C-Glycoside based mimics of D-myo-inositol 1,4,5-trisphosphate

Article abstract of DOI:10.1016/S0008-6215(00)00175-0

Epimeric C-glycoside based polyphosphates, α- and β-D-glucopyranosylmethanol 3,4,1'-trisphosphates (8 and 9) were prepared from D-glucose. The key intermediate, allyl 2,6-di-O-benzyl-α-D-glucopyranoside, was prepared in five steps (67% yield) from allyl α-D-glucopyranoside without the need for chromatography. Compounds 8 and 9 were shown to be full agonists at the Ins(1,4,5)P₃ receptors of permeabilised hepatocytes, but with markedly different potencies. Such C-glycoside analogues are worthy of further development as Ins(1,4,5)P₃ receptor ligands. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00175-0

www.elsevier.nl/locate/carres
Carbohydrate Research 329 (2000) 7–16
C-Glycoside based mimics of
D-myo-inositol
1,4,5-trisphosphate
Heidi J. Rosenberg a, Andrew M. Riley a, Vanessa Correa b, Colin W. Taylor b,
Barry V.L. Potter a,*
a Department of Pharmacy and Pharmacology, Wolfson Laboratory of Medicinal Chemistry, Uni6ersity of Bath,
Cla6erton Down, Bath BA2 7AY, UK
b Department of Pharmacology, Tennis Court Road, Uni6ersity of Cambridge, Cambridge CB2 1QJ, UK
Received 13 March 2000; accepted 1 June 2000
Abstract
Epimeric C-glycoside based polyphosphates, a- and b-
were prepared from -glucose. The key intermediate, allyl 2,6-di-O-benzyl-a-
steps (67% yield) from allyl a- -glucopyranoside without the need for chromatography. Compounds 8 and 9 were
D
-glucopyranosylmethanol 3,4,1%-trisphosphates (8 and 9)
D
D
-glucopyranoside, was prepared in five
D
shown to be full agonists at the Ins(1,4,5)P3 receptors of permeabilised hepatocytes, but with markedly different
potencies. Such C-glycoside analogues are worthy of further development as Ins(1,4,5)P3 receptor ligands. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Adenophostin; myo-Inositol; Selective protection
1. Introduction
motifs of Ins(1,4,5)P3/adenophostin, respec-
tively. However, slight variations in the third
phosphate group are tolerated [7].
D
-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,
5)P3, 1] is an intracellular signalling molecule
that stimulates mobilisation of intracellular
Ca2+ stores by binding to the Ins(1,4,5)P3
receptor, an intracellular Ins(1,4,5)P3-gated
Ca2+ channel [1]. In 1993 the glyconucleotides
adenophostins A and B (2 and 3) were isolated
from Penicillium bre6icompactum and found to
be very potent agonists of Ins(1,4,5)P3 receptors
[2–6]. Adenophostins A and B are 10–100 times
more potent than Ins(1,4,5)P3 in both
binding affinity for the receptor and the ability
to mobilise Ca2+ in cells. Structure–activity
investigations have established that high-
affinity Ins(1,4,5)P3 receptor ligands must con-
tain the 4,5-(bisphosphate)/3¦,4¦-(bisphos-
phate) and adjacent 6-hydroxyl/2¦-hydroxyl
* Corresponding author. Fax: +44-1225-826114.
E-mail address: b.v.l.potter@bath.ac.uk (B.V.L. Potter).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00175-0

8
H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
using a less flexible chain. It is clearly imprac-
We and other groups have synthesised vari-
tical to attempt synthesis of the chain-short-
ened version of 4, therefore a C-glycoside
derivative was an attractive target. On the
basis of all the above considerations, we des-
ous carbohydrate-based ligands of Ins(1,4,5)P3
receptors that are related to adenophostins
but lack the adenine base (4–7) [8–14]. All of
these compounds were similar to, or less po-
tent than, Ins(1,4,5)P3, suggesting that the
adenine base may play an essential role in the
increased potencies of the adenophostins.
However, the third phosphate group in the
above compounds may not be in the appropri-
ate position for optimal binding, particularly
in 4 and 5.
igned the syntheses of a- and b- -glucopyrano-
D
sylmethanol 3,4,1%-trisphosphate analogues (8
and 9) based on 4 but with a shorter side
chain. In 8 and 9, the third phosphate is
attached to a carbon centre fixed in the a and
b positions. Biological evaluation of these
compounds, together with previous results,
may help to clarify the structural requirements
for the significant biological activity of
adenophostins and their carbohydrate based
derivatives.
2. Results and discussion
The glycopyranose intermediate 15 [14a,b]
was prepared from -glucose (Scheme 1). Fis-
D
cher glycosylation with allyl alcohol in the
presence of HCl gave a mixture of pyranosides
from which the a anomer 10 was isolated
[14a,b]. Although stannylene mediated direct
dibenzylation of positions 2 and 6 in 10 has
been reported [14b], the overall yield was less
than 44% on a 2 g scale and less than 15% on
35 g scale. Moreover, stannylene mediated
dibenzylation requires extensive purification
of the product by flash chromatography,
therefore limiting the scale of the synthesis.
We therefore designed a different synthetic
strategy. Even though the proposed route was
longer it was hoped that the overall yield
would be higher and no purification by chro-
matography would be required.
The lower affinity of 4 compared with
Ins(1,4,5)P3 was thought to be due to the
conformational flexibility of the side chain
perhaps preventing the third phosphate from
achieving an optimal interaction with the re-
ceptor [14]. We therefore envisaged that it
would be of interest to design a monosaccha-
ride polyphosphate Ins(1,4,5)P3 mimic, having
the third phosphate closer to the ring and

H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
9
Selective acylation of C-2 and C-6 was
achieved by the treatment of 10 with trimethyl-
acetylchloride (pivaloyl chloride) at 0 °C in
pyridine [15]. One major product was formed,
and the crude material obtained from this
reaction was carried forward to the next step.
Positions C-3 and C-4 were then protected by
an isopropylidene acetal using 2-methoxy-
propene in THF in the presence of a catalytic
amount of p-toluenesulphonic acid [16]
and the product 12 was used immediately in
the next step without purification. The tri-
methylacetyl groups were removed and re-
placed by benzyl protecting groups using
sodium hydride and benzyl bromide in DMF
and again the product of this reaction was
used with no purification. Finally, the iso-
propylidene group was removed by stirring
the crude intermediate with 1 M HCl for 30
min, and crystallisation from ether–hexane
gave pure 13 [14b] (67% yield over five steps).
This method was found to be the most conve-
nient and efficient method to produce 13 in
multigram quantities. Similar reactions on
xylose to produce selectively allyl 2-O-benzyl-
-xylopyranoside (to be reported else-
D
-
a-
D
where), confirmed the general applicability of
this method.
The diol 13 was easily converted in high
yield into 14 [14b], a selectively protected in-
termediate without the labile trans isopropyli-
dene group. Thus, p-methoxybenzylation with
sodium hydride and p-methoxybenzyl chloride
in DMF gave the fully protected product 14.
The allyl glycoside was then cleaved by stir-
ring 14 vigorously with palladium chloride in
methanol for 4 h to give 15. The reaction
mixture becomes increasingly acidic as the
reaction proceeds, and it must be neutralised
before workup to prevent the formation of
Scheme 1. (i) PivCl, pyridine, 0 °C, 2.5 h; (ii) 2-methoxypropene, PTSA, THF, 30 min; (iii) NaOH, MeOH, reflux, 1 h; (iv) NaH,
BnBr, DMF, 0 °C, 90 min; (v) 1 M HCl–MeOH, 1:10, rt, 30 min (67%, over five steps); (vi) NaH, PMBCl, DMF, rt, 12 h (77%);
(vii) PdCl2, MeOH, rt, 4 h (71%); (viii) (a) oxalyl chloride, CH2Cl2, −78 °C. (b) Me2SO added dropwise. (c) 15 in CH2Cl2 added
dropwise. (d) NEt3 (67%); (ix) pyridine, THF, Tebbe reagent (1.1 equiv) added dropwise −45 °C for 30 min 65%; (x) 9-BBN,
THF 0 °C, 3 h, then H2O2, 5% KOH (58%) or BH3-THF, 0 °C, 2 h, then H2O2, 5% KOH (74%); (xi) CF3COOH, CH2Cl2 (80%);
(xii) (a) (BnO)2PNPri2, tetrazole. (b) MCPBA, −78 °C to RT (78%); (xiii) H2, Pd–C, 50 psi, MeOH–water, 24 h. All=allyl,
Piv=(CH3)3CCO, (Pivaloyl), PMB=p-methoxybenzyl, Bn=benzyl.

10
H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
Table 1
The PMB protecting groups were removed
with 10% trifluoroacetic acid in dichloro-
methane. The products of this reaction were
then easily separated by flash chromatography
to give the corresponding triols 20 and 21 in
good yields. These were phosphitylated with
tetrazole-activated bis(benzyloxy)diisopropyl-
aminophosphine in dichloromethane [20] and
oxidation of the intermediate phosphites with
m-chloroperbenzoic acid (MCPBA) gave the
fully protected compounds 22 and 23, which
were obtained in high yields after purification
by flash chromatography. Deprotection by hy-
drogenation over Pd/C yielded trisphosphates
8 and 9. The final products were purified on Q
Sepharose Fast Flow resin eluting with a 0–1
mol dm−3 gradient of triethylammonium hy-
drogen carbonate (pHꢀ7.5), to give the tri-
ethylammonium salts of 8 and 9, which were
quantified by total phosphate assay [21] before
biological evaluation.
The biological activities of 8 and 9 were
assessed by measuring 45Ca2+ release from
permeabilised rat hepatocytes. Maximally ef-
fective concentrations (10 mM) of Ins(1,4,5)P3
and 8 released the same fraction of the intra-
cellular Ca2+ stores, 4792% (n=5) and
4295% (n=3), respectively; half-maximal ef-
fects (EC50) occurred with 14496 nM and
2.4190.17 mM, respectively (Table 1). At a
concentration of 10 mM, 9 released only 199
3% (n=5) of the stores. Higher concentra-
tions of 9 were not examined, but its inability
to antagonise the response to a submaximal
concentration of Ins(1,4,5)P3 (Table 2) sug-
gests that it is not a partial agonist. The
results are therefore consistent with 9 being a
full agonist with an EC50 of about 10 mM.
Thus, trisphosphate 8 was similar in potency
to 4 and 5, while 9 was considerably weaker.
Previous studies [9,13] have clearly demon-
strated that sugar-based polyphosphates (e.g.
6 and 7) are able to approach the potency of
Ins(1,4,5)P3 but not of adenophostin. Such
molecules possess a glucopyranosyl 3,4-bis-
phosphate with an auxiliary phosphate, which
is accommodated in an optimal position for
45Ca2+ release data for Ins(1,4,5)P3, 8 and 9 a
EC50 (nM)
h
% release
n
with 10 mM
Ins(1,4,5)P3
8
14496
1.6890.26 4792
5
3
5
3
24149173 4.9991.84 4295
9
nd
nd
1993
4 b
1867964
2.7690.10 4793
a The EC50 values and Hill coefficients (h) were separately
determined for n independent experiments by fitting results to
logistic equations. Results are shown as means9S.E.M.
b Data for 4 obtained in an independent study [9] when
Ins(1,4,5)P3 had an EC50 value and Hill coefficient (h) of
153911 and 2.2590.20 nM, respectively.
Table 2
45Ca2+ release data for combined stimulation with
Ins(1,4,5)P3 and 9 a,b
% Ca2+
release
Ins(1,4,5)P3 (150 nM)
9 (10 mM)
Ins(1,4,5)P3 (150 nM) with 9 (10 mM)
2294
2192
3891
a The percentage of the intracellular Ca2+ stores released
by a submaximal concentration of Ins(1,4,5)P3 alone or in
combination with 9 are shown.
b Results are shown as means9S.E.M. for three indepen-
dent experiments.
side products if acid-labile groups such as
p-methoxybenzyl ethers are present. The pro-
tected glycopyranose intermediate 15 was oxi-
dised under Swern conditions to give the
lactone 16, which was purified by flash chro-
matography with a yield of 67%. Elaboration
to the exo-methylene derivative 17 was carried
out by the Tebbe reaction [17–19]. The
product 17 was obtained in 65% yield after
purification by flash chromatography.
The a and b epimers of the C-glucosyl-
methanol were then synthesised by hydrobora-
tion reactions [18]. Hydroboration of 17 using
9-BBN produced exclusively 2,6-di-O-benzyl-
3,4-di-O-(p-methoxybenzyl)-b- -glucopyrano-
D
sylmethanol (18), while hydroboration of 17
with a borane–THF complex produced a mix-
ture of a and b products 19 and 18 in an
approximate ratio of 1:2. The two products
could not be separated by flash chromatogra-
phy at this stage.
binding using
a
second ring as in
adenophostin. A challenge therefore is to ex-
plore the possibility of optimal positioning of

H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
11
interaction of 9 with the receptor protein due
to the extra CH2 group. The results show that
the anomeric oxygen atom in 4 is not essential
for biological activity and confirm that the
three-dimensional location of the third phos-
phate group plays an important role in strong
binding to the receptor.
These C-glycoside based polyphosphate
analogues represent steps in designing high
potency ligands using insights gained from the
adenophostins and are worthy of further
development.
the third phosphate without the need for the
second ring. This would also be useful in the
design of related molecules since it would
avoid problems of coupling strategies (e.g. in
the synthesis of 6 and 7).
While this work was in progress a report
appeared [8b] on a synthesis of the 2%-deoxy
derivative of 5. Not surprisingly, this molecule
was some 2000-fold less potent than
Ins(1,4,5)P3 in mobilising intracellular Ca2+
,
showing the expected parallel with deletion of
the 6-hydroxyl group of Ins(1,4,5)P3 [7]. It is
encouraging to see that the C-glycosides mim-
ics prepared here (8 and 9) do possess
Ins(1,4,5)P3-like Ca2+-mobilising activity, al-
though neither has attained the activity of 6 or
7. The differential potencies of 8 and 9 are of
interest and indicate that 8 presents the auxil-
iary phosphate group in the more favourable
position. However, a comparison of the activ-
ity of 8 with that of 4 (Table 1) indicates that
both molecules possess similar potency, imply-
ing no significant advantage of 8 over 4 in
binding to the Ins(1,4,5)P3 receptor.
3. Experimental
General methods.—Chemicals were pur-
chased from Aldrich, Sigma and Fluka. DMF
was distilled from barium oxide under reduced
,
pressure and then stored over 4 A molecular
sieves. Pyridine, Me2SO, CH2Cl2, THF, tolu-
ene and dioxane were purchased in anhydrous
forms. Ins(1,4,5)P3 was from American Radio-
labeled Chemicals.
Moitessier et al. [8a] synthesised 2%,3,4-
Thin-layer chromatography (TLC) was per-
formed on precoated plates (E. Merck alu-
minum sheets Silica 60 F254, Art. No. 5554).
Products were visualised by dipping plates
into phosphomolybdic acid in MeOH, fol-
lowed by heating. Flash chromatography was
carried out on silica gel (particle size 40–63
mm).
trisphosphates of (2-hydroxyethyl) a- and b- -
D
xylopyranosides and 3%,3,4-trisphosphates of
-xylopyra-
(3-hydroxypropyl) a- and b-
D
nosides. It was found that three of the mimics
were comparable and released approximately
the same amount of intracellular Ca2+
,
roughly with tenfold lower potency than
Ins(1,4,5)P3, with only the larger and more
flexible b-hydroxypropyl mimic having a
much lower potency. On the basis of these
results, it might be reasoned that the C-gly-
cosides with a less flexible chain should both
behave similarly, while based upon the struc-
ture of Ins(1,4,5)P3, one might expect the b
modified C-glycoside to have higher potency.
However, on examination of molecular mod-
els this is clearly not the case. The differential
activity of 8 and 9 can be qualitatively ex-
plained by the fact that the a-C-glycoside
phosphate group could in 8 readily access
some of the conformational space described
by the 1-phosphate of Ins(1,4,5)P3. While this
is not impossible for the b-C-glycoside phos-
phate group in 9, it does seem to be more
difficult and we also cannot exclude poten-
tially disfavourable steric or missing electronic
13
1H and C NMR spectra were recorded on
JEOL JMN GX-270 or EX-400 NMR spec-
trometers. Unless otherwise stated, chemical
shifts were measured in ppm relative to inter-
nal tetramethylsilane. 31P NMR chemical
shifts were measured in ppm and denoted
positive downfield from external 85% H3PO4.
J values are given in Hz. Melting points (un-
corrected) were determined using a Reichert–
Jung
Thermo
Galen
Kofler
block.
Microanalysis was carried out at the Univer-
sity of Bath Microanalysis Service. FAB Mass
spectra [m-nitrobenzyl alcohol (mNBA)] were
recorded at the University of Bath Mass Spec-
trometry Service using a VG Analytical Au-
tospec Mass Spectrometer. Optical rotations
were measured at ambient temperature using
an Optical Activity Ltd. AA-10 polarimeter,
and [h]D values are given in 10−1 deg cm2

12
H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
−1. Ion-exchange chromatography was per-
jected to flash chromatography (3:1 eluent
g
formed on an LKB-Pharmacia Medium Pres-
sure Ion-Exchange Chromatograph using Q
Sepharose Fast Flow resin and gradients of
Et2O–hexane) to give the title compound as
an oil.
[h]2D0 +90.6° (c 1.2, CHCl3); 1H NMR
(CDCl3; 400 MHz): l 5.79–5.89 (m, 1 H,
triethylammonium
hydrogen
carbonate
2
3
(TEAB) as eluent. Compounds containing
phosphates were assayed by a modification of
the Briggs phosphate test [21].
CH2CHꢀCH2), 5.28 (dd, 1 H, J 1.5, J 17.1
2
Hz, CH2CHꢀCHcisHtrans), 5.17 (dd, 1 H, J
3
0.9, J 10.3 Hz, CH2CHꢀCHcisHtrans), 5.02 (d,
45Ca2+ release from permeabilised rat hepa-
tocytes.—Permeabilized hepatocytes were
loaded to steady state (5 min at 37 °C) with
45Ca2+ in a cytosol-like medium (CLM: KCl,
140 mM, NaCl, 20 mM, 2 mM MgCl2, 1 mM
EGTA, 300 mM CaCl2, 20 mM Pipes, pH 7.0)
containing ATP (1.5 mM), creatine phosphate
(5 mM) creatine phosphokinase (5 units/mL)
and FCCP (10 mM). After 5 min, thapsigargin
(1.25 mM) was added to the cells (still at
37 °C) to inhibit further Ca2+ uptake, 30 s
later the cells were added to appropriate con-
centrations of the agonists and after a further
60 s the 45Ca2+ contents of the stores were
determined by rapid filtration.
1 H, J1,2 3.9 Hz, H-1), 4.61 (dd, 1 H, J2,3 9.8
Hz, H-2), 4.42 (dd, 1 H, J6a,5 4.9 Hz, H-6a),
4.33 (dd, 1 H, J6b,5 1.9, J6b,6a 12.2 Hz, H-6b),
4.14–4.19 (m, 1 H, CH2CHꢀCH2), 3.92–4.09
(m, 2 H, H-3, CH2CHꢀCH2), 3.80–3.85 (m, 1
H, H-5), 3.38 (t, 1 H, J4,3 9.3, Hz, H-4) and
1.22 (s, 18 H, 2×CMe3); 13C NMR (CDCl3;
67 MHz): l 179.4 and 178.5 (2×CꢀO), 133.4
(CH2CHꢀCH2), 117.6 (CH2CHꢀCH2), 95.1
(C-1), 69.8, 70.7, 71.5, 72.9 (4×CH), 68.4
(CH2CHꢀCH2), 63.2 (C-6), 38.9 (CMe3), 27.2
(3×Me) and 27.0 (3×Me); Anal. Calcd for
C19H32O8: C, 58.75; H, 8.3. Found: C, 58.8; H,
8.32%.
Allyl 2,6 - di - O - trimethylacetyl - 3,4 - O - iso-
Concentration–response relationships were
fitted to a logistic equation using Kaleide-
graph software (Synergy Software, PA) from
which the maximal response, half-maximally
effective agonist concentration (EC50) and Hill
slope (h) were determined. All results are ex-
pressed as means9SEM. Cytosol-like
medium (CLM); ethylene glycol-bis(b-amino-
ethyl ether)N,N,N%,N%-tetraacetic acid (EGTA);
piperazine - N,N% - bis[2 - ethanesulfonic acid]
(PIPES); adenosine 5%-triphosphate (ATP);
carbonyl cyanide p-trifluoromethoxyphenyl-
hydrazone (FCCP); concentration causing half
the maximal effect (EC50); Hill coefficient (h).
propylidene-h-
D
-glucopyranoside
(12).—A
soln of crude 11 (8.82 g, 22.0 mmol), 2-
methoxypropene (5.5 mL, 56 mmol) and p-
toluenesulphonic acid (100 mg) in THF (100
mL) was stirred for 2 h at rt under N2. TLC
(1:1 Et2O–hexane) indicated consumption of
the starting material. Diethyl ether (250 mL)
was added and the mixture was washed with
satd NaHCO3 (250 mL). The organic layer
was dried (MgSO4) and filtered, and a few
drops of Et3N were added before the organic
layer was concentrated. The product of this
reaction was used directly in the next step
without purification. A small sample was
purified by flash chromatography for analysis.
[h]2D0 +114° (c 1.2, CHCl3); 1H NMR
(CDCl3; 400 MHz): l 5.78–5.92 (m, 1 H,
CH2CHꢀCH2), 5.28 (dd, 1 H, J 1.7, J 17.2
Hz, CH2CHꢀCHcisHtrans), 5.17–5.20 (m, 2 H,
H-1, CH2CHꢀCHcisHtrans), 4.83 (dd, 1 H, J2,1
3.7, J2,3 10.6 Hz, H-2), 4.37 (dd, 1 H, J6b,5 2.2,
J6b,6a 11.9 Hz, H-6b), 4.10–4.20 (m, 1 H,
H-6a), 3.91–4.18 (m, 4 H, H-3, H-5,
CH2CHꢀCH2), 3.30–3.37 (m, 1 H, H-4), 1.44
(s, 3 H, CMe2), 1.42 (s, 3 H, CMe2), and 1.23
(s, 9 H, CMe3), 1.22 (s, 9 H, CMe3); 13C NMR
(CDCl3; 67 MHz): l 177.9 (2×CꢀO), 133.4
Allyl 2,6-di-O-trimethylacetyl-h- -gluco-
D
pyranoside (11).—Compound 10 (5.0 g, 22
mmol) was dissolved in dry pyridine (50 mL)
under N2. The solution was cooled to 0 °C
and trimethylacetyl chloride (5.6 mL, 45
mmol) was added dropwise over 1 h. Stirring
was continued for a further hour. The mixture
was poured into ice water (200 mL) and ex-
tracted with Et2O (200 mL). The organic soln
was washed successively with 1 M HCl (200
mL) and satd NaHCO3 (200 mL), dried
(MgSO4) and concentrated to give crude 11,
which was used in the next step without fur-
ther purification. A small sample was sub-
2
3

H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
13
stirred vigorously with PdCl2 (304 mg, 1.7
mmol) for 3 h. TLC (10:1 CH2Cl2–acetone)
showed consumption of the starting material.
Triethylamine (1 mL) was added to neutralize
the reaction. It was filtered through Celite, the
solvents were evaporated off and the residue
was subjected to flash chromatography (eluent
10:1 CH2Cl2–acetone) to give a pale-yellow
oil, which was crystallised from Et2O to give
the title compound as a white solid (3.59 g,
70.5 %).
(CH2CHꢀCH2), 117.8 (CH2CHꢀCH2), 111.0
(CMe2), 95.3 (C-1), 69.8, 70.7, 71.5, 72.9 (4×
CH), 69.8 (CH2CHꢀCH2), 63.5 (C-6) 38.8
(2×CMe3), 27.0 and 27.1 (CMe3) and 26.4
and 26.7 (2×Me); Anal. Calcd for C22H36O8:
C, 61.66; H, 8.47. Found: C, 61.9; H, 8.35.
Allyl 2,6-di-O-benzyl-h- -glucopyranoside
D
(13).—A soln of crude 12 (9.77 g, 22 mmol)
and NaOH (3.6 g, 90 mmol) in MeOH (50
mL) was heated under reflux for 1 h. The
mixture was cooled to rt and the pH was
adjusted to 8 by careful addition of solid CO2.
The solvents were evaporated off and the
residue was partitioned between Et2O (100
mL) and water (50 mL). The aq layer was
back-extracted with Et2O (10 mL) and the
combined organic fractions were dried
(MgSO4), filtered and evaporated. The oil ob-
tained was dissolved in dry DMF (40 mL) and
was stirred at 0 °C with NaH (2.25 g of an
60% w/w dispersion in mineral oil, 56 mmol)
and benzyl bromide (6.1 mL, 56 mmol) was
added slowly. The mixture was stirred at rt for
90 min. Water (50 mL) was added and stirring
continued for 60 min. The solvents were evap-
orated and the residue was dissolved in Et2O
(200 mL). The extract was washed with water
(500 mL), dried (MgSO4), filtered and evapo-
rated. The oil was dissolved in MeOH (50 mL)
and stirred with 1 M HCl (5 mL) for 30 min.
TLC (1:2 Et2O–hexane) indicated consump-
tion of the starting material. Solid NaHCO3
was added until the mixture was neutral. The
solvents were evaporated off and the residue
was partitioned between CH2Cl2 (100 mL) and
water (50 mL). The aq layer was back-
extracted with CH2Cl2 (200 mL) and the com-
bined organic layers were dried (MgSO4),
filtered and concentrated. The product was
crystallised from Et2O–hexane to give the title
compound (6.1 g, 67% over the previous five
steps from 10).
2,6-Di-O-benzyl-3,4-di-O-(p-methoxybenzyl)-
D-glucono-1,5-lactone (16).—Dry CH2Cl2 (1
mL) was placed into a 100 mL flask under an
atmosphere of N2. A soln of oxalyl chloride
in dry CH2Cl2 (1.7 mL, 3.4 mmol of 2 M
soln) was injected and the flask was cooled to
−78 °C. Anhyd Me2SO (0.5 mL, 0.7 mmol)
dissolved in dry CH2Cl2 was added dropwise
(care evolution of gas) and stirring was con-
tinued for 5 min. A soln of 15 (2.0 g, 3.3
mmol in 2 mL dry CH2Cl2) was added drop-
wise and stirring was continued for 20 min.
Triethylamine (1.8 mL, 13 mmol) was added
dropwise and the reaction was stirred for a
further 5 min, then the mixture was allowed to
heat up to rt. The mixture was stirred with
water (40 mL) for 10 min, then CH2Cl2 (80
mL) was added. The organic layer was sepa-
rated and the aq layer was re-extracted with
CH2Cl2 (80 mL). The combined organic layers
were washed successively with satd NaCl (80
mL), 1% HCl (80 mL) and 10% aq NaHCO3
(80 mL), dried and concentrated. The residue
was subjected to flash chromatography (eluent
1:1 Et2O–hexane) to give the title compound
as an oil (1.33 g, 67%).
[h]2D0 +64.7° (c 0.5, CHCl3); IR  1755
cm−1 (carbonyl); 1H NMR (CDCl3; 400
MHz): l 7.07–7.39 (m, 14 H, Ph, 4×ortho H
of PMB rings), 6.80–6.86 (m, 4 H, 4×meta H
of PMB rings), 4.98 (0.5×AB, 1 H, JAB 11.42
Hz, ArCH2O), 4.40–4.67 (m, 8 H, H-5, 3.5×
ArCH2O), 4.09 (d, 1 H, J2,3 6.2, H-2), 3.85–
3.95 (m, 2 H, H-3, H-4), 3.79 (s, 3 H, OMe),
3.78 (s, 3 H, OMe), 3.69 (dd, 1 H, J6a,6b 10.8,
J6b,5 2.3 Hz, H-6b) and 3.63 (dd, 1 H, J6a,5 3.5
Hz, H-6a); 13C NMR (CDCl3, 100 MHz): l
169.5, 159.5 (2×para C of PMB ring), 137.2,
137.8 (2×ipso C of Ph), 129.9, 129.8, 128.6,
128.5, 128.3, 128.0 (aromatic CH), 114.1
[h]2D0 +76.3° (c 0.8, CHCl3); Lit. [h]2D0
+
76.4° (c 0.8, CHCl31) [15]; mp 75–77 °C; Lit.
mp 74–77 °C [14b]. H NMR identical to Ref.
[14b].
2,6-Di-O-benzyl-3,4-di-O-(p-methoxybenzyl)-
h- -glucopyranoside (15).—Compound 14
D
was prepared from 13 in 77% yield as de-
scribed by Jenkins et al. [14b]. Compound 14
(5.5 g, 8.6 mmol) in methanol (50 mL) was

14
H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
NaCl, dried (MgSO4), filtered and concen-
trated. The residue was purified by flash chro-
matography (eluent 2:3 EtOAc–hexane) to
give the title compound (60 mg, 58%).
(meta C of PMB ring), 98.7 (C-1), 80.9, 78.5,
77.7 and 77.7 (4×CH), 75.9, 74.0, 73.8 and
73.7 (4×ArCH2O), 68.5 (C-6) and 55.6
(OMe); FABMS m/z calcd for C36H38O8
598.2566. Found m/z 598.2518.
[h]2D0 +4.5° (c 0.2, CHCl3); 1H NMR
(CDCl3; 400 MHz): l 7.04–7.72 (m, 14 H,
10×Ph, 4×ortho H of PMB rings), 6.80–
6.90 (m, 4 H, 2×H-3 and 2×H-5, PMB
ring), 4.84–4.88 (1.5×AB, 3 H, PhCH2O),
4.75, 4.66 (AB, 2 H, JAB 12.0 Hz, PhCH2O),
4.59, 4.54 (AB, 2 H, JAB 10.5 Hz, PhCH2O),
4.48 (AB, 1 H, PhCH2O), 3.86 (dd, 1 H, J 2.3,
J 12.0 Hz, H-1%b), 3.79 (2×s, 6 H, 2×OMe),
3.60–3.74 (m, 4 H, H-1%a, H-3, H-6a, H-6b),
3.51–3.57 (2×t, 2 H, H-2, H-4), 3.45 (ddd, 1
H, H-5) and 3.34 (ddd, 1 H, H-1); 13C NMR
(CDCl3; 100 MHz): l 159.4 and 159.3 (2×
para C of PMB rings), 138.2, 138.0 (2×ipso
C of Ph rings), 130.9, 130.4 (2×ipso C of
PMB ring), 129.8, 129.6, 128.7, 128.6, 128.2,
128.1 and 127.9 (aromatic CH), 114.1, 114.0
(2×meta C of PMB ring), 87.0 (C-3), 79.5
(C-1), 78.9, 78.4 and 78.3 (C-5, C-4, C-2),
75.6, 75.4, 75.0 and 73.77 (4×PhCH2O),
69.44 (C-6), 62.4 (C-1%) and 55.6 (OMe); Anal.
Calcd for C37H42O8: C, 72.29; H, 6.89. Found:
C, 72.1; H, 6.89.
2,6-Anhydro-3,7-di-O-benzyl-4,5-di-O-(p-
methoxybenzyl)-1-deoxy- -glucohept-1-enitol
D
(17).—To a soln of 16 (1.07 g, 1.8 mmol) in
dry toluene (3.5 mL) and dry THF (1.5 mL),
pyridine (26 mL) was added. The mixture was
cooled to −45 °C and Tebbe Reagent (3.9
mL, 2 mmol) was added slowly. The mixture
was stirred at −40 °C to −45 °C for 1 h and
then at 0 °C for a further 30 min. It was then
cooled to −10 °C to −15 °C and NaOH (1
mL, 15% w/v) was added. The cooling bath
was removed and the reaction mixture was
diluted with Et2O (50 mL). The inorganic
residue was removed by filtration through
Celite and the filter cake was washed with
excess Et2O. The solvents were evaporated off
and the residue was subjected to flash chro-
matography (eluent 1:3 Et2O–hexane) to give
the title compound, which was recrystallised
from hexane (691 mg, 65.1%).
Mp 52–54 °C; [h]2D0 +42.8° (c 0.5, CHCl3);
1H NMR (CDCl3; 400 MHz): l 7.03–7.36 (m,
14 H, ArH, 4×ortho H of PMB rings), 6.77–
6.86 (m, 4 H, 2×meta H of PMB rings),
4.39–4.81 (m, 11 H, H-6, H2-1, 4×ArCH2O),
3.93 (d, 1 H, J3,4 7.2 Hz, H-3), 3.79 (s, 3 H,
OMe), 3.77 (s, 3 H, OMe) and 3.71–3.74 (m,
2,6-Di-O-benzyl-3,4-di-O-(p-methoxybenzyl-
i and h- -glucopyranosylmethanol (18 and
D
19).—To compound 17 (570 mg, 0.9 mmol)
was added a 1.0 M soln of BH3ꢁTHF in THF
(2.9 mL, 2.9 mmol) at 0 °C. It was stirred for
2 h and allowed to heat up to rt. H2O2 (0.9
mL, 30%) was added and stirring continued
for 30 min. Water (10 mL) and Et2O (20 mL)
were added. The organic layer was washed
with satd NaCl (10 mL), dried (MgSO4),
filtered and concentrated. The residue was
purified by flash chromatography (eluent 2:1
Et2O–hexane) to give the title compounds as a
mixture in a ratio approximately 1:2 a:b (587
mg, 73.5%).
13
4 H, H-4, H-5, H2-7); C NMR (CDCl3; 100
MHz): l 159.2 and 156.3 (2×para C of PMB
ring and C-2), 137.9 (ipso C of Ph), 130.5,
130.3 (2×ipso C of PMB), 129.6, 128.4,
128.4, 127.9, 127.8, 127.6 (aromatic CH),
113.8, 113.7 (2×meta C of PMB ring), 94.6
(C-1), 84.4, 78.9, 78.6 and 77.2 (4×CH), 74.1,
73.5 and 72.8 (4×ArCH2O), 68.7 (C-7) and
55.3 (OMe); FABMS: m/z calcd for C37H40O7
[M+H]+ 597.2852. Found m/z 597.2833;
Anal. Calcd for C37H40O7: C, 74.47; H, 6.75.
Found: C, 74.3; H, 6.75.
2,6-Di-O-benzyl-i and h- -glucopyranosyl-
D
methanol (20 and 21).—The mixture of 18 and
19 (87 mg, 0.14 mmol) was dissolved in
CH2Cl2 (10 mL), TFA (1 mL) was added and
the mixture was stirred for 20 min. TLC (7:3
Et2O–hexane) showed that the reaction had
gone to completion. Saturated aq NaHCO3
(50 mL) was added to neutralize the reaction.
The products were extracted with CH2Cl2
2,6-Di-O-benzyl-3,4-di-O-(p-methoxyben-
zyl)-i-D-glucopyranosylmethanol
(18).—9-
BBN (0.5 M in THF, 1.68 mL, 0.8 mmol) was
added to 17 (100 mg, 0.2 mmol) at 0 °C. The
mixture was stirred for 3 h. H2O2 (3 mL, 30%)
and 5% aq KOH were added. The product
was extracted into Et2O and washed with satd

H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
15
mL), dried (MgSO4), filtered and concen-
trated. The concentrate was purified by flash
chromatography (eluent 3:2 EtOAc–hexane)
to give the title compound as an oil (137 mg,
99.1%).
(2×50 mL), the organic layer was dried
(MgSO4), filtered and concentrated to give a
white solid. It was purified by flash chro-
matography (eluent 4:1 EtOAc–hexane) to
give 20 (20.5 mg, 39%).
[h]2D0 −5.5° (c 1.8, CHCl3); 1H NMR
(CDCl3; 400 MHz): l 7.04–7.46 (m, 40 H,
Ph), 4.72–5.09 (m, 13 H, PhCH2O), 4.63–4.70
(m, 1 H, H-3), 4.48–4.55 (m, 1 H, H-4), 4.44
(0.5×AB, 1 H, JAB 11.1 Hz, PhCH2O), 4.40
(0.5×AB, 1 H, JAB 12.3 Hz, PhCH2O), 4.25–
4.3 (m, 2 H, H-1%b, PhCH2O), 4.07–4.12 (m, 1
H, H-1%a), 3.79 (dd, 1 H, J6b,5 1.5, J6b,6a 11.1
Hz, H-6b), 3.66 (dd, 1 H, J6a,5 5.3 Hz, H-6a),
3.51–3.65 (m, 2 H, H-2, H-5) and 3.43–3.46
Mp 68–70 °C; [h]2D0 +6.3° (c 0.2, CHCl3);
1H NMR (CDCl3; 400 MHz): l 7.25–7.38 (m,
10 H, Ph), 4.78, 4.72 (AB, 2 H, JAB 11.4 Hz,
PhCH2O), 4.57, 4.54 (AB, 2 H, JAB 12.0 Hz,
PhCH2O), 3.87 (dd, 1 H, J 2.3 Hz, H–1%b),
3.67–3.7 (m, 3 H, H-6a, H-6b, H-1%a), 3.4–
3.64 (m, 4 H, H-2, H-3, H-4, H-5) and 3.32–
3.39 (m, 1 H, H-1); 13C NMR (CDCl3; 100
MHz): l 138.2 and 137.6 (2×ipso C of Ph
ring). 128.8, 128.7, 128.3, 128.2, 128.1 and
128.03 (Ph), 77.62, 78.7 and 79.28 (3×CH),
77.0, 75.0 (2×PhCH2O), 73.9 (CH), 72.2
(CH), 70.5 (C-6) and 62.4 (C-1%); FABMS:
m/z calcd for C21H26O6 [M+H]+ 375.1807.
Found: m/z 375.1805.
13
(m, 1 H, H-1); C NMR (CDCl3; 100 MHz):
l 138.2 and 137.6 (ipso C of Ph rings), 136.2,
135.9, 135.8, 128.7 and 128.7 (Ph) and 74.6,
73.5, 70.2, 70.1, 69.8, 69.7, 69.0 and 66.2
31
(CH2); P NMR (CDCl3; 109 MHz): lp 0.02,
Further elution gave 21 (22 mg, 41.2%).
[h]2D0 +5.6° (c 0.17, CHCl3); 1H NMR
(CDCl3; 400 MHz): l 7.20–7.37 (m, 10 H,
Ph), 4.64, 4.63 (AB, 2 H, JAB 11.7 Hz,
PhCH2O), 4.58, 4.54 (AB, 2 H, JAB 12.0 Hz,
PhCH2O), 4.17 (ddd, 1 H, J1,2 5.9 Hz, J1,1%a
4.1, J1,1%b Hz, 9.1 Hz, H-1), 3.90 (dd, 1 H,
J1%b,1%a 12.3 Hz, H-1%b), 3.77 (dd, 1 H, H-1%a),
3.63–3.73 (m, 5 H, H-3, H-4, H-5, H-6a,
H-6b) and 3.60 (dd, 1 H, J2,3 9.4 Hz, H-2); 13C
NMR (CDCl3; 100 MHz): l 137.6 and 137.5
(2×ipso C of Ph ring). 128.8, 128.7, 128.4,
128.1 and 128.0 (Ph), 78.5 and 77.0 (2×CH),
74.6, 74.0 (2×PhCH2O), 73.5 (CH), 72.1 (C-
6), 71.8 (CH), 70.8 (CH) and 58.8 (C-1%);
FABMS: m/z calcd for C21H26O6 [M+H]+
375.1807. Found: m/z 375.1810.
−0.19, −0.79; FABMS: m/z calcd for
C63H65O15P3 [M+H]+1155.3614: Found: m/z
1155.3628.
2,6-Di-O-benzyl-h- -glucopyranosylmetha-
D
nol 3,4,1%-tris(dibenzylphosphate) (23).—The
title compound (90.5 mg, 78%) was obtained
from compound 21 (42 mg, 0.112 mmol) as
described above for 22.
[h]2D0 +7.7° (c 1.81, CHCl3); 1H NMR
(CDCl3; 400 MHz): l 7.16–7.35 (m, 40 H,
Ph), 4.86–5.11 (m, 12 H, PhCH2O), 4.76–4.81
(m, 1 H, H-3), 4.61–4.67 (m, 1 H, H-4), 4.57
(AB, 1 H, JAB 11.7 Hz, PhCH2O), 4.29–4.44
(m, 4 H, H-1%b, PhCH2O), 4.04–4.14 (m, 2 H,
H-1, H-1%a), 3.94–3.98 (m, 1 H, H-5), 3.64–
3.70 (m, 2 H, H-6a, H-2) and 3.58 (dd, 1 H,
J6b,5 2.9, J6b,6a 10.8 Hz, H-6b); 13C NMR
(CDCl3; 100 MHz): l 138.1 and 137.3 (ipso C
of PH ring), 135.9, 135.8, 128.7 and 128.7 (Ph)
77.7 and 77.6 (2×CH), 73.8, 73.8 (2×CH2),
73.4, 71.2, 71.8 (3×CH) and 69.9, 69.8, 69.7,
69.6, 68.1 and 64.1 (CH2); 31P NMR (CDCl3;
162 MHz): lp 0.21, −0.594, −1.07; FABMS:
m/z calcd for C63H65O15P3 [M+H]+
1155.3614. Found: m/z 1155.3648.
2,6-Di-O-benzyl-i- -glucopyranosylmetha-
D
nol 3,4,1%-tris(dibenzylphosphate) (22).—A
mixture of bis(benzyloxy)(diisopropylamino)-
phosphine (276 mg, 0.8 mmol), tetrazole (84
mg, 1.2 mmol) and dry CH2Cl2 (5 mL) was
vigorously stirred at rt for 30 min under N2,
whereupon 20 (50 mg, 0.1 mmol) was added
and stirring was continued for 30 min. The
mixture was cooled to 0 °C and MCPBA (460
mg, 1.6 mmol) was added. The mixture was
stirred at rt for 10 min, and was then diluted
with CH2Cl2 (50 mL). The soln was washed
successively with 10% (w/v) aq Na2SO3, satd
aq NaHCO3 (25 mL) and satd aq NaCl (25
h- -Glucopyranosylmethanol 3,4,1%-trisphos-
D
phate (8).—10% palladium on activated char-
coal (50%, 200 mg) was added to a soln of
compound 23 (90 mg, 0.08 mmol) in MeOH
(20 mL) and water (5 mL) and the mixture
was hydrogenated at 50 psi at rt for 24 h, after

16
H.J. Rosenberg et al. / Carbohydrate Research 329 (2000) 7–16
which it was filtered. The filtrate was concen-
trated to give a glassy clear solid. The residue
was dissolved in de-ionised water and purified
by ion-exchange chromatography on Q Sep-
harose Fast Flow resin, eluting with a gradient
of TEAB buffer (0–1 M), pH 8. The triethy-
lammonium salt of 8 eluted between 80 and
92% TEAB. Fractions containing compound
8, as judged by phosphate assay, were com-
bined and concentrated to give a residue from
which MeOH (3×100 mL) was evaporated to
give the title trisphosphate as its triethylam-
monium salt (58 mmol, 73%).
Support (045491 to B.V.L.P. and 039662 to
C.W.T.). We also thank Professor J.G.
Buchanan for useful discussions.
References
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1
[h]2D0 +19.8° (c 0.9, MeOH); H NMR
(CD3OD; 400 MHz): l 4.35 (q, 1 H, J 8.8 Hz,
H-3), 4.12–4.26 (m, 3 H, H-1, H-1%a, H-1%b),
4.05–4.10 (q, 1 H, H-4), 3.90 (dd, 1 H, J6a,5
4.1, J6a,6b 12.6 Hz, H-6a), 3.79–3.86 (m, 2 H,
31
H-2, H-5) and 3.72 (d, 1 H, H-6b); P NMR
(CD3OD; 162 MHz): l 2.70, 2.11, 2.10; S;
FABMS: m/z calcd for C7H16O15P3 [M−H]−
432.9657. Found: m/z 432.9694.
i- -Glucopyranosylmethanol 3,4,1%-trisphos-
D
phate (9).—The title compound (69 mmol,
86%) as its triethylammonium salt was ob-
tained from compound 22 (90 mg, 0.08
mmol), as described above for the synthesis of
8.
[h]2D0 −3.7° (c 1.3, MeOH); 1H NMR
(CD3OD; 400 MHz): l 4.15–4.22 (m, 2 H,
H-3, H-1%b), 3.96–4.07 (m, 2 H, H-4, H-1%a),
3.86 (dd, 1 H, J6a,5 3.8, J6a,6b 12.6 Hz, H-6a),
3.77 (dd, 1 H, J6b,5 1.8 Hz, H-6b), 3.50 (t, 1 H,
J 8.8 Hz, H-2), 3.39–3.43 (m, 1 H, H-5) and
31
3.29–3.36 (m, 1 H, H-1); P NMR (CD3OD;
162 MHz):
l
−5.90, −5.85, −5.38;
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Soc., 100 (1978) 3611–3613.
FABMS: m/z calcd for C7H16O15P3 [M−H]+
432.9657. Found: m/z 432.9684.
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Acknowledgements
We thank the Wellcome Trust for a Prize
Studentship (H.J.R.) and Programme Grant
.