1063 cmꢁ1; 1H NMR
(CDCl3) d 7.45–7.17 (15H, m, Ar), 5.26 (0.7H, br s), 4.71
(0.3H, d, J 9.2 Hz), 4.01–3.76 (2H, m), 3.41–3.33 (3H, m),
2.18–1.97 (1H, m), 1.63–1.43 (1H, m); 13C NMR (CDCl3)
d 143.6 (2), 143.4, 128.6, 128.5, 127.9, 127.8, 127.2,
127.1, 94.1, 91.9, 87.3, 87.0, 741, 73.8, 71.2, 70.2, 64.7,
64.4, 39.5, 37.0; HRMS (ES) Calcd for C25H25O5 (MꢁH+)
405.1702 found 405.1721.
0
J5–6 10.4 Hz, a, H-5), 3.81 (0.6H, t, J3–4,4–5 9.2 Hz, b, H-
4), 3.68–3.58 (2.6H, m, b/a, H-2, b/a, H-3, b, H-6), 3.46
0
(0.4H, dd, J6–6 1.8 Hz, J5–6 10.1 Hz, a, H-6), 3.42 (0.6H,
0
ddd, J5–6 1.7 Hz, J5–6 3.9 Hz, J4–5 9.8 Hz, 0b, H-5), 3.27
0
0
(0.6H, dd, J5–6 4.1 Hz, J6–6 10.1 Hz, b, H-6 ), 3.19 (0.4H,
dd, J5–6 4.5 Hz, J6–6 10.1 Hz, a, H-60); 13C NMR (CDCl3)
d 144.0, 138.8, 138.6, 138.3, 138.0, 137.9, 137.4, 137.1,
128.9, 128.7, 128.5, 128.4 (2), 128.2 (2), 128.1, 127.8,
127.7 127.6, 127.0, 102.3 (b, C-1), 94.7(a, C-1), 86.4 (b/
a, C(Ph)3), 84.8 (b, C-2 or C-3), 82.6 (b, C-3 or C-2), 82.4
(a, C-4), 80.3 (a, C-2 or C-3), 78.2 (a, C-2 or C-3), 78.0
(b, C-4), 76.0 (b/a, 2, O–CH2–Ph), 75.0 (b/a, 2, O–CH2–
Ph), 74.7 (C-5), 73.0 (b/a, O–CH2–Ph), 70.7 (b/a, O–
CH2–Ph), 70.6 (C-5), 68.7 (b/a, O–CH2–Ph), 62.5 (C-6);
LRMS (ES) (M+Na+) 783.0.
0
0
4.1.3. Benzyl 2,3,4-tri-O-benzyl-D-glucopyranoside 18.18
To a solution of benzyl-6-O-trityl-2,3,4-tri-O-benzyl-D-
glucopyranoside 16 (4.27 g, 5.81 mmol) in dry DCM
(60 mL) was added aluminium trichloride (0.78 g,
5.81 mmol) diluted in dry Et2O (60 mL). The resulting
solution turned bright yellow almost immediately. The reac-
tion mixture was then stirred for 1 h. The reaction mixture
was then diluted with Et2O (100 mL) and NaHCO3
(100 mL). The aqueous layer was then extracted with Et2O
(75 mLꢂ2). The combined organic layers were then washed
with NaHCO3 (70 mLꢂ2), water (70 mLꢂ2) and brine
(70 mLꢂ2), dried, filtered and concentrated under reduced
pressure to afford the crude sugar (4.32 g) as a colourless
oil, which turned into a waxy solid overnight. Purification
by flash chromatography (90/10/50/50, v/v, PE/EtOAc)
afforded three fractions of pure sugar: fraction 1 (834 mg)
4.1.5. Benzyl 6-O-trityl-3,4-di-O-benzyl-2-deoxy-D-gluco-
pyranose 17. Sodium hydride (552 mg of 60% suspension

E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
10047
in mineral oil, 13.8 mmol) was added portionwise to a solu-
tion of 6-O-trityl-2-deoxy-D-glucopyranoside 15 (1.4 g,
3.45 mmol), benzyl bromide (1.64 mL, 13.8 mmol) and tet-
rabutylammonium iodide (130 mg, 0.35 mmol) in dry DMF
at (40 mL) 0 ꢀC. The resulting suspension was allowed to
warm up to room temperature and stirred overnight. The re-
action mixture was then diluted with Et2O (75 mL) and
NH4Cl(aq) (75 mL) was added to the resulting solution.
The organic layer was washed with NH4Cl(aq) (50 mL), wa-
ter (50 mL), brine (50 mL), dried, filtered and concentrated
under vacuum to afford the crude sugar as a waxy yellowish
solid (4.50 g). Purification by flash chromatography (100/
0/50/50, v/v, PE/EtOAc) afforded pure compound 17 as
a waxy solid (1.82 g, 78%). nmax (liquid film) 3018, 2934,
(Ar), 127.8 (Ar), 127.6 (Ar), 98.9 (C-1), 79.2, 78.1, 75.3,
75.0, 71.4, 70.9, 62.5, 36.7 (C-2); HRMS (ES) Calcd for
C27H30O5Na (M+Na+) 457.1991 found 457.1994. a-
anomer: nmax (liquid film) 3466, 2925, 1496, 1367, 1213,
1059 cmꢁ1; [a]D20 +46.1 (c 0.015, CHCl3) (lit.20a [a]D20 53
1
(c 1.5, CHCl3)); H NMR (CDCl3) d 7.28–7.20 (15H, m,
Ar), 4.93 (1H, d, J 2.9 Hz, H-1), 4.87 (1H, d, J 11.0 Hz,
CH2–Ph), 4.62–4.55 (4H, m, CH2–Ph), 4.36 (1H, d, J
11.9 Hz, CH2–Ph), 3.99 (1H, ddd, J 5.0, 8.9, 11.4 Hz, H-
3), 3.74 (3H, m), 3.46 (1H, t, J 9.3 Hz, H-4), 2.26 (1H,
ddd, J 0.8, 4.9, 13.0 Hz, H-2), 1.61 (1H, ddd, J 3.7, 11.5,
13.1 Hz, H-20); 13C NMR (CDCl3) d 138.6 (Ar), 138.3,
137.5 (Ar), 128.4 (2, Ar), 128.3 (Ar), 128.0 (Ar), 127.9
(Ar), 127.8 (Ar), 127.7 (Ar), 127.6 (Ar), 96.7 (C-1), 78.2,
75.0, 71.8, 71.5, 68.9, 62.2, 35.5 (C-2); HRMS (ES) Calcd
for C27H30O5Na (M+Na+) 457.1991 found 457.1995.
2400, 1492, 1214, 1077, 769 cmꢁ1 1H NMR (CDCl3)
;
d 7.57–7.50 (6H, m, Ar), 7.41–7.18 (22H, m, Ar), 6.93–
6.90 (2H, m, Ar), 5.10 (0.35H, d, J 2.9 Hz, a, H-1), 5.02
(0.65H, d, J 11.6 Hz, CH2–Ph), 4.77–4.63 (3.65H, m, CH2–
Ph), 4.58 (0.65H, d, J 6.9 Hz, b, H-1), 4.51 (0.65H, d, J
11.8 Hz, CH2–Ph), 4.39–4.32 (1H, m, CH2–Ph), 4.01
(0.35H, ddd, J 5.1, 9.1, 11.6 Hz, a, H-3), 3.88 (0.35H, ddd,
J 1.5, 4.5, 9.9 Hz, a, H-6), 3.73 (0.65H, t, J 9.1 Hz, b, H-4),
3.67–3.57 (1.65H, m, b, H-6, b, H-5, a, H-5), 3.51 (0.35H,
dd, J 1.7, 10.0 Hz, a, H-60), 3.37 (0.65H, ddd, J 1.4, 4.2,
9.0 Hz, b, H-3), 3.29 (0.65H, dd, J 4.1, 9.8 Hz, b, H-60),
3.26 (0.35H, dd, J 4.8, 11.7 Hz, a, H-4), 2.43–2.34 (1H, m,
a and b, H-2), 1.86–1.75 (1H, m, a and b, H-20); 13C NMR
(CDCl3) d 144.1, 144.0, 138.3, 138.1, 137.4, 128.9, 128.5,
128.4 (2), 128.1 (2), 127.9, 127.8, 127.7 (2), 127.5 (2),
126.9 (2), 98.5, 96.1, 86.3 (2), 79.5, 78.8, 78.3, 77.9, 75.0,
72.1, 71.7, 71.3, 70.0, 68.5, 63.0, 62.9, 37.9, 35.6; HRMS
(ES) Calcd for C46H44O5Na (M+Na+) 699.3086 found
699.3088.
4.1.7. Benzyl 2,3,4-tri-O-benzyl-5-C-(hydroxymethyl)-D-
glucopyranoside 22a/b. To a solution of oxalyl chloride
(740 mL, 8.48 mmol) in dry DCM (25 mL) cooled to
ꢁ78 ꢀC was added DMSO (1.2 mL, 16.96 mmol) diluted
in dry DCM (25 mL) and the resulting solution was stirred
for 40 min. To the reaction mixture was then slowly added
a solution of benzyl-2,3,4-tri-O-benzyl-a-D-glucopyrano-
side 18 (1.15 g, 2.12 mmol) in dry DCM (5 mL). Stirring
was continued at ꢁ78 ꢀC for 2 h. Et3N (4.73 mL,
33.9 mmol) was added at ꢁ78 ꢀC and after 10 min the solu-
tion was warmed up to room temperature. Water (50 mL)
was then added, the aqueous layer was extracted with
Et2O (2ꢂ75 mL). The combined organic layers were washed
with NH4Cl(aq), brine, dried, filtered and concentrated under
vacuum to afford the crude aldehyde 20a/b (1.11 g, 100%)
as a yellow oil. This crude aldehyde was co-evaporated with
toluene (four times) in order to remove residual water and
used without any further purification. Formaldehyde
(3.1 mL of 37 wt % solution in water) and NaOH (7.0 mL
of 1.0 M aqueous solution) were added to a solution of crude
aldehyde (1.11 g, 2.12 mmol) in 1,4 dioxane (20 mL). The
resulting reaction mixture was kept under stirring at room
temperature for 48 h. After dilution with EtOAc (40 mL),
the reaction mixture was neutralised with hydrochloric
acid (7.0 mL of 1.0 M solution). The aqueous layer was
then extracted with EtOAc (50 mLꢂ2). Combined organic
layers were then washed with water (40 mL), NaHCO3
(40 mL) and brine (40 mL), dried, filtered and concentrated
under reduced pressure to afford the crude diol as an orange
oil. Purification by flash chromatography (75/25/50/50,
v/v, PE/ EtOAc) afforded the diol 22 (0.64 g, 53%) as a col-
ourless oil. nmax (liquid film) 3445 (br), 2929, 1722, 1043
4.1.6. Benzyl 3,4-di-O-benzyl-2-deoxy-D-glucopyranoside
19.20
A solution of aluminium trichloride (80 mg,
0.60 mmol) in dry Et2O (5 mL) was added to a solution of
benzyl-6-O-trityl-3,4-di-O-benzyl-2-deoxy-D-glucopyrano-
side 17 (400 mg, 0.60 mmol) in dry DCM (20 mL). The re-
action mixture turned bright yellow. After 25 min under
stirring the reaction mixture was quenched by the addition
of NaHCO3 (10 mL). After dilution with Et2O (40 mL),
the organic layer was washed with water (50 mL) and brine
(50 mL), then dried, filtered and concentrated under vacuum
to afford the crude sugar as an orange oil (370 mg). Purifica-
tion by flash chromatography (75/25/50/50, v/v, PE/
EtOAc) afforded three fractions of the pure detritylated
sugar 19 as a colourless oil: fraction 1: 98 mg, b-anomer;
fraction 2: 49 mg, mixture of anomers and fraction 3:
40 mg, a-anomer. b-Anomer: nmax (liquid film) 3464, 2922,
1491, 1365, 1211, 1059 cmꢁ1; [a]D20 ꢁ22.1 (c 0.021, CHCl3)
1
(br) cmꢁ1; [a]D20 ꢁ22.0 (c 0.03, CHCl3); H NMR (CDCl3)
d 7.45–7.25 (20H, m, Ar), 5.07 (1H, d, J 10.8 Hz, CH2–
Ph), 4.99 (1H, d, J 11.0 Hz, CH2–Ph), 4.96 (1H, d, J
4.0 Hz, H-1), 4.89 (1H, d, J 10.8 Hz, CH2–Ph), 4.85 (1H,
d, J 12.4 Hz, CH2–Ph), 4.69 (1H, d, J 11.0 Hz, CH2–Ph),
4.65 (1H, d, J 11.7 Hz, CH2–Ph), 4.64 (1H, d, J 12.4 Hz,
CH2–Ph), 4.52 (1H, d, J 11.7 Hz, CH2–Ph), 4.35 (1H, t, J
9.7 Hz, H-3), 4.16 (1H, dd, J 2.7, 12.0 Hz, H-6), 3.93 (1H,
d, J 9.6 Hz, H-4), 3.80–3.67 (3H, m, H-60, H-7, H-70), 3.53
(1H, dd, J 4.0, 9.9 Hz, H-2); 13C NMR (CDCl3) d 138.5
(Ar), 137.8 (Ar), 137.7 (Ar), 136.6 (Ar), 128.3 (Ar), 128.2
(Ar), 128.1 (Ar), 127.9 (Ar), 127.7 (2, Ar), 127.6 (2, Ar),
127.4 (Ar), 96.2 (C-1), 80.0 (C-2), 79.8 (C-4), 79.6 (C-5),
78.6 (C-3), 75.9 (O–CH2–Ph), 75.4 (O–CH2–Ph), 72.9
1
(lit.20b [a]2D0 ꢁ26.7 (c 1.1, CHCl3)); H NMR (CDCl3) d
7.38–7.29 (15H, m, Ar), 4.94 (1H, d, J 11.0 Hz, CH2–Ph),
4.86 (1H, d, J 11.9 Hz, CH2–Ph), 4.69 (1H, d, J 12.7 Hz,
CH2–Ph), 4.68 (1H, d, J 11.9 Hz, CH2–Ph), 4.67 (1H, d, J
11.0 Hz, CH2–Ph), 4.67 (1H, d, J 11.0 Hz, CH2–Ph), 4.60
(1H, d, J 12.7 Hz, CH2–Ph), 4.58 (1H, dd, J 1.9, 9.5 Hz,
H-1), 3.88 (1H, dd, J 2.8, 11.7 Hz, H-6), 3.74 (1H, dd, J
4.8, 11.8 Hz, H-60), 3.68 (1H, ddd, J 5.0, 8.7, 11.7 Hz, H-
3), 3.50 (1H, appt, J 9.1 Hz, H-4), 3.31 (1H, ddd, J 3.0,
4.8, 9.5 Hz, H-5), 2.38 (1H, ddd, J 1.9, 5.0, 12.6 Hz, H-2),
1.68 (1H, ddd, J 9.9, 11.8, 12.4 Hz, H-20); 13C NMR
(CDCl3) d 138.2 (Ar), 137.3, 128.4 (Ar), 128.1 (Ar), 127.9

10048
E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
(O–CH2–Ph), 70.2 (O–CH2–Ph), 64.5 (C-6/C7), 64.3 (C-6/
C-7); HRMS (ES) Calcd for C35H42O7N (M+NH+4)
588.2956 found 588.2953.
4.1, 13.2 Hz, H-20); 13C NMR (CDCl3) d 138.4 (Ar), 138.0
(Ar), 128.5 (Ar), 128.4 (Ar), 127.9 (Ar), 127.7 (Ar), 127.6
(Ar), 97.4 (C-1), 80.2 (2, C-5 and C-4), 75.9 (O–CH2–Ph),
74.1 (C-3), 71.9 (O–CH2–Ph), 70.2 (O–CH2–Ph), 65.0 (C-
6), 64.6 (C-7), 35.7 (C-2); HRMS (ES) Calcd for
C28H36O6N (M+NH+4) 482.2537 found 482.2541.
4.1.8. Benzyl 3,4-di-O-benzyl-5-C-(hydroxymethyl)-2-
deoxy-D-glucopyranoside 23a/b. To a solution of oxalyl
chloride (1.75 mL, 20.0 mmol) in dry DCM (20 mL) cooled
to ꢁ78 ꢀC was added DMSO (2.83 mL, 40.0 mmol) diluted
in dry DCM (20 mL) and the resulting solution was stirred
for 40 min. To the reaction mixture was then slowly added
a solution of benzyl 3,4-di-O-benzyl-2-deoxy-D-glucopyra-
noside 19 (2.22 g, 5.0 mmol) in dry DCM (20 mL). Stirring
was continued at ꢁ78 ꢀC for 2 h. Et3N (11.15 mL,
80.0 mmol) was added at ꢁ78 ꢀC and after 10 min the solu-
tion was warmed up to room temperature. Water (40 mL)
was then added, the aqueous layer was extracted with
Et2O (2ꢂ40 mL). The combined organic layers were washed
with NH4Cl(aq), brine, dried, filtered and concentrated under
vacuum to afford the crude aldehyde 21 (2.26 g, 98%) as
a yellow oil. This crude aldehyde was co-evaporated with
toluene (four times) in order to remove residual water and
used without any further purification. Formaldehyde
(0.73 mL of 37 wt % solution in water) and NaOH (1.5 mL
of 1.0 M aqueous solution) were added to a solution of crude
aldehyde (220 mg, 0.5 mmol) in 1,4 dioxane (10 mL). The
resulting reaction mixture was stirred at room temperature
for 48 h. After dilution with EtOAc (30 mL), the reaction
mixture was neutralised with hydrochloric acid (2.0 mL of
1.0 M solution). The aqueous layer was then extracted
with EtOAc (40 mLꢂ2). The combined organic layers
were then washed with water (20 mL), NaHCO3 (20 mL)
and brine (20 mL), dried, filtered and concentrated under re-
duced pressure to afford the crude diol as a yellowish oil. Pu-
rification by flash chromatography (75/25/50/50, v/v, PE/
EtOAc) afforded the diol 23 (100 mg, 43% overall) as a col-
ourless oil 40 mg first fraction (a-anomer) and 60 mg second
fraction (b-anomer). a-Anomer: nmax (liquid film) 3468 (br),
3018, 2934, 1216, 1026 (br), 756 cmꢁ1; [a]D20 +15.3 (c 0.018,
4.1.9. Benzyl 6-methylene-2,3,4-tri-O-benzyl-D-glucopyr-
anoside 24. To a solution of oxalyl chloride (1.48 mL,
16.93 mmol) in dry DCM (20 mL) cooled to ꢁ78 ꢀC was
added dimethylsulfoxide (2.4 mL, 33.86 mmol) diluted in
dry DCM (20 mL) and the resulting solution was stirred
for 40 min. To the reaction mixture was then added slowly
a solution of benzyl 2,3,4-tri-O-benzyl-D-glucopyranoside
(2.26 g, 4.23 mmol) in dry DCM (20 mL) and stirring was
continued at ꢁ78 ꢀC for 2 h. Et3N (9.4 mL, 67.73 mmol)
was then added still at ꢁ78 ꢀC and after 10 min reaction
mixture was then allowed to warm up to room temperature.
Water (30 mL) was added, the aqueous layer was extracted
with diethyl ether (2ꢂ40 mL), combined organic layers
were washed with NH4Cl(aq), brine, dried, filtered and con-
centrated under vacuum to afford the crude aldehyde as a yel-
low oil. This crude aldehyde was co-evaporated with toluene
(four times) to remove residual water and use without any
further purification.
n-BuLi (5.06 mL of a 2.5 M solution in hexane, 12.65 mmol)
was slowly added to a suspension of methyltriphenylphos-
phonium bromide (4.52 g, 12.65 mmol) in dry THF
(50 mL), cooled to ꢁ78 ꢀC. After 10 min under stirring at
ꢁ78 ꢀC the resulting orange solution was warmed up to
0 ꢀC and stirred for 1 h. The crude aldehyde diluted in dry
THF (50 mL) was then slowly added to the ylide still at
0 ꢀC. The reaction mixture was stirred overnight and allowed
to slowly warm up to room temperature. The reaction mixture
was quenched by the addition of NH4Cl(aq) (50 mL). The
aqueous layer was extracted with Et2O (2ꢂ40 mL). The com-
bined organic layers were dried, filtered and concentrated un-
der vacuum to afford the crude alkene. Purification by flash
chromatography (100/0/90/10, v/v, PE/EtOAc) gave the
alkene 24 (1.19 g, 53%) as a yellowish oil. b-Anomer: nmax
(liquid film) 3030, 2906 (br), 1951, 1874, 1809, 1725,
1605, 1216, 1068 (br), 756 cmꢁ1; [a]D20 ꢁ23.9 (c 0.021,
1
CHCl3); H NMR (CDCl3) d 7.36–7.26 (15H, m, Ar), 4.96
(1H, d, J 11.1 Hz, CH2–Ph), 4.91 (1H, dd, J 1.4, 9.1 Hz,
H-1), 4.83 (1H, d, J 11.9 Hz, CH2–Ph), 4.83 (1H, d, J
11.9 Hz, CH2–Ph), 4.68 (1H, d, J 11.7 Hz, CH2–Ph), 4.65
(1H, d, J 11.1 Hz, CH2–Ph), 4.60 (1H, d, J 11.3 Hz, CH2–
Ph), 4.59 (1H, d, J 11.3 Hz, CH2–Ph), 3.98–3.93 (1H, m,
H-3), 3.86–3.82 (1H, m, H-6), 3.83 (1H, d, J 8.2 Hz, H-4),
3.75–3.64 (3H, m, H-60, H-7 and H-70), 2.42–2.36 (1H, m,
H-2), 1.68 (1H, m, H-20); 13C NMR (CDCl3) d 138.1 (Ar),
138.0 (Ar), 137.4 (Ar), 128.5 (Ar), 127.9 (2, Ar), 127.7
(Ar), 127.6 (Ar), 95.3 (C-1), 80.0 (C-4), 78.2 (C-3), 77.2
(C-5), 76.0 (O–CH2–Ph), 71.5 (O–CH2–Ph), 70.8 (O–
CH2–Ph), 65.4 (C-6), 63.3 (C-7), 36.4 (C-2); HRMS (ES)
Calcd for C28H36O6N (M+NH+4) 482.2537 found 482.2541.
b-Anomer: nmax (liquid film) 3467 (br), 3016, 2932, 1214,
1028 (br), 755 cmꢁ1; [a]D20 ꢁ28.0 (c 0.02, CHCl3); 1H
NMR (CDCl3) d 7.38–7.28 (15H, m, Ar), 5.04 (1H, dd, J
1.5, 4.0 Hz, H-1), 5.00 (1H, d, J 11.1 Hz, CH2–Ph), 4.77
(1H, d, J 12.0 Hz, CH2–Ph), 4.69 (1H, d, J 11.4 Hz, CH2–
Ph), 4.67 (1H, d, J 11.1 Hz, CH2–Ph), 4.66 (1H, d, J
11.4 Hz, CH2–Ph), 4.49 (1H, d, J 12.0 Hz, CH2–Ph), 4.32
(1H, ddd, J 4.7, 9.3, 11.3 Hz, H-3), 4.10 (1H, dd, J 3.1,
12.0 Hz, H-6), 3.85 (1H, d, J 9.3 Hz, H-4), 3.77–3.68 (2H,
m, H-6 and H-7), 2.99 (1H, dd, J 3.2, 10.1 Hz, H-70), 2.34
(1H, ddd, J 1.5, 4.7, 13.2 Hz, H-2), 1.71 (1H, ddd, J 3.5,
1
CHCl3); H NMR (CDCl3) d 7.40–7.18 (20H, m, Ar), 5.98
(1H, ddd, J 5.9, 10.5, 17.1 Hz, H-6), 5.47 (1H, dt, J 1.5,
17.3 Hz, H-7), 5.30 (1H, dt, J 1.4, 10.4 Hz, H-70), 4.97 (1H,
d, J 11.9 Hz, CH2–Ph), 4.96 (1H, d, J 10.8 Hz, CH2–Ph),
4.90 (1H, d, J 10.9 Hz, CH2–Ph), 4.80 (1H, d, J 10.8 Hz,
CH2–Ph), 4.77 (1H, d, J 10.6 Hz, CH2–Ph), 4.73 (1H, d, J
10.9 Hz, CH2–Ph), 4.68 (1H, d, J 11.9 Hz, CH2–Ph), 4.62
(1H, d, J 10.6 Hz, CH2–Ph), 4.57 (1H, d, J 7.7 Hz, H-1),
3.79 (1H, appdd, J 6.0, 9.6 Hz, H-5), 3.65 (1H, appt, J
9.1 Hz, H-4), 3.52 (1H, dd, J 7.8, 9.2 Hz, H-2), 3.33 (1H,
appt, J 9.3 Hz, H-3); 13C NMR (CDCl3) d 138.6 (Ar),
138.4 (Ar), 138.0 (Ar), 137.4 (Ar), 134.6 (C-6), 128.4 (2,
Ar), 128.3 (2, Ar), 128.1 (Ar), 128.0 (Ar), 127.9 (2, Ar),
127.8 (Ar), 127.6 (2, Ar), 117.8 (C-7), 102.5 (C-1), 84.4
(C-4), 82.3 (C-3), 82.1 (C-2), 75.8 (CH2–Ph), 75.6 (CH2–
Ph), 75.1 (C-5), 74.9 (CH2–Ph), 71.2 (CH2–Ph); C35H36O5
Calcd C 78.33, H 6.76 found C 78.31, H 6.81.
4.1.10. Benzyl-6-methylene-3,4-di-O-benzyl-2-deoxy-D-
glucopyranoside 25. To a solution of oxalyl chloride

E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
10049
(1.75 mL, 20.0 mmol) in dry DCM (20 mL) cooled at
ꢁ78 ꢀC was added DMSO (2.83 mL, 40.0 mmol) diluted
in dry DCM (20 mL) and the resulting solution was stirred
for 40 min. To the reaction mixture was then slowly added
a solution of benzyl 3,4-di-O-benzyl-2-deoxy-D-glucopyra-
noside (2.22 g, 5.0 mmol) in dry DCM (20 mL). Stirring
was continued at ꢁ78 ꢀC for 2 h. Et3N (11.15 mL,
80.0 mmol) was added at ꢁ78 ꢀC and after 10 min the reac-
tion mixture was allowed to warm up to room temperature.
Water (40 mL) was then added, the aqueous layer was ex-
tracted with Et2O (2ꢂ40 mL). The combined organic layers
were washed with NH4Cl(aq), brine, dried, filtered and con-
centrated under vacuum to afford the crude aldehyde 21
(2.26 g, 98%) as a yellow oil. This crude aldehyde was co-
evaporated with toluene (four times) in order to remove
residual water and used without any further purification.
solutionꢂ2), water (20 mLꢂ2) and brine (20 mL), then
dried, filtered and concentrated under reduced pressure to af-
ford the crude diol 26 as a colourless oil 285 mg, 100%),
which turned into a waxy solid upon standing overnight.
nmax (liquid film) 3468, 3017, 2880 (br), 1454, 1216, 1068
1
(br), 756 cmꢁ1; [a]D20 +2.36 (c 0.023, CHCl3); H NMR
(CDCl3) d 7.40–7.26 (20H, m), 5.06 (1H, d, J 10.8 Hz,
CH2–Ph), 5.01 (1H, d, J 10.9 Hz, CH2–Ph), 4.80 (1H, d, J
10.8 Hz, CH2–Ph), 4.77 (1H, d, J 3.9 Hz, H-1), 4.75 (1H,
d, J 10.9 Hz, CH2–Ph), 4.66 (1H, d, J 12.0 Hz, CH2–Ph),
4.64 (1H, d, J 11.9 Hz, CH2–Ph), 4.54 (1H, d, J 11.9 Hz,
CH2–Ph), 4.53 (1H, d, J 12.0 Hz, CH2–Ph), 4.10 (1H, t, J
9.6, H-3), 3.86 (1H, dd, J 5.4, 9.7 Hz, H-5), 3.80 (1H, dd,
J 4.4, 9.3 Hz, H-6), 3.66 (1H, dd, J 4.4, 11.6 Hz, H-7),
3.58 (1H, dd, J 4.1, 7.4 Hz, H-70), 3.55 (1H, t, J 9.6 Hz, H-4),
3.50 (1H, dd, J 3.7, 9.6 Hz, H-2); 13C NMR (CDCl3) d 138.4,
137.9, 137.3, 136.8, 128.7, 128.5, 128.4, 128.2, 128.0, 127.9,
127.7, 94.7 (C-1), 82.2 (C-3), 80.1 (C-5 or C-2), 80.0 (C-5 or
C-2), 75.6 (CH2–Ph), 75.0 (CH2–Ph), 72.9 (CH2–Ph), 72.7
(C-6), 70.1 (C-7), 69.1 (CH2–Ph), 62.9 (C-4); Calcd C
73.66, H 6.71 found C 73.75, H 6.87; LRMS (ES)
(M+Na+) Calcd 593.0 found 593.1; HRMS (ES) Calcd for
C35H42O7N (M+NH+4) 588.2956 found 588.2951.
n-BuLi (4.0 mL of a 2.5 M solution in Hex, 10.0 mmol) was
slowly added to a suspension of methyltriphenylphospho-
nium bromide (3.57 g, 10.0 mmol) in dry THF (20 mL),
cooled to ꢁ78 ꢀC. After 10 min under stirring at ꢁ78 ꢀC
the resulting orange solution was warmed up to 0 ꢀC and
stirred for 1 h. The crude aldehyde (0.76 g, 3.33 mmol)
diluted in dry THF (20 mL) was then slowly added to the
ylide solution still at 0 ꢀC. The reaction mixture was allowed
to slowly warm up to room temperature and kept under
stirring overnight. The reaction mixture was quenched
with NH4Cl(aq) (50 mL); the aqueous layer was extracted
with Et2O (2ꢂ50 mL), combined organic layers were
washed with NH4Cl (50 mL), water (50 mL) and brine
(50 mL), dried, filtered and concentrated under reduced
pressure to afford the crude alkene. Purification by flash
chromatography (75/25, v/v, PE/EtOAc) yielded the alkene
25 (0.63 g, 44%) as a yellowish oil. nmax (liquid film)
3033, 2912 (br), 1955, 1878, 1809, 1723, 1605, 1219,
4.1.12. Benzyl 3,4-di-O-benzyl-glycero-a-D-2-deoxy-
gluco-heptopyranoside 27. A small crystal of osmium
tetroxide was added to a solution of alkene 25 (200 mg,
0.46 mmol) and N-methylmorpholine N-oxide (126 mg,
0.93 mmol) in DCM (20 mL). The resulting solution was
stirred overnight. The reaction mixture was then diluted
with DCM (20 mL) and sodium metabisulfite (20 mL of
a 10% solution) was added. The resulting biphasic solution
was then kept under stirring for 10 min. The aqueous layer
was then extracted with DCM (25 mLꢂ3). The combined
organic layers were then washed with sodium metabisulfite
(20 mL of 10% solutionꢂ2), water (20 mLꢂ2) and brine
(20 mL), then dried, filtered and concentrated under reduced
pressure to afford the crude diol 27 as a colourless oil
(200 mg, 98%), which turned into a waxy solid upon stand-
ing overnight. nmax (liquid film) 3465, 3018, 2875 (br), 1458,
1
1058 (br), 756 cmꢁ1; H NMR (CDCl3) d 7.35–7.25 (15H,
m, Ar), 6.00 (1H, ddd, J 6.3, 10.5, 17.0 Hz, H-6), 5.42
(1H, dt, J 1.5, 10.5 Hz, H-7), 5.27 (1H, dt, J 1.3, 17.3 Hz,
H-70), 5.02 (1H, d, J 2.9 Hz, H-1), 4.84 (1H, d, J 10.7 Hz,
CH2–Ph), 4.67–4.64 (3H, m, CH2–Phꢂ3), 4.44 (1H, d, J
11.9 Hz, CH2–Ph), 4.16 (1H, dd, J 6.5, 9.6 Hz, H-5), 4.04
(1H, ddd, J 5.1, 8.8, 11.4 Hz, H-3), 3.27 (1H, appt, J
9.2 Hz, H-4), 2.33 (1H, ddd, J 1.2, 5.1, 13.0 Hz, H-2), 1.73
(1H, ddd, J 3.7, 11.5, 13.0 Hz, H-20); 13C NMR (CDCl3)
d 139.4 (Ar), 138.7(Ar), 137.6 (Ar), 135.7 (C-6), 128.3 (2)
(Ar), 128.0 (Ar), 127.9 (Ar), 127.6 (Ar), 127.5(Ar), 117.7
(C-7), 96.5 (C-1), 82.8 (C-4), 77.0 (O–CH2–Ph), 75.1 (O–
CH2–Ph), 72.2 (C-5), 72.1 (C-3), 68.8 (O–CH2–Ph), 35.7
(C-2); Calcd C 78.11, H 7.02 found C 78.06, H 7.06;
HRMS (ES) Calcd for C28H34O4N (M+NH+4) 448.2482
found 448.2468.
1
1216, 1063 (br), 758 cmꢁ1; H NMR (CDCl3) d 7.29–7.21
(15H, m, Ar), 4.99 (0.75H, d, J 11.0 Hz), 4.97 (0.25H, d, J
11.0 Hz), 4.90 (0.75H, d, J 3.1 Hz), 4.73 (0.25H, d,
J 11.9 Hz), 4.64 (0.75H, d, J 11.6 Hz), 4.62 (0.75H, d,
J 11.0 Hz), 4.59 (0.75H, d, J 11.4 Hz), 4.51 (0.75H, d, J
11.4 Hz), 4.50–4.38 (1.25H, m), 4.35 (1H, d, J 11.8 Hz),
4.05–4.00 (1H, m), 3.85–3.74 (2.25H, m), 3.65 (0.75H, dd,
J 4.6, 11.6 Hz), 3.69–3.62 (0.25H, m), 3.57 (0.75H,
dd, J 3.8, 11.6 Hz), 3.58–3.54 (0.25H, m), 3.48 (0.75H,
appt, J 9.1 Hz), 3.46 (0.25H, appt, J 8.9 Hz), 3.30 (0.25H,
dd, J 5.4, 9.6 Hz), 3.13 (0.75H, m), 2.32 (0.25H, ddd, J
1.8, 5.0, 13.0 Hz), 2.28 (0.75H, dd, J 5.0, 13.1 Hz), 1.60
(1H, app ddd, J 3.5, 11.4, 12.9 Hz); 13C NMR (CDCl3)
d 138.2, 137.9, 137.6, 137.5, 137.3, 137.2, 128.6, 128.5,
128.4 (2), 128.3 (2), 128.1 (2), 128.0, 127.9 (2), 127.8 (2),
127.7 (2), 127.6, 99.1, 96.1, 80.7, 80.2, 79.6, 77.9, 74.9
(2), 74.8, 72.9, 72.4, 71.4, 71.2, 70.9, 70.6, 68.9, 63.8,
63.0, 62.8, 61.2, 54.1, 53.9, 36.5, 35.2; HRMS (ES) Calcd
for C28H36O6N (M+NH+4) 482.2537 found 482.2551.
4.1.11. Benzyl 2,3,4-tri-O-benzyl-glycero-a-D-gluco-hep-
topyranoside 26. A small crystal of osmium tetroxide was
added to a solution of alkene 24 (240 mg, 0.45 mmol) and
N-methylmorpholine N-oxide (121 mg, 0.90 mmol) in
DCM (20 mL). The resulting solution was kept stirring over-
night. The reaction mixture was then diluted with DCM
(20 mL) and sodium metabisulfite (20 mL of a 10% solu-
tion) was added. The resulting biphasic solution was then
stirred for 10 min. The aqueous layer was then extracted
with DCM (25 mLꢂ3). The combined organic layers were
then washed with sodium metabisulfite (20 mL of 10%
4.1.13. Benzyloxydichlorophosphine. To a solution of
phosphorus trichloride (6.1 mL, 70 mmol) in dry CH3CN
(25 mL) was added benzyl alcohol (1.0 mL, 10 mmol)

10050
E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
diluted in dry CH3CN (25 mL). The resulting reaction solu-
tion was stirred for 1 h. The crude reaction mixture was con-
centrated under reduced pressure to afford the crude
benzyloxydichlorophosphine as a colourless oil (2.05 g,
added. The resulting reaction mixture was then kept under
stirring for 30 min. The solution was then concentrated un-
der reduced pressure to afford the crude cyclic phosphate
triester as a yellowish oil. The residue was then purified
through a short silica column (75/25/0/100, v/v, PE/
EtOAc) to afford the pure cyclic phosphate triester 29 as col-
1
98%). H NMR (CDCl3) d 7.39 (5H, br), 5.25 (2H, d, J
8.0 Hz); 13C NMR (CDCl3) d 134.8 (d, JPOCC 7.6 Hz),
126.7, 126.3, 125.7, 63.2 (d, JPOC 5.4 Hz), 43.4 (d, JPOC
6.3 Hz), 21.1 (d, JPNCC 2.0 Hz); 31P (CDCl3) d 178.5.
1
ourless oil (54 mg, 71%). H NMR (CDCl3) d 7.45–7.29
(20H, m, Ar), 5.20–4.23 (13H, m), 2.45 (1H, m), 1.93–1.78
(1H, m); 13C NMR (CDCl3) d 137.9, 137.6, 137.4, 137.3,
137.1 (2), 135.6, 128.6 (2), 128.5, 128.4 (2), 128.3, 128.1,
128.0, 127.9, 127.8, 127.7, 127.6, 97.0, 95.9, 79.0, 75.4,
74.9, 74.7, 74.5 (d, JPOCC 7.6 Hz), 73.9, 73.8, 73.7, 72.6 (d,
JPOCC 6.5 Hz), 71.7, 71.6 (d, JPOC 7.3 Hz), 71.3 (d, JPOC
6.4 Hz), 70.3, 69.9 (d, JPOC 5.7 Hz), 68.7 (d, JPOC 5.2 Hz),
35.5, 33.8; 31P NMR (CDCl3) d ꢁ4.0, ꢁ7.1; HRMS (FABꢁ)
Calcd for C35H37O8P (M+) 617.2304 found 617.2301.
4.1.14. (3R,4S,5S)-2,3,4,5,9-Pentakis-benzyloxy-1,8,10-
trioxa-9-phospha-spiro[5.5]undecane 9-oxide 28. To a so-
lution of diol 22 (200 mg, 0.35 mmol) in dry DCM (5 mL)
under stirring was added Et3N (195 mL, 1.4 mmol). The re-
sulting solution was stirred for 5 min before the addition of
benzyloxydichlorophosphine (80 mg, 0.38 mmol; freshly
prepared) diluted in dry DCM (5 mL). The resulting reaction
mixture was then kept stirring until TLC indicated the
complete conversion of the starting material into a more
lipophilic material (75/25, EtOAc/PE, v/v). The reaction
mixture was then concentrated under reduced pressure to af-
ford the crude cyclic phosphite as a colourless oil. The re-
sulting residue was passed through a short silica pad (75/
25, EtOAc/PE, v/v) to afford the pure cyclic phosphite as
a colourless oil. The purity of the cyclic phosphite was
checked by 31P NMR (mixture of diastereoisomers). The cy-
clic phosphite was then rediluted in dry DCM (10 mL), and
tert-butylhydroperoxide (230 mL of a 3.0 M solution in tol-
uene, 0.70 mmol) was added. The resulting reaction mixture
was then kept under stirring for 30 min. The solution was
then concentrated under reduced pressure to afford the crude
cyclic phosphate triester as a yellowish oil. The residue was
then purified through a short silica pad (75/25/0/100, v/v,
PE/EtOAc) to afford the pure cyclic phosphate triester 28 as
colourless oil (180 mg, 71%). 1H NMR (CDCl3) d 7.45–7.24
(25H, m, Ar), 5.20–4.23 (14H, m), 4.06–3.32 (4H, m); 13C
NMR (CDCl3) d 137.8, 137.6, 136.9, 136.8, 136.7, 133.0,
129.9, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0 (2),
127.9 (2), 127.8, 127.7, 127.3, 126.8, 98.1, 97.3, 82.2, 81.5,
81.4, 80.7, 78.0, 75.5, 75.4, 75.0, 74.7, 74.5, 74.1, 73.9 (d,
JPOCC 7.3 Hz), 73.3 (d, JPOCC 6.6 Hz), 72.8 (d, JPOC
6.2 Hz), 71.9 (d, JPOC 6.2 Hz), 71.0, 70.9, 70.1 (d, JPOC
5.4 Hz), 68.9 (d, JPOC 5.1 Hz), 66.9 (d, JPOC 5.4 Hz), 66.3
(d, JPOC 6.7 Hz); 31P NMR (CDCl3) d ꢁ4.3, ꢁ7.3; HRMS
(FABꢁ) Calcd for C42H43O9P (M+)722.2723found 722.2702.
4.1.16. (3R,4S,5S,6S)-2,3,4,5-Tetrakis-benzyloxy-6-(2-
benzyloxy-2-oxo-2-l-5-[1,3,2]dioxaphospholan-4-yl)-tet-
rahydropyran 30. 2,4-Dinitrophenol (26 mg, 0.140 mmol)
diluted in dry DCM (5 mL) was added to a solution of diol
26 (54 mg, 0.093 mmol) and benzyloxy-bis(diisopropylami-
no)phosphine (34 mg, 0.102 mmol) in dry DCM (5 mL),
under stirring. The resulting reaction mixture was kept under
stirring until only one peak corresponding to the cyclic
phosphite was observed by 31P NMR. The reaction mixture
was cooled down to ꢁ78 ꢀC prior to the addition of tert-
butylhydroperoxide (37 mL of 5.0 M solution in decane,
0.186 mmol). After 10 min stirring at ꢁ78 ꢀC, the reaction
was allowed to warm up to room temperature and sodium
thiosulfate (5 mL of 10% aqueous solution) was added.
The reaction mixture was then diluted with Et2O (20 mL).
The organic layer was washed with sodium thiosulfate
(15 mL), water (15 mLꢂ2), dried, filtered and concentrated
under reduced pressure to afford the crude cyclic phosphate
triester 30 as a yellow oil. Purification by reversed-phase
chromatography (C18-RP Supelcosyl HPLC column;
CH3CN/water) gave the pure cyclic phosphate triester
(31 mg, 46%) as a colourless oil. 1H NMR (CDCl3)
d 7.38–7.26 (25H, m, Ar), 5.12 (2H, d, JPOC 4.9 Hz, CH2–
Phꢂ2), 5.04 (1H, d, J 10.9 Hz, CH2–Ph), 5.00 (1H, d, J
8.1 Hz, H-1), 4.84 (1H, d, J 12.0 Hz, CH2–Ph), 4.81 (1H,
d, J 11.0 Hz, CH2–Ph), 4.74 (1H, d, J 12.1 Hz, CH2–Ph),
4.70 (1H, s, CH2–Ph), 4.61 (1H, d, J 13.6 Hz, CH2–Ph),
4.70–4.50 (1H, m), 4.58 (1H, d, J 11.7 Hz, CH2–Ph), 4.50
(1H, d, J 11.9 Hz, CH2–Ph), 4.12 (1H, dd, J 7.2, 14.3 Hz,
H-4), 4.07 (1H, dd, J 8.7, 9.4 Hz, H-2), 3.98 (1H, dd, J
1.7, 10.5 Hz, H-7), 3.50 (1H, ddd, J 7.6, 8.0, 15.6 Hz, H-
5), 3.47 (1H, dd, J 3.7, 9.6 Hz, H-3), 3.20 (1H, dd, J 8.6,
10.4 Hz, H-70); 13C NMR (CDCl3) d 138.5, 137.8, 137.3,
136.7, 135.8, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4,
128.3, 128.0, 127.9, 127.8, 127.7, 127.6, 127.0, 94.2, 82.4,
79.7, 76.3, 75.8, 75.5, 74.4, 72.7, 70.1, 69.5, 69.3, 68.8;
31P NMR (CDCl3) d 17.4; HRMS (FABꢁ) Calcd for
C42H44O9P (M+H+) 723.2723 found 723.2702.
4.1.15. (4R,5S)-2,4,5,9-Tetrakis-benzyloxy-1,8,10-trioxa-
9-phospha-spiro[5.5]undecane 9-oxide 29. To a stirred so-
lution of diol 23 (55 mg, 0.108 mmol) in dry DCM (5 mL)
was added Et3N (60 mL, 0.43 mmol). The resulting solution
was stirred for 5 min before the addition of benzyloxydi-
chlorophosphine (25 mg, 0.12 mmol; freshly prepared) di-
luted in dry DCM (5 mL). The resulting reaction mixture
was then kept stirring until TLC indicated the complete con-
version of the starting material into a more lipophilic mate-
rial (75/25, EtOAc/PE, v/v). The reaction mixture was then
concentrated under reduced pressure to afford the crude cy-
clic phosphite as a colourless oil. The resulting residue was
passed through a short silica column (75/25, EtOAc/PE, v/v)
to afford the pure cyclic phosphite as a colourless oil. The
purity of the cyclic phosphite was checked by 31P NMR
(mixture of diastereoisomers). The cyclic phosphite was
then rediluted in dry DCM (10 mL), and tert-butylhydroper-
oxide (74 mL of a 3.0 M solution in toluene, 0.22 mmol) was
4.1.17. (2S,3S,4R)-3,4,6-Tris-benzyloxy-2-(2-benzyloxy-
2-oxo-2-l-5-[1,3,2]dioxaphospholan-4-yl)-tetrahydro-
pyran 31. To a solution of diol 27 (50 mg, 0.12 mmol) in dry
DCM (5 mL) under stirring was added Et3N (75 mL,
0.48 mmol). The resulting solution was stirred for 5 min be-
fore the addition of benzyloxydichlorophosphine (28 mg,
0.13 mmol; freshly prepared) diluted in dry DCM (5 mL).

E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
10051
The resulting reaction mixture was then stirred until TLC in-
dicated the complete conversion of the starting material into
a more lipophilic material (75/25, EtOAc/PE, v/v). The reac-
tion mixture was then concentrated under reduced pressure
to afford the crude cyclic phosphite as a colourless oil.
The resulting residue was passed through a short silica col-
umn (75/25, EtOAc/PE, v/v) to afford the pure cyclic phos-
phite as a colourless oil. The purity of the cyclic phosphite
was checked by 31P NMR (mixture of diastereoisomers).
The cyclic phosphite was then rediluted in dry DCM
(10 mL), and tert-butylhydroperoxide (74 mL of a 3.0 M so-
lution in toluene, 0.22 mmol) was added. The resulting reac-
tion mixture was then kept stirring for 30 min. The solution
was then concentrated under reduced pressure to afford the
crude cyclic phosphate triester as a yellowish oil. The resi-
due was then purified through a short silica column (75/
25/0/100, v/v, PE/EtOAc) to afford the pure cyclic phos-
(O–CH2–Ph), 69.1 (C-5), 60.9 (C-6), 33.9 (C-7); HRMS
(FAB+) Calcd for C35H38O6 (M+) 554.2668 found 554.2669
4.1.19. Phosphoric acid dibenzyl ester 2-((2R,3R,4S,5R)-
3,4,5,6-tetrakis-benzyloxy-tetrahydro-pyran-2-yl)-ethyl
ester (benzyl 6-deoxy-2,3,4-tri-O-benzyl-D-gluco-hepto-
pyranoside 7-dibenzylphosphate) 36. t-BuLi (423 mL of
a 1.7 M solution in hexanes, 0.72 mmol) was added to a
solution of benzyl 6-deoxy-2,3,4-tri-O-benzyl-D-gluco-
heptopyranoside (200 mg, 0.36 mmol) diluted in THF
(15 mL) cooled down at ꢁ78 ꢀC. The resulting solution
was stirred at ꢁ78 ꢀC for 10 min. A solution of TBBP
(554 mg, 0.72 mmol) in THF (10 mL) was then added to
the reaction mixture at ꢁ78 ꢀC. The reaction mixture was
stirred for 1 h at ꢁ78 ꢀC, before being allowed to warm up
to room temperature. After 3 h under stirring at room tem-
perature, the reaction mixture was then diluted with CHCl3
(30 mL) and NH4Cl(aq) (30 mL) was then slowly added.
The aqueous layer was extracted with CHCl3 (30 mLꢂ2).
The combined organic layers were then washed with
NH4Cl(aq) (50 mL), water (50 mL) and brine (50 mL), dried,
filtered and concentrated under reduced pressure to afford
the crude phosphorylated sugar. Purification by flash chro-
matography (75/25/55/45, v/v, PE/EtOAc) afforded pure
1
phate triester 31 as colourless oil (54 mg, 71%). H NMR
(CDCl3) d 7.45–7.29 (20H, m, Ar), 5.17–4.98 (10H, m),
4.23–4.17 (1H, m), 4.10–3.98 (2H,m), 3.60–3.54 (1H, m),
3.20–3.07 (1H), 2.36–2.30 (1H, m), 1.68–1.59 (1H, m); 13C
NMR (CDCl3) d 138.3, 138.2, 137.5, 137.3 (2), 137.0,
128.8 (2), 128.7, 128.6, 128.5, 128.4, 128.2, 128.0, 127.8
(2), 127.7, 127.6, 96.0, 95.8, 78.0, 77.9, 75.8, 75.7, 74.3
(2), 71.7 (d, JPOCC 7.7 Hz), 70.3 (d, JPOCC 5.5 Hz), 70.1 (d,
JPOC 6.0 Hz), 70.0 (d, JPOC 5.5 Hz), 69.5 (d, JPOC 5.9 Hz),
68.8, 65.0, 64.9, 35.1, 35.0; 31P NMR (CDCl3) d 18.23,
18.8; HRMS (ES) Calcd for C35H38O8P (M+H+) 617.2304
found 617.2310.
1
36 as a colourless oil (138 mg, 47%). H NMR (CDCl3)
d 7.52–7.34 (30H, m, Ar), 5.18 (0.3, d, J 7.9 Hz), 5.12
(0.7H, d, J 10.5 Hz), 5.11–5.07 (0.6H, m), 5.06 (1H, d, J
12.0 Hz, CH2–Ph), 4.99 (1H, d, J 11.4 Hz, CH2–Ph), 4.92
(0.7H, d, J 10.8 Hz, CH2–Ph), 4.89 (0.7H, d, J 10.9 Hz,
CH2–Ph), 4.86 (0.7H, d, J 3.6 Hz), 4.83 (0.7H, d, J
10.8 Hz, CH2–Ph), 4.81–4.51 (6.3H, m), 4.14 (0.7H, appt,
J 9.3 Hz), 4.02 (0.3H, dt, J 2.8, 9.6 Hz), 3.87 (1H, appt, J
5.8 Hz), 3.81–3.79 (1H, m), 3.74 (0.3H, appt, J 9.0 Hz),
3.44 (0.3H, appt, J 9.2 Hz), 3.36 (0.7H, appt, J 9.3 Hz),
1.86–1.72 (2H, m); 13C NMR (CDCl3) d 138.7 (Ar), 138.4
(Ar), 138.3 (Ar), 138.1 (Ar), 138.0 (2, Ar), 137.9 (Ar),
137.1 (Ar), 136.8 (Ar), 135.8 (2, Ar), 128.7 (Ar), 128.5
(Ar), 128.4 (2, Ar), 128.1 (2, Ar), 128.0 (2, Ar), 127.9
(Ar), 127.8 (3, Ar), 127.7 (Ar), 127.6 (2, Ar), 102.7, 94.9,
84.6, 82.3, 81.8, 81.6, 81.3, 80.0, 75.7, 75.3, 75.2, 74.9,
73.9, 73.0, 71.4, 70.4, 69.2, 69.0, 60.8, 60.3, 34.0, 33.9;
31P NMR (CDCl3) d 0.6, 0.5. MS (ES) Calcd for
C49H51O9P (M+Na+) 838; (Mꢁ261+)(phosphate) 577.
4.1.18. Benzyl 6-deoxy-2,3,4-tri-O-benzyl-D-gluco-hepto-
pyranoside. To a solution of alkene 24 (536 mg, 1 mmol)
in dry THF (10 mL), was added a solution of 9-BBN
(10 mL of 0.5 M solution in THF, 5 mmol). The resulting so-
lution was heated under reflux for 5 h. The reaction mixture
was then allowed to cool down to room temperature, and then
cooled down to 0 ꢀC. NaOH (12 mL of a 10% solution,
30 mmol) and followed by H2O2 (12 mL of a 30% solution,
105 mmol) were added slowly (exothermic reaction). The re-
sulting biphasic reaction mixture was stirred for 30 min, then
diluted with CHCl3 (50 mL). The aqueous layer was then ex-
tracted with CHCl3 (25 mLꢂ2). Combined organic layers
were washed with sodium metabisulfite solution (40 mL
of 10% solution), water (40 mL), and brine (40 mL),
then dried, filtered and concentrated under reduced pressure
to afford the crude heptose as a yellowish oil (950 mg). Puri-
fication by flash chromatography (85/15/60/40, v/v, PE/
EtOAc) afforded the alcohol (505 mg, 91%) as a colourless
oil. nmax (liquid film) 3465, 3012, 2880 (br), 1454, 1214,
4.1.20. 2-Benzyloxy-2-((1S,2R,3S)-1,2,3,4-tetrakis-ben-
zyloxy-butyl)-propane-1,3-diol 32. An extensive synthetic
sequence starting from D-glucose was developed to access
this glucitol derivative. However, only the full characterisa-
1
tion of this compound is provided here. H NMR (CDCl3)
1
1068 (br), 756 cmꢁ1; H NMR (CDCl3) d 7.40–7.23 (20,
d 7.23–7.14 (25H, m, Ar), 4.66 (1H, d, J 11.1, CH2–Ph),
4.64 (1H, d, J 11.6, CH2–Ph), 4.58 (2H, s, H-6/H-60 or H-
7/H-70), 4.55 (1H, d, J 11.3, CH2–Ph), 4.53 (1H, d, J 11.4,
CH2–Ph), 4.49 (2H, d, J 11.8, CH2–Ph), 4.28 (2H, s, H-6/
H-60 or H-7/H-70), 3.95 (1H, appt, J 4.5, H-3), 3.89 (1H, d,
J 4.2, H-4), 3.82 (1H, appdd, J 4.9, 10.5, H-2), 3.80 (1H,
d, J 12.3, CH2–Ph), 3.76 (1H, d, J 12.4, CH2–Ph), 3.70
(1H, d, J 12.4, CH2–Ph), 3.68 (1H, d, J 12.1, CH2–Ph),
3.59 (1H, dd, J 4.2, 10.3, H-1), 3.50 (1H, dd, J 5.7, 10.2,
H-10); 13C NMR (CDCl3) d 138.6 (Ar), 138.4 (Ar), 138.4
(Ar), 138.1 (Ar), 137.9, 128.3 (2, Ar), 127.9 (Ar), 127.6
(2, Ar), 127.5 (Ar), 80.8, 79.4, 78.5, 76.6, 74.9, 74.0, 73.1,
73.0, 70.6, 64.8, 62.8, 62.3; LRMS (FAB) Calcd for
C42H46O7 (M+Na+) 685.0 found 685.0.
m, Ar), 4.99 (1H, d, J 10.8 Hz, CH2–Ph), 4.90 (1H, d, J
11.0 Hz, CH2–Ph), 4.80 (1H, d, J 10.8 Hz), 4.75 (1H, d,
J 3.7 Hz, H-1), 4.70 (1H, d, J 12.3 Hz), 4.66 (1H, d, J
11.9 Hz, CH2–Ph), 4.59 (1H, d, J 11.0 Hz, CH2–Ph), 4.54
(1H, d, J 11.9 Hz, CH2–Ph), 4.51 (1H, d, J 12.3 Hz, CH2–
Ph), 4.02 (1H, appt, J 9.2 Hz, H-3), 3.90 (1H, dt, J 2.9,
9.5 Hz, H-5), 3.71–3.65 (2H, m, H-7, H-70), 3.50 (1H, dd, J
3.7, 9.5 Hz, H-2), 3.25 (1H, appt, J 9.3 Hz, H-4), 2.07–2.00
(1H, m, H6), 1.66–1.60 (1H, m, H-60); 13C NMR (CDCl3)
d 138.7 (Ar), 138.1(Ar), 136.9 (Ar), 128.5 (Ar), 128.4 (Ar),
128.3 (Ar), 128.0 (Ar), 127.9 (2, Ar), 127.8 (2, Ar), 127.6
(Ar), 95.1 (C-1), 81.9 (C-3), 81.7 (C-4), 80.0 (C-2), 75.7
(O–CH2–Ph), 75.3 (O–CH2–Ph), 73.0 (O–CH2–Ph), 70.5

10052
E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
4.1.21. Benzyl 6-dihydroxymethylene-1,2,3,4,5-penta-O-
benzyl-D-glucitol 33. An extensive synthetic sequence
from D-glucose was developed to access this glucitol deriva-
tive. However, only the full characterisation of this com-
pound is provided here. nmax (liquid film) 3463, 3013, 2880
J 5.4), 4.50–3.81 (4H, m), 3.21–3.02 (2H, m), 2.16–1.97
(2H, m); 13C NMR (D2O) d 106.2, 105.6, 82.9, 80.2, 72.8,
72.6, 71.8, 71.5, 71.4, 56.5, 56.4, 50.2, 43.4, 42.4; 31P
NMR (D2O) d ꢁ1.8 (br s); LRMS (ES) Calcd for
C7H13O8P (Mꢁ1) 255.1 found 255.0; HRMS (ES) Calcd
for C7H12O8P (MꢁHꢁ) 255.0270 found 255.0265.
1
(br), 1454, 1217, 1063 (br), 756 cmꢁ1; H NMR (CDCl3)
d 7.33–7.25 (23H, m, Ar), 7.20–7.18 (2H, m, Ar), 4.76 (1H,
d, J 11.6 Hz, CH2–Ph), 4.75 (1H, d, J 11.3 Hz, CH2–Ph), 4.71
(1H, d, J 11.3 Hz, CH2–Ph), 4.66 (1H, d, J 11.3 Hz, CH2–Ph),
4.65 (1H, d, J 11.3 Hz, CH2–Ph), 4.56 (1H, d, J 11.6 Hz,
CH2–Ph), 4.55 (1H, d, J 11.5 Hz, CH2–Ph), 4.51 (2H, d, J
2.4, 12.0 Hz, CH2–Ph), 4.37 (1H, d, J 11.5 Hz, CH2–Ph),
4.08 (1H, dd, J 2.6, 5.8 Hz, H-7), 3.90–3.84 (3H, m, H-3,
H-4 and H-5), 3.75 (1H, dd, J 3.8, 11.3 Hz, H-2), 3.69 (1H,
dd, J 2.6, 7.2 Hz, H-70), 3.67–3.63 (2H,m, H-10 and H-6),
3.57 (1H, dd, J 5.5, 10.3 Hz, H-1); 13C NMR (CDCl3)
d 138.2 (2), 138.1 (2), 138.0, 128.4, 128.3 (2), 128.1,
128.0, 127.8, 127.7 (2), 81.1, 80.0, 79.6, 78.5, 7.8, 74.6,
73.3, 72.9, 72.7, 71.1, 69.9, 63.9; HRMS (ES) Calcd for
C42H47O7 (M+H+) 663.3316 found 663.3324.
4.1.24. (3R,4S,5S,6S)-6-(2-Hydroxy-2-oxo-2-l-5-[1,3,2]-
dioxaphospholan-4-yl)-tetrahydro-pyran-2,3,4,5-tetraol
13. Sodium metal (100 mg) was added to a solution of cyclic
phosphate triester 30 (60 mg, 0.083 mmol) in liquid ammo-
nia. The resulting reaction mixture was stirred at ꢁ78 ꢀC for
2 h. MeOH (10 mL) and NH4Cl (350 mg) were then sequen-
tially added. The resulting reaction mixture was warmed up
to room temperature overnight to allow evaporation of the
ammonia. The crude reaction mixture was then concentrated
under reduced pressure to afford the crude cyclic phosphate
diester 13 (mixture of a and b-anomers) (450 mg, heavily
contaminated with salts). Attempts to desalt the title com-
1
pound proved unsuccessful, hence the lack of H NMR
data. 31P NMR (D2O) d 19.4, 19.3; LRMS (ES) Calcd for
C7H13O9P (Mꢁ1) 271.1 found 271.0; HRMS (ES) Calcd
for C7H12O9P (MꢁHꢁ) 271.0219 found 271.0213.
4.1.22. (3R,4S,5S)-9-Oxo-1,8,10-trioxa-9-l-5-phospha-
spiro[5.5]undecane-2,3,4,5,9-pentaol 9. A suspension of
Pd/C (50 mg, 10% Pd/C), potassium carbonate (5.5 mg,
38.7 mmol) and cyclic phosphate triester 28 (55 mg,
77.5 mmol) in MeOH (10 mL) was stirred overnight under
a positive pressure of H2 (balloon). The reaction mixture
was then filtered and concentrated under reduced pressure.
31P and 1H NMR spectroscopic analyses (D2O) showed par-
tial deprotection. The crude reaction mixture was resus-
pended in MeOH (10 mL). More catalyst (50 mg, Pd/C
10%) and K2CO3 were added and the resulting reaction mix-
ture was then stirred overnight under a positive pressure of
H2 (balloon). This cycle was repeated until NMR analysis
showed complete removal of the benzyl protecting group.
Three cycles were needed to fully deprotect 28. The crude
9 was then filtrated and lyophilised to afford 9 as an hydro-
scopic powder (21.0 mg, 98%). 1H NMR (D2O) d 4.76–4.73
(1H, m), 4.40–4.25 (4H, m), 3.82–3.70 (1H, m), 3.41–3.34
(1H, m), 3.19–3.06 (1H, m); 13C NMR (D2O) d 93.2, 92.1,
75.8, 75.0, 74.1, 74.0, 72.8, 72.7, 72.1, 71.7, 69.3 (2), 64.1
(2); 31P NMR (D2O) d ꢁ0.9 (s), ꢁ2.2 (s); LRMS (ES) Calcd
for C7H13O9P (Mꢁ1) 271.1 found 271.0; HRMS (ES) Calcd
for C7H12O9P (MꢁHꢁ) 271.0219 found 271.0215.
4.1.25. (1S,2R)-1-(2,5-Dihydroxy-2-oxo-2-l-5-[1,3,2]di-
oxaphosphinan-5-yl)-butane-1,2,3,4-tetraol 11. Sodium
borohydride (4.6 mg, 0.12 mmol) was added to a stirred so-
lution of 9 (30 mg, 0.11 mmol) in D2O (5 mL). The reaction
was monitored by 31P NMR. After 2 h the starting material
was completely consumed. The reaction was quenched by
the addition of DowexÒ (H+ form). The crude reaction mix-
ture was freeze-dried after filtration to afford glucitol 11. 1H
NMR (D2O) d 4.76–4.73 (2H, m), 4.40–4.25 (4H, m), 3.82–
3.70 (1H, m), 3.41–3.34 (1H, m), 3.19–3.06 (1H, m); 13C
NMR (D2O) d 80.1, 75.8, 74.1, 72.8, 72.1, 69.3, 64.1; 31P
NMR (D2O) d ꢁ2.1; LRMS (ES) Calcd for C7H14O9P
(Mꢁ1) 273.1 found 273.2; HRMS (ES) Calcd for
C7H14O9P (MꢁHꢁ) 273.0319 found 273.0313.
4.1.26. (1S,2R)-1-(2,5-Dihydroxy-2-oxo-2-l-5-[1,3,2]-
dioxaphosphinan-5-yl)-butane-1,2,4-triol 12. Sodium
borohydride (3.3 mg, 0.086 mmol) was added to a stirred
solution of 10 (20 mg, 0.078 mmol) in D2O (5 mL). The re-
action was monitored by 31P NMR. After 2 h the starting
material was completely consumed. The reaction was
quenched by the addition of DowexÒ (H+ form). The crude
reaction mixture was freeze-dried after filtration to afford
4.1.23. (4R,5S)-9-Oxo-1,8,10-trioxa-9-l-5-phospha-spiro-
[5.5]undecane-2,4,5,9-tetraol 10. A suspension of Pd/C
(50 mg, 10% Pd/C), potassium carbonate (5.5 mg,
38.7 mmol) and cyclic phosphate triester 29 (54 mg,
87.7 mmol) in MeOH (10 mL) was stirred overnight under
a positive pressure of H2 (balloon). The reaction mixture
was then filtered and concentrated under reduced pressure.
31P and 1H NMR spectroscopic analysis (D2O) showed par-
tial deprotection. The crude reaction mixture was resus-
pended in MeOH (10 mL). More catalyst (50 mg, Pd/C
10%) and K2CO3 were added and the resulting reaction mix-
ture was then stirred overnight under a positive pressure of
H2 (balloon). This cycle was repeated until NMR analysis
showed the complete removal of the benzyl protecting
group. Two cycles were needed to fully deprotect 29. The
crude reaction mixture was then filtrated and lyophilised to
afford 10 as a hygroscopic powder (22.1 mg, 98%). 1H
NMR (D2O) d 5.20 (0.6H, dd, J 4.6, 4.8), 5.04 (0.4H, d,
1
glucitol 12. H NMR (D2O) d 4.50–3.81 (4H, m), 3.21–
3.02 (4H, m), 2.16–1.97 (2H, m); 13C NMR (D2O) d 82.9,
80.2, 72.8, 72.6, 56.5, 50.2, 42.4; 31P NMR (D2O) d ꢁ1.8
(br s); LRMS (ES) Calcd for C7H15O8P (Mꢁ1) 255.1 found
255.10; HRMS (ES) Calcd for C7H14O8P (MꢁHꢁ)
255.0265 found 255.0275.
4.2. In vitro assay of isolated human MIP synthase
4.2.1. Purification of recombinant human MIP synthase.
Human MIP synthase was purified as previously de-
scribed.16 Briefly, Escherichia coli strain containing the re-
combinant pRSETA/hINO1 construct was grown at 37 ꢀC
to an A550 of 0.4. Production of recombinant protein was
induced by 1 mM isopropyl-1-thio-D-galactopyranoside for
3 h. Cells were lysed by sonication, and supernatant was

E. J. Amigues et al. / Tetrahedron 63 (2007) 10042–10053
10053
loaded to Ni2+ column (invitrogen). The column was then
washed five times with buffer A (50 mM Na2PO4, 0.5 M
NaCl, 20 mM imidazole, pH 7.0), and the protein was eluted
with buffer B (50 mM Na2PO4, 0.5 M NaCl, 250 mM imid-
azole, pH 8.0).
2. Bootman, M. D.; Collins, T. J.; Peppiatt, C. M.; Prothero, L. S.;
MacKenzie, L.; De Smet, P.; Travers, M.; Tovey, S. C.; Seo,
J. T.; Berridge, M. J.; Ciccolini, F.; Lipp, P. Semin. Cell Dev.
Biol. 2001, 12, 3.
3. Salman, M.; Lonsdale, J. T.; Besra, G. S.; Brennan, P. J.
Biochim. Biophys. Acta-Mol. Cell Biol. Lipids 1999, 1436, 437.
4. Movahedzadeh, F.; Smith, D. A.; Norman, R. A.; Dinadayala,
P.; Murray-Rust, J.; Russell, D. G.; Kendall, S. L.; Rison,
S. C. G.; McAlister, M. S. B.; Bancroft, G. J.; McDonald,
N. Q.; Daffe, M.; Av-Gay, Y.; Stoker, N. G. Mol. Microbiol.
2004, 51, 1003.
5. Haites, R. E.; Morita, Y. S.; McConville, M. J.; Jacobe, H. B.
J. Biol. Chem. 2005, 280, 10981.
6. Jin, X. S.; Geiger, J. H. Acta Crystallogr., Sect. D: Biol.
Crystalallogr. 2003, 59, 1154.
7. Migaud, M. E.; Frost, J. W. J. Am. Chem. Soc. 1995, 117, 5154.
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9. Migaud, M. E.; Frost, J. W. J. Am. Chem. Soc. 1996, 118, 495.
10. Tian, F.; Migaud, M. E.; Frost, J. W. J. Am. Chem. Soc. 1999,
121, 5795.
4.2.2. MIP Synthase assay. Purified MIP synthase activity
was determined by the rapid colorimetric method of Barnett
et al.21 with minor modification. Purified protein was sus-
pended in the reaction buffer (100 mM Tris acetate, pH
8.0, 0.8 mM NAD, 2 mM dithiothreitol, 14 mM NH4Cl)
containing the inhibitor present in various concentrations
(0, 50 nM, 5mM and 5 mM). After addition of 5 mM glucose
6-phosphate to a final volume of 150 mL, the reaction mix-
ture was incubated for 1 h at 37 ꢀC. The reaction was termi-
nated by the addition of 50 mL of 20% (w/v) trichloroacetic
acid and kept on ice for 10 min. The precipitated protein was
removed by centrifugation. Supernatant (200 mL) was incu-
bated with 200 mL of NaIO4 for 1 h. Na2SO3 (200 mL of
1 M) was then added to the supernatant to remove the excess
NaIO4. For the measurement of phosphate, a 600-mL reagent
mixture (240 mL of H2O, 120 mL of 2.5% ammonium mo-
lybdate, 120 mL of 10% ascorbic acid, and 120 mL of 6 N
sulfuric acid) was added and incubated for 1 h at 37 ꢀC.
The absorbance was measured at 820 nm, and activity was
determined by the amount of inorganic phosphate liberated.
For each assay, a second aliquot of the sample was measured
for phosphates not released by NaIO4 treatment to control
for phosphatase activity. This value was subtracted from
the experimental sample to obtain MIP synthase activity.
Data represent the average of two independent experiments.
11. Jin, X. S.; Foley, K. M.; Geiger, J. H. J. Biol. Chem. 2004, 279,
13889.
12. Stein, A. J.; Geiger, J. H. J. Biol. Chem. 2002, 277, 9484.
13. Schaeffer, R. J. Am. Chem. Soc. 1959, 81, 5452.
14. Mazur, A. W.; Hiler, G. D. J. Org. Chem. 1997, 62, 4471.
15. Amigues, E. J.; Migaud, M. E. Tetrahedron Lett. 2004, 45,
1001.
16. Ju, S. L.; Shaltiel, G.; Shamir, A.; Agam, G.; Greenberg, M. L.
J. Biol. Chem. 2004, 279, 21759.
17. Yu, B.; Xie, J.; Deng, S.; Hui, Y. J. Am. Chem. Soc. 1999, 121,
12196.
18. (a) Lubineau, A.; Thierry, A.; Veyrieres, A. Carbohydr. Res.
1976, 46, 143; (b) Fernandez, C.; Nieto, O.; Fontenela, J. A.;
Rivas, E.; de Ceballos, M. L.; Ferandez-Mayoralas, A. Org.
Biomol. Chem. 2003, 5, 767.
19. Lievre, C.; Frechou, C.; Demailly, G. Carbohydrate Res. 1997,
303, 1.
Acknowledgements
We wish to thank the EPSRC for funding E.J.A. Ph.D.
studentship.
20. (a) Lemarechal, P.; Froussios, C.; Level, M.; Azerad, R.
Carbohydrate Res. 1981, 94, 1; (b) Paulsen, H.; Schnell, D.;
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References and notes
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