Probing multivalency for the inhibition of an enzyme: Glycogen phosphorylase as a case study

Article abstract of DOI:10.1039/b812540f

Glycogen phosphorylase is involved in the hepatic glucose production and appears an emerging biological target for the treatment of type 2 diabetes. Two distinct trivalent inhibitors of GP were synthesized either through Cu(i)-assisted 1,3-dipolar cycload

Full text of DOI:10.1039/b812540f

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Probing multivalency for the inhibition of an enzyme: glycogen
phosphorylase as a case studyw
a
a
b
b
´
Samy Cecioni, Oana-Andreea Argintaru, Tibor Docsa, Pal Gergely,
a
a
´
Jean-Pierre Praly and Sebastien Vidal*
Received (in Montpellier, France) 22nd July 2008, Accepted 21st August 2008
First published as an Advance Article on the web 23rd October 2008
DOI: 10.1039/b812540f
Glycogen phosphorylase is involved in the hepatic glucose production and appears an emerging
biological target for the treatment of type 2 diabetes. Two distinct trivalent inhibitors of GP were
synthesized either through Cu(I)-assisted 1,3-dipolar cycloaddition or through formation of a tris-
oxadiazole derivative. A biological study of the inhibiting properties of these trivalent inhibitors
of GP have shown that the valency of the molecules influences slightly the inhibition of the
enzyme whereas the presence of a spacer arm between the core and the pharmacophore moieties
does not. The possible modes of binding of these multivalent inhibitors to the enzyme are
discussed.
displaying a large number of sites for the inhibition of this
Introduction
enzyme.8 A series of GP inhibitors have been described pre-
viously with various heterocyclic structures9 but our work10 is
focusing on carbohydrate-based inhibitors of GP7b,c,f which
are capable of binding selectively at the catalytic site of GP.
These glycomimetic approaches are based on structural modi-
fications at the molecular level for improving the binding to
the enzyme and therefore affording valuable GP inhibitors.
Glycogen phosphorylase and diabetes
Diabetus mellitus affects about 3% of the world population
and up to 6% for the adult population of developed countries.
Diabetes, one of the major causes of death worldwide, is
characterized by elevated glycaemia causing heart and kidney
failures as well as visual impairment problems.1 Type 2
diabetes is non-insulino-dependent and arises from insulin
signalling inefficiency resulting in insufficient or even late
insulin secretion. A series of biological targets have been
identified for anti-diabetic therapy2 such as peroxisome
proliferator-activated receptors a/g (PPARs a/g),3 glucagon-
like peptide-1 (GLP-1),4 dipeptidyl peptidase IV (DPP-IV)5 or
protein tyrosine phosphatase 1B (PTP 1B).6 Glycogen phos-
phorylase (GP) has recently appeared as an enzyme of interest
for the treatment of type 2 diabetes.7 This enzyme catalyses
glycogen depolymerisation to release glucose-1-phosphate
according to the schematic equation:
Multivalency and inhibition of enzymes
Another approach for the inhibition of GP could take advan-
tage of multivalency. This strategy may provide additional
opportunities in the field of drug discovery for the design of
potent enzyme inhibitors particularly by reaching higher
affinities and probably better selectivities. A few examples of
multivalent inhibition of an enzyme are reported in the
literature where multimeric species of a specific drug are
capable of improving the inhibition in comparison to the
monomeric molecule.11 The binding of one ligand subunit
from the multivalent molecule to the enzyme generates an
increase in local concentration of ligands, thus creating an
apparent cooperativity causing an enhancement in inhibition.
Dimeric inhibitors of influenza virus neuraminidase have been
developed by MacDonald et al.12 and displayed up to a
100-fold increase in inhibition along with improved pharmaco-
kinetic properties. In an additional study of the same
group,13 a set of trimeric and tetrameric inhibitors displayed
improved antiviral activities and long-lasting protective activ-
ities against influenza virus. More recently, the group of
J. Gervay-Hague has described the synthesis of a series of
trivalent zanamivir derivatives via click chemistry although no
biological activity has been reported yet.14 Inhibition of
acetylcholinesterase by dimeric molecules resulted in up to
3000-fold increases in potency and selectivity compared to
the monomeric inhibitor.15 Glycosidases inhibition16 with
tethered dimeric azasugars was investigated and the molecules
displayed interesting inhibitions of these enzymes but more
(Glucose)n - (Glucose)nꢀ1 + Glucose-1-phosphate
The inhibition of GP is expected to slow down glycogenolysis
and to lower the production of glucose from the liver therefore
allowing for a better control over hyperglycaemia. GP has
been extensively studied and crystallographic data analyses are
a
´
Universite de Lyon, Lyon, Universite Lyon 1, Villeurbanne, CNRS,
UMR5246, Institut de Chimie et Biochimie Moleculaires et
´
´
´
Supramoleculaires, Laboratoire de Chimie Organique
2 - Glycochimie, 43 Boulevard du 11 Novembre 1918, F-69622
Villeurbanne, France. E-mail: sebastien.vidal@univ-lyon1.fr;
Fax: +33 472 448 349; Tel: +33 472 448 349
b Cell Biology and Signalling Research Group of the Hungarian
Academy of Sciences, Department of Medical Chemistry, Research
Centre for Molecular Medicine, University of Debrecen, Nagyerdei
krt. 98, Debrecen, H-4032, Hungary. E-mail: gpal@dote.hu;
Fax: +36 52 412 566; Tel: +36 52 412 345
w Electronic supplementary information (ESI) available: Determina-
tion of Ki values and detailed atom numbering of molecules. See DOI:
10.1039/b812540f
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importantly a selectivity for two enzymes out of the seven
tested.16a However, tetravalent 1-azafagomine inhibitors dis-
played no improved inhibition compared to the monovalent
structure but rather a strong decrease in activity.16b Finally,
bivalent inhibitors of tetrameric b-tryptase constructed on a
cyclodextrin scaffold have shown increased inhibitions for this
enzyme.17
effect is observed and the IC50 value observed will be similar to
1/3 IC50mono (Fig. 1, Case 1). Nevertheless, the IC50 measured
can be improved with a lower value in comparison to 1/3
IC50mo (Fig. 1, Case 2). If each binding site of the enzyme is
occupied by a ligand of the multivalent inhibitor, the forma-
tion of 3:1 complexes would afford aggregates of proteins. If
the size of the aggregates remains small enough to maintain a
good solubility of the complex, the IC50 observed will be
similar to 1/3 IC50mono (Fig. 1, Case 3). Nevertheless, if the
size of the multivalent inhibitor-enzyme clusters becomes large
enough to cause their precipitation, the quantity of enzyme
present in the solution will diminish and therefore the IC50
value measured will be lower than 1/3 IC50mono (Fig. 1, Case
4). In this case, the IC50 value measured will be a ‘‘virtual’’
value because of the lower quantity of the enzyme available in
solution.
Inspired by the above mentioned results for the multivalent
neuraminidase inhibition and by the fact that a multimeric
enzyme offers additional possibilities for improved inhibition
through multivalency, we have designed synthetic routes to
trivalent carbohydrate-based GP inhibitors. A single case of a
dimeric inhibitor of GP has been reported with a bis(5-
chloroindole-2-carboxamide) derivative inhibiting human liver
GPa (HLGPa) with an IC50 value of 6 nM compared to 12.5 mM
for the parent monovalent inhibitor (2000-fold increase).18
This result highlights a productive and cooperative binding
of both ends of the bivalent inhibitor to two binding sites. The
co-crystallization of HLGPa with the divalent inhibitor
demonstrated that a single molecule of inhibitor was capable
of interacting with each monomeric unit of GP by linking the
two binding sites across the interface of GP homodimeric
structure. This result encouraged us to further investigate this
approach for the design of multivalent inhibitors of GP.
A closer look at the modes of binding of multivalent
inhibitors to an enzyme reveals several possibilities as depicted
in Fig. 1. A dimeric enzyme such as GP can interact with two
monomeric molecules of inhibitor in a 1:1 complex (2:2 at the
molecular level) providing a reference IC50mono value for
monovalent inhibitors. When considering the same inhibitor
repeated three times on a molecular scaffold, four main
possible cases can then be envisaged. The inhibition of a
trivalent inhibitor must be divided by three in order to
consider the contribution of each residue in its comparison
to a monovalent inhibitor. If a 1:1 complex is formed in
solution, a simple statistical effect can be invoked if no positive
Results and discussion
Synthesis of trivalent inhibitors
We have recently reported the preparation of 3-C-glycosyl-
5-aryl-1,2,4-oxadiazoles which displayed good inhibition
towards GP.10b In this context, we synthesized an alkyne-
terminated 3-C-glycosylated 1,2,4-oxadiazole which could then
be involved in a 1,3-dipolar cycloaddition with a tris-azido-
functionalized derivative to obtain a multivalent GP inhibitor
candidate. A more condensed trimeric inhibitor was also
prepared by direct coupling of an amidoxime with a tris-acyl
chloride and subsequent dehydrative cyclization to the corres-
ponding trivalent C-glycosylated oxadiazole. These inhibitors
were designed in order to determine the influence of a spacer
arm between the core and the pharmocophore ligands on the
inhibition of the enzyme.
Synthesis of the trivalent inhibitor with a spacer arm
The perbenzoylated glucosyl cyanide 119 was reacted with
hydroxylamine hydrochloride in pyridine to afford the desired
amidoxime 2 (Scheme 1). In our previous work,10b the crude
product obtained was rather difficult to purify by silica gel
column chromatography. The amidoxime 2 could be obtained
pure without chromatography simply by diluting the crude
product in ethyl acetate and then washing the organic layer
with 1 M aqueous HCl to remove pyridine and excess of
hydroxylamine, followed by saturated aqueous NaHCO3 and
brine. This simple chromatography-free purification process
afforded the expected amidoxime 2 in 99% yield and high
purity. The formation of the O-acyl-amidoxime 3 was
achieved with 4-pentynoic acid in the presence of EDCI/HOBt
as coupling agents.
We previously observed that reactions times lasting from a
few hours to a few days were required for the thermal
cyclodehydration of O-acyl-amidoximes. In order to optimize
both time and yield, we performed this reaction under TBAF
catalysis20 and/or microwaves activation21 (Table 1). We
observed that the use of TBAF catalysis at room temperature
provided the cyclic oxadiazole 4 within 1 day (entry 1) while
thermal activation combined with TBAF catalysis drastically
shortened the reaction time to 10 minutes (entry 2). No
Fig. 1 Possible modes of binding for a mono- and trivalent inhibitors
with a dimeric enzyme.
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Scheme 1 Reagents and conditions: (a) NH2OHꢂHCl, C5H5N, 50 1C, 5 h, 99%; (b) HCRC(CH2)2CO2H, EDCI, HOBt, CH2Cl2/DMF (9:1), ꢀ8 1C
then r.t., 16 h, 67%; (c) PhMe, TBAF 10 mol%, mW (150 1C, 200 W, 5 min), 97%; (d) NaOMe, MeOH then Amberlite IR-120 (H+ form);
(e) PhCH2N3, CuI, Et3N, mW (110 1C, 150 W, 15 min), 88%; (f) C6H3(CH2N3)3, CuI, Et3N, mW (110 1C, 150 W, 15 min), 98%.
Table 1 Cyclodehydration of O-acyl-amidoxime 3 to the 1,2,4-oxadiazole 4 in toluene
Entry
Catalyst
T/1C
Microwave condition
Time
Yield (%)
1
2
3
4
5
6
10% TBAF
10% TBAF
None
None
10% TBAF
10% TBAF
25
110
150
175
150
150
None
None
100 W
200 W
200 W
200 W
24 h
99
97
10 min
1 h
2 h
5 min
30 min
No reaction
Decomposition
97
66
reaction or decomposition of the starting material was ob-
served when applying microwaves activation without TBAF
catalysis (entries 3 and 4). Nevertheless, the association of
TBAF catalysis and microwaves activation performed on a
short timescale (entry 5 and 6) provided a result comparable to
that observed under conventional heating (entry 2). These
results underline the beneficial influence of TBAF catalysis.
The alkyne-terminated oxadiazole 4 was then engaged in a
Huisgen’s Cu(I)-catalyzed 1,3-dipolar cycloaddition22 reaction
under microwaves activation with benzyl azide to afford the
desired 1,4-disubstituted 1,2,3-triazole 6 in excellent yield.
Debenzoylation of compounds 4 and 6 afforded two hydro-
xylated GP inhibitor candidates 5 and 7. Similarly, the reac-
tion of 1,3,5-tris(azidomethyl)benzene23 with the alkyne
derivative 4 under microwaves activation and Cu(I) catalysis
afforded the cycloadduct 8. The saponification of the benzoate
esters provided the fully hydroxylated macromolecule 9.
by mass spectrometry (m/z = 2035.4 [M + H]+). Interest-
ingly, molecular ions could be observed neither for compound
13 nor the mono-oxadiazole intermediate. Saponification of
the ester groups of 11 resulted in the concomitant cleavage of
the O-acyl amidoxime function and afforded the benzoic acid
derivative 12 whose structure was clearly demonstrated by
mass spectrometry (m/z = 581 [M ꢀ H]ꢀ) and NMR analyses.
The triple thermal cyclodehydration of 10 was then performed
under microwaves activation and TBAF catalysis for
40 minutes. The tris-oxadiazole derivative 13 was isolated in
72% yield as the only product of the reaction highlighting
again the positive influence of TBAF catalysis and microwaves
activation for this cyclodehydration process. Deprotection
under Zemplen conditions afforded the expected hydroxylated
´
trivalent GP inhibitor candidate 14.
Inhibition of glycogen phosphorylase
The inhibition of GP was determined, as previously repor-
ted,10b for the three monovalent C-glycosylated oxadiazoles
(5, 7 and 1510b) and the two trivalent derivatives 9 and 14
(Fig. 2, Table 2). The enzymatic assays were performed at two
concentrations and most molecules displayed poor inhibition
properties at a concentration of 625 mM and moderate to good
inhibition at higher concentration (2.5 mM). In addition,
Ki values could be estimated only for trivalent derivatives 9
and 14 (see ESIw).
Synthesis of the trivalent inhibitor without spacer arm
We next prepared a more condensed trifunctional macro-
molecule were the C-glucosyl-oxadiazole moiety was directly
attached to a benzene ring (Scheme 2). Condensation of
amidoxime 2 with 1,3,5-benzenetricarbonyl trichloride af-
forded the corresponding triester 10 in 73% yield. At first,
compound 10 was subjected to cyclodehydration under
thermal conditions (reflux in 1,4-dioxane). The product obtained
was not the expected tris-oxadiazole 13 but the bis-oxadiazole
11 with one unreacted O-acyl amidoxime moiety as evidenced
The alkyne-terminated C-glycosylated oxadiazole derivative
5 displayed no inhibition at 625 mM and poor activity at
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Scheme 2 Reagents and conditions: (a) C6H3(COCl)3, 1,4-dioxane, r.t., 24 h, 73%; (b) 1,4-dioxane, 100 1C, 4 days; (c) NaOMe, MeOH then
Amberlite IR-120 (H+ form); (d) PhMe, TBAF 30 mol%, mW (150 1C, 200 W, 40 min), 72%.
Table 2 Inhibition of GP observed for monovalent and trivalent
inhibitors at two concentrations
Inhibition (%)
Inhibitor
Valency
At 625 mM
At 2.5 mM
Ki/mM
5
7
15
9
14
1
1
1
3
3
0
0
10
22 ꢃ 4
0
n.d.a
n.d.a
n.d.a
n.d.a
30 ꢃ 5
35 ꢃ 5
b
56 ꢃ 5
480 ꢃ 45b
535 ꢃ 50b
62 ꢃ 5
a
n.d. = not determined. Estimated.
2.5 mM. The inhibition properties disappeared completely
when a spacer arm was added such as in the structure of 7.
Interestingly, the trivalent analogue 9 of the non-active deri-
vative 7 was now inhibiting GP with values of 30% at 625 mM
and 56% at 2.5 mM. The valency of the molecule is therefore
responsible for an increase in inhibition from 0 to 56% when
comparing 7 and 9 at 2.5 mM. The C-glycosylated oxadiazole
derivative 15 bearing a phenyl group on the 5-position of the
oxadiazole ring displayed 10% inhibition at 625 mM.10b The
inhibition was again increased to 35% at 625 mM for trivalent
analogue 14. We anticipated that the distance between the core
and the carbohydrate moiety would influence for the binding
to the enzyme. Nevertheless, this was not the case based on the
inhibition measured for 9 and 14.
The increase of valency from monovalent to trivalent species
is responsible for an increase in inhibition of GP. The inhibi-
tion per residue for trivalent molecules is always similar the
Fig. 2 Structure of monovalent and trivalent GP inhibitors tested.
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corresponding monovalent analogue in accordance with the
statistical effect model proposed (Fig. 1, Case 1). The 3:1
complex non soluble mode of binding (Fig. 1, Case 4) can be
ruled out for these trivalent inhibitors of GP to the dimeric
enzyme since no precipitate was observed under the concen-
trations of trivalent inhibitors and enzyme used for the
inhibition studies. The inhibition observed for the trivalent
species was never better than 1/3 of the inhibition (in %)
observed for the corresponding monovalent molecules. In
conclusion, two modes of binding are possible with either
1:1 or 3:1 complexes (Fig. 1, Case 1 or Case 3, respectively)
resulting in an observed inhibition close to 1/3 of the inhibition
for the parent monovalent inhibitor.
were inspected by UV light (l = 254 nm) and developed by
treatment with a mixture of 10% H2SO4 in EtOH/H2O
(1:1 v/v) followed by heating. Silica gel column chromato-
graphy was performed with Gedurans silica gel Si 60 (40–63 mm)
purchased from Merck (Darmstadt, Germany). Reactions
under microwave activation were performed on a CEM
Discover system. HRMS (LSIMS) mass spectra were recorded
in the positive mode using a Thermo Finnigan Mat 95 XL
spectrometer. MS (ESI) mass spectra were recorded in the
positive mode using a Thermo Finnigan LCQ spectrometer.
1H and 13C NMR spectra were recorded at 23 1C using Bruker
¨
Advance DRX300 or DRX500 spectrometers with the residual
solvent as the internal standard. The following abbreviations
are used to explain the observed multiplicities: s, singlet; d,
doublet; dd, doublet of doublet; ddd, doublet of doublet of
doublet; t, triplet; td, triplet of doublet; q, quadruplet; m,
multiplet; br, broad; p, pseudo. Structure elucidation was
deduced from 1D and 2D NMR spectroscopy which allowed,
in most cases, complete signal assignments based on COSY,
HSQC, and HMBC correlations. NMR solvents were
purchased from Euriso-Top (Saint Aubin, France). Atom
numbering of the molecules is presented in the ESI.w
In the present study, the expected binding sites of these
glucose-based multivalent inhibitors are the catalytic site of
GP homodimer which are separated from each other by a long
distance and pointing into opposite directions.8d The structure
of the trivalent inhibitors tested did not permit such an intra-
molecular interaction with both catalytic sites on the same
GP dimer, but rather an interaction with two independent
GP dimers. In comparison, the bis(5-chloroindole-2-carboxamide)
derivative is binding simultaneously at each indole binding site
near the interface between the monomeric units of the GP
dimer.17 The linker is composed of 12 atoms between two
indole aromatic units which are therefore available for inter-
acting with the binding site of each monomer of GP.
Syntheses
1,3,5-Tris(azidomethyl)benzene. A solution of 1,3,5-tris(bro-
momethyl)benzene (3.07 g, 8.6 mmol) and sodium azide
(3.36 g, 51.6 mmol) in DMF (100 mL) was stirred at 65 1C
for 24 hours. The solution was cooled to room temperature
then poured into water (400 mL). The aqueous layer was
extracted with Et2O (3 ꢄ 250 mL). The combined organic
layers were washed with water (2 ꢄ 400 mL) and brine
(300 mL). The organic layer was dried (Na2SO4), filtered
and evaporated with extreme care (water-bath at room tem-
perature, reduced pressure and Plexiglas shield) to afford 1,3,5-
tris(azidomethyl)benzene (2.05 g, 98%) as a colorless oil. Rf =
The designed trivalent molecules could also bind to the enzyme
on a different site. The large aromatic appendage present in the
aglycon, composed of oxadiazole, phenyl and triazole rings,
might interact with the surface of the protein through hydro-
phobic interactions. The stability of the inhibitor-protein com-
plex would therefore be lower than complex involving an internal
binding site such as the catalytic site. The observed inhibitions
would therefore be weak as currently observed in the present
study. Nevertheless, we do not possess any experimental data
confirming or denying such a mode of interaction.
1
0.83 (PE/EtOAc, 8:2). H NMR (300 MHz, CDCl3) d = 4.39
(s, 6H, CH2N3), 7.25 (s, 3H, H-ar).
Conclusions
C-(2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl)-formami-
doxime (2). A solution of 2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl cyanide 1 (3.00 g, 4.96 mmol) and hydroxylamine
hydrochloride (0.86 g, 12.4 mmol) in pyridine (10 mL) was
stirred at 50 1C for 5 hours. The mixture was diluted with
EtOAc (250 mL) and washed with 100 mL portions of water,
1 M HCl, saturated NaHCO3, water and brine successively.
The organic layer was dried (MgSO4), filtered and evaporated
to obtain the pure amidoxime 2 (3.24 g, 99%) as a white foam.
Rf = 0.48 (PE/EtOAc, 1:1). 1H NMR (300 MHz, CDCl3)
d 4.21 (ddd, 1H, J = 9.7 Hz, J = 5.1 Hz, J = 2.7 Hz, H-5),
4.31 (d, 1H, J = 9.8 Hz, H-1), 4.47 (dd, 1H, J = 5.1 Hz, J =
12.4 Hz, H-6a), 4.62 (dd, 1H, J = 2.7 Hz, J = 12.4 Hz, H-6b),
4.76 (bs, 2H, NH2), 5.69 (t, 1H, J = 9.8 Hz, H-2), 5.73 (t, 1H,
J = 9.8 Hz, H-4), 5.96 (t, 1H, J = 9.8 Hz, H-3), 7.24–7.43
(m, 10H, H-ar), 7.47–7.57 (m, 2H, H-ar), 7.81–8.04 (m, 8H, H-ar).
In conclusion, we have designed two kinds of multivalent
inhibitors of GP based on the acylation of an amidoxime
intermediate followed by thermal dehydrative cyclization to
the corresponding oxadiazole. The introduction of an alkyne
residue at the 5-position of the oxadiazole ring allowed the
coupling to a trivalent azido-functionalized benzene ring
leading to an extended trivalent inhibitor candidate. The
enzyme inhibition assays revealed poor to moderate inhibitory
effect of these analogues. But, more important was the fact
that multivalent inhibitors were always superior to their
monovalent counterparts. This study provides one of the few
examples of multivalent inhibition for an enzyme, even though
the inhibitions observed remain modest.
Experimental
O-(Pent-40-ynoyl)-3-C-(2,3,4,6-tetra-O-benzoyl-b-D-gluco-
pyranosyl)-formamidoxime (3). A solution of 4-pentynoic acid
(27 mg, 0.27 mmol) in CH2Cl2/DMF (4 mL, 9:1) was cooled to
ꢀ8 1C before addition of 1-hydroxybenzotriazole (HOBt)
General methods
Thin-layer chromatography (TLC) was carried out on aluminum
sheets coated with silica gel 60 F254 (Merck). TLC plates
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(36.5 mg, 0.27 mmol), 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDCI) (52 mg, 0.27 mmol) and
amidoxime 2 (146 mg, 0.23 mmol). The mixture was kept at
ꢀ8 1C for 30 minutes then stirred at room temperature for
16 hours. The solvents were evaporated off and the crude
product was purified by flash silica gel column chromato-
graphy (PE then PE/EtOAc, 1:1) to afford the O-acylamidoxime
3 (110 mg, 67%) as a white foam. Rf = 0.53 (PE/EtOAc, 1:1).
[a]D = ꢀ2.9 (c = 1.00/CH2Cl2). 1H NMR (300 MHz, CDCl3)
d 1.94 (t, 1H, J = 2.5 Hz, H-50), 2.40–2.63 (m, 4H, H-20 H-30),
4.31 (ddd, 1H, J = 2.7 Hz, J = 5.1 Hz, J = 9.8 Hz, H-5), 4.54
(d, 1H, J = 9.8 Hz, H-1), 4.53–4.59 (m, 1H, H-6a), 4.67
(dd, 1H, J = 2.7 Hz, J = 9.5 Hz, H-6b), 5.36 (s, 2H, NH2),
5.76 (t, 1H, J = 9.8 Hz, H-4), 5.80 (t, 1H, J = 9.8 Hz, H-2),
6.01 (t, 1H, J = 9.8 Hz, H-3), 7.25–7.57 (m, 12H, H-ar),
7.83–8.07 (m, 8H, H-ar). 13C NMR (75 MHz, CDCl3) d 14.3
(C-30), 32.1 (C-20), 63.0 (C-6), 69.2 (C-4), 69.3 (C-50), 70.0
(C-2), 73.7 (C-3), 75.6 (C-1), 76.7 (C-5), 82.5 (C-40), 128.40,
128.45, 128.5 (3s, 8C, CH-ar), 128.77, 128.82, 129.4 (3s, 4C,
CIV-ar), 129.8, 129.9, 130.00, 130.04 (4s, 8C, CH-ar), 133.4,
133.5, 133.7 (3s, 4C, CH-ar), 153.7 (NQCR–NH2), 165.3,
165.6, 165.7, 166.3 (4s, 4C, COPh), 169.0 (C-10). ESI-MS
(positive mode) m/z: 719.2 [M + H]+, 741.3 [M + Na]+,
786.9 [M + HCOOH + Na]+, 1436.9 [2M + H]+, 1458.9
[2M + Na]+, 1504.5 [2M + HCOOH + Na]+. HR-ESI-MS
(positive mode) m/z: calcd. for C40H34N2O11 [M + H]+
719.2241, found 719.2244.
was stirred at room temperature for 4 hours. The solution
was neutralized with a cation exchange resin (Amberlite IR-120,
H+ form) and the resin washed with MeOH (3 ꢄ 5 mL).
The filtrate was evaporated off and the residue was dissolved in
MeOH then precipitated with CH2Cl2. The resulting solid was
washed with CH2Cl2 (2 ꢄ 5 mL) and dried under vacuum
to afford the hydroxylated oxadiazole 5 (103 mg, 98%) as a
white foam. Rf = 0.26 (CH2Cl2/MeOH, 9:1). [a]D = +9.6
1
(c = 0.51/H2O). H NMR (300 MHz, CD3OD) d 2.36 (t, 1H,
J = 2.6 Hz, H-100), 2.73 (td, 2H, J = 2.6 Hz, J = 7.3 Hz, H-300),
3.18 (t, 2H, J = 7.3 Hz, H-400), 3.42–3.53 (m, 3H, H-30 H-40 H-50),
3.65–3.73 (m, 2H, H-20 H-60a), 3.87 (dd, 1H, J o1.0 Hz, J = 12.0
Hz, H-60b), 4.44 (d, 1H, J = 9.7 Hz, H-10). 13C NMR (75 MHz,
CD3OD) d 16.6 (C-300), 27.1 (C-400), 62.8 (C-60), 71.2 (C-40), 71.3
(C-100), 73.3 (C-20), 74.8 (C-10), 79.2 (C-30), 82.3 (C-200), 82.6 (C-50),
169.2 (C-3), 180.5 (C-5). ESI-MS (positive mode) m/z: 285.0
[M + H]+, 307.1 [M + Na]+, 590.9 [2M + Na]+. HR-ESI-MS
(positive mode) m/z: calcd. for C12H16N2O6Na [M + Na]+
307.0906, found 307.0907.
5-[200-(10 00-Benzyl-10 0 0,20 0 0,30 00-triazol-40 0 0-yl)ethyl]-3-C-
(20,30,40,60-tetra-O-benzoyl-b-D-glucopyranosyl)-1,2,4-oxa-
diazole (6). In a CEM Discover 5 mL vial was introduced a
solution of benzyl azide (110 mg, 0.828 mmol), alkyne 4
(193 mg, 0.276 mmol), copper iodide (26 mg, 0.138 mmol)
and DIPEA (240 mL, 1.38 mmol) in toluene (5 mL). The
solution was sonicated for 1 min then heated at 110 1C for 15
min upon microwave irradiation (150 W). The solvent was
evaporated off and the residue purified by flash silica gel
column chromatography (PE/EtOAc, 1:1) to afford the cyclo-
adduct 6 (202 mg, 88%) as a colorless oil. Rf = 0.24
5-(But-100-yn-400-yl)-3-C-(20,30,40,60-tetra-O-benzoyl-b-D-gluco-
pyranosyl)-1,2,4-oxadiazole (4). In a CEM Discover 5 mL vial
was introduced a solution of O-acylamidoxime 3 (369 mg,
0.5 mmol) and TBAF (50 mL, 50 mmol, 1 M in THF) in toluene
(5 mL). The reaction vial was heated at 150 1C for 5 min upon
microwave irradiation (200 W). The solvent was evaporated
and the residue purified by flash silica gel column chromato-
graphy (PE then PE/EtOAc, 1:1) to afford the oxadiazole 4
(348 mg, 97%) as a white foam. Rf = 0.65 (PE/EtOAc, 7:3).
[a]D = +8.3 (c = 1.12/CH2Cl2). 1H NMR (300 MHz, CDCl3)
d 1.84 (t, 1H, J = 2.6 Hz, H-100), 2.65 (td, 2H, J = 2.6 Hz, J =
7.5 Hz, H-300), 3.10 (t, 2H, J = 7.5 Hz, H-400), 4.33 (ddd, 1H,
J = 3.4 Hz, J = 5.1 Hz, J = 9.8 Hz, H-50), 4.53 (dd, 1H, J =
5.2 Hz, J = 12.4 Hz, H-60a), 4.65 (dd, 1H, J = 3.0 Hz, J =
12.4 Hz, H-60b), 5.09 (d, 1H, J = 9.5 Hz, H-10), 5.82 (t, 1H,
J = 9.5 Hz, H-40), 6.00 (m, 2H, H-20 H-30), 7.29–7.58 (m, 12 H,
H-ar), 7.81–8.02 (m, 8H, H-ar). 13C NMR (75 MHz, CDCl3) d
16.0 (C-300), 26.2 (C-400), 63.3 (C-60), 69.4 (C-40), 70.2 (C-100),
70.6 (C-20), 72.4 (C-10), 74.1 (C-30), 77.0 (C-50), 80.8 (C-200),
128.3 (s, 2C, CH-ar), 128.4 (s, 4C, CH-ar), 128.5 (s, 2C, CH-
ar), 128.7, 128.7, 128.8, 129.5 (4s, 4C, CIV-ar), 129.7, 129.8,
129.8, 129.9 (4s, 8C, CH-ar), 133.2, 133.3, 133.4, 133.5 (4s, 4C,
CH-ar), 164.6, 165.2, 165.8, 166.3 (s, 4C, COPh), 166.2 (C-3),
179.0 (C-5). ESI-MS (positive mode) m/z: 701.1 [M + H]+,
723.2 [M + Na]+, 1400.9 [2M + H]+, 1422.9 [2M + Na]+.
HR-ESI-MS (positive mode) m/z: calcd. for C40H32N2O10Na
[M + Na]+ 723.1955, found 723.1954.
1
(PE/EtOAc, 1:1). [a]D = +1.4 (c = 1.02/CH2Cl2). H NMR
(500 MHz, CDCl3) d 3.22 (bs, 2H, H-200), 3.28 (bs, 2H, H-100),
4.37 (ddd, 1H, J = 3.0 Hz, J = 5.3 Hz, J = 9.7 Hz, H-50), 4.56
(dd, 1H, J = 5.3 Hz, J = 12.4 Hz, H-60a), 4.68 (dd, 1H, J =
3.0 Hz, J = 12.4 Hz, H-60b), 5.10 (d, 1H, J = 9.7 Hz, H-10),
5.48 (s, 2H, NCH2Ph), 5.86 (t, 1H, J = 9.7 Hz, H-40), 5.97
(t, 1H, J = 9.7 Hz, H-20), 6.06 (t, 1H, J = 9.7 Hz, H-30),
7.24–7.56 (m, 18H, H-500 0 H-ar), 7.80–8.03 (m, 8H, H-ar). 13C
NMR (125 MHz, CDCl3) d 21.8 (C-200), 26.6 (C-100), 53.6
(NCH2Ph), 62.9 (C-60), 68.1 (C-40), 69.8 (C-20), 71.7 (C-10),
73.4 (C-30), 76.5 (C-50), 122.2 (C-50 00), 127.8, 127.9, 128.0,
128.4, 128.5, 128.9, 129.7, 129.8 (8s, 25C, CH-ar), 133.4, 133.6,
133.8, 133.9 (4s, 4C, CIV-ar), 134.7 (CIV-ar), 145.3 (C-40 00),
164.7, 165.0, 165.6, 165.3 (4s, 4C, COPh), 165.6 (C-3), 180.1
(C-5). ESI-MS (positive mode) m/z: 834.1 [M + H]+, 856.1
[M + Na]+, 1666.4 [2M + H]+. HR-ESI-MS (positive mode)
m/z: calcd. for C47H40N5O10 [M + H]+ 834.2775, found
834.2781.
5-[200-(10 00-Benzyl-10 0 0,20 0 0,30 00-triazol-40 0 0-yl)-ethyl]-3-C-(b-D-
glucopyranosyl)-1,2,4-oxadiazole (7). A solution of benzoy-
lated cycloadduct 6 (128 mg, 0.153 mmol) and NaOMe
(5 mg, 0.09 mmol) in CH2Cl2/MeOH (5.5 mL, 10:1) was
stirred at room temperature for 4 hours. The solution was
neutralized with a cation exchange resin (Amberlite IR-120,
H+ form) and resin washed with MeOH (3 ꢄ 5 mL). The
filtrate was evaporated off and the residue purified by
flash silica gel column chromatography (CH2Cl2 then
3-C-(b-D-Glucopyranosyl)-5-(but-100-yn-400-yl)-1,2,4-oxadiazole (5).
A solution of benzoylated oxadiazole 4 (261 mg, 0.37 mmol)
and NaOMe (5 mg, 0.09 mmol) in CH2Cl2/MeOH (5 mL, 2:3)
ꢁc
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New J. Chem., 2009, 33, 148–156 | 153

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1
CH2Cl2/MeOH, 8:2 then EtOAc/MeOH, 8:2) to afford the
hydroxylated cycloadduct 7 (63 mg, 98%) as a white foam.
Rf = 0.68 (EtOAc/MeOH, 4:1). [a]D = +5.5 (c = 0.95/
(45 mg, 84%) as a white foam. H NMR (300 MHz, D2O) d
3.02–3.19 (m, 6H, H-100), 3.20–3.30 (m, 6H, H-200), 3.51–3.86
(m, 18H, H-20 0 0 0 H-30 0 0 0 H-40 0 0 0 H-50 0 0 0 H-60 0 0 0 a H-60 0 0 0 b), 4.54
(d, 3H, J = 8.8 Hz, H-10 0 0 0 ), 5.39 (s, 6H, NCH2C6H3), 6.98
(s, 3H, H-2 H-4 H-6), 7.73 (s, 3H, H-50). 13C NMR (125 MHz,
D2O) d 22.0 (s, 3C, C-100), 26.3 (s, 3C, C-200), 53.4 (s, 3C,
NCH2C6H3), 61.1 (s, 3C, C-60 0 0 0 ), 72.1 (s, 3C, C-20 0 0 0 ), 73.2
(s, 3C, C-10 0 0 0 ), 69.6, 77.1, 80.6 (3s, 9C, C-30 0 0 0 C-40 0 0 0 C-50 0 0 0 ),
124.2 (s, 3C, C-50), 127.3 (s, 3C, C-2 C-4 C-6), 137.2 (s, 3C, C-1
C-3 C-5), 146.5 (s, 3C, C-40), 167.5 (s, 3C, C-30 0 0), 181.0 (s, 3C,
C-50 0 0). ESI-MS (positive mode) m/z: 1118.2 [M + Na]+.
HR-ESI-MS (positive mode) m/z: calcd. for C45H57N15NaO18
[M + Na]+ 1118.3904, found 1118.3918.
1
MeOH). H NMR (300 MHz, CD3OD) d 3.18–3.23 (m, 2H,
H-200), 3.24–3.36 (m, 2H, H-100), 3.44–3.52 (m, 3H, H-30 H-40
H-50), 3.68–3.74 (m, 2H, H-20 H-60a), 3.88 (dd, 1H, J o1.0 Hz,
J = 11.4 Hz, H-60b), 4.44 (d, 1H, J = 9.7 Hz, H-10), 5.55
(s, 2H, NCH2Ph), 7.28–7.38 (m, 5H, H-ar), 7.79 (s, 1H, H-500 0).
13C NMR (75 MHz, CD3OD) d 23.3 (C-200), 27.2 (C-100), 54.9
(NCH2Ph), 62.8 (C-60), 71.3, 79.2, 82.6 (3s, 3C, C-30 C-40
C-50), 73.4 (C-20), 74.9 (C-10), 123.9 (C-50 00), 129.1, 129.5,
130.0 (3s, 5C, CH-ar), 136.8 (CIV-ar), 146.9 (C-40 0 0), 169.2
(C-3), 180.9 (C-5). ESI-MS (positive mode) m/z: 418.1 [M +
H]+, 440.1 [M + Na]+, 856.6 [2M + Na]+. HR-ESI-MS
(positive mode) m/z: calcd. for C19H23N5NaO6 [M + Na]+
440.1546, found 440.1549.
N,N0,N00-1,3,5-Tris(benzoyloxy)-C-(20,30,40,60-tetra-O-benzoyl-
b-D-glucopyranosyl)tricarboximidamide (10).
A solution of
1,3,5-benzenetricarbonyl trichloride (102 mg, 0.38 mmol) and
amidoxime 2 (794 mg, 1.24 mmol) in 1,4-dioxane (15 mL) was
stirred at room temperature for 24 hours. The solvent was then
evaporated off and the mixture was diluted with EtOAc (150 mL).
The organic layer was washed by 100 mL portions of saturated
NaHCO3, water and brine successively. The organic layer was
dried (MgSO4), filtered and evaporated. The crude product was
purified by flash silica gel column chromatography (EtOAc) to
afford the O-acylamidoxime 10 (571 mg, 73%) as a white foam.
1,3,5-Tris-40-200-[300 0-C-(20 0 0 0 ,30 0 0 0 ,40 0 0 0 ,60 0 0 0 -tetra-O-benzoyl-b-
D-glucopyranosyl)-100 0,200 0,400 0-oxadiazol-500 0-yl]-ethyl-10,20,30-
triazol-10-ylmethylbenzene (8). In a CEM Discover 5 mL vial
was introduced a solution of 1,3,5-tris(azidomethyl)benzene
(POTENTIALLY EXPLOSIVE, 4.9 mg, 20 mmol), alkyne 4
(63 mg, 90 mmol), copper iodide (1.9 mg, 10 mmol) and DIPEA
(17 mL, 100 mmol) in toluene (6 mL). The solution was
sonicated for 1 min then heated at 110 1C for 15 min upon
microwave irradiation (150 W). The solvent was evaporated
off and the residue purified by flash silica gel column chromato-
graphy (PE/EtOAc, 1:1 then EtOAc) to afford the tris-
cycloadduct 8 (46 mg, 98%) as a colorless oil. Rf = 0.67
1
Rf = 0.75 (PE/EtOAc, 3:7). [a]D = ꢀ48.6 (c = 1/CH2Cl2). H
NMR (300 MHz, CDCl3) d 4.23–4.33 (m, 3H, H-50), 4.49–4.57
(m, 6H, H-10 H-60a), 4.58–4.65 (m, 3H, H-60b), 5.46 (bs, 6H,
NH2), 5.70 (t, 3H, J = 9.6 Hz, H-20), 5.76 (t, 3H, J = 9.6 Hz,
H-40), 5.98 (t, 3H, J = 9.6 Hz, H-30), 7.22–7.58 (m, 36H, H-ar),
7.80–8.04 (m, 24H, H-ar), 8.52 (s, 3H, H-2 H-4 H-6). 13C NMR
(75 MHz, CDCl3) d 62.9 (C-60), 69.0 (C-40), 70.0 (C-20), 73.5
(C-30), 75.6 (C-10), 76.8 (C-50), 128.3, 128.4, 128.5, 128.7, 129.3,
129.7, 129.8, 129.90, 129.94, 130.2 (10s, 20C, CH-ar), 133.3,
133.4, 133.6, 134.1 (4s, 4C, C-ar), 154.5 (H2NCQNO), 161.5
(NOCQO), 165.2, 165.58, 165.63, 166.1 (4s, 4C, COPh). LSIMS
(positive mode, thioglycerol) m/z: 2071.6 [M + H]+. HR-ESI-
MS (positive mode) m/z: calcd. for C114H91N6O33 [M + H]+
2071.5627, found 2071.5651.
1
(EtOAc). H NMR (300 MHz, CDCl3) d 3.10–3.32 (m, 12H,
H-100 H-200), 4.34–4.40 (m, 3H, H-50 0 0 0 ), 4.51 (dd, 3H, J = 5.1
Hz, J = 12.4 Hz, H-60 0 0 0 a), 4.66 (dd, 3H, J = 2.7 Hz, J = 12.4
Hz, H-60 0 0 0 b), 5.12 (d, 3H, J = 9.4 Hz, H-10 0 0 0 ), 5.31 (s, 6H,
NCH2C6H3), 5.87 (t, 3H, J = 9.4 Hz, H-40 0 0 0 ), 5.97 (t, 3H, J =
9.4 Hz, H-20 0 0 0 ), 6.05 (t, 3H, J = 9.4 Hz, H-30 0 0 0 ), 6.93 (s, 3H,
H-2 H-4 H-6), 7.23–7.50 (m, 36H, H-ar), 7.74–8.00 (m, 24H,
13
H-ar). C NMR (75 MHz, CDCl3) d 22.5 (s, 3C, C-100), 26.6
(s, 3C, C-200), 53.1 (NCH2C6H3), 63.3 (s, 3C, C-60 0 0 0 ), 69.3
(s, 3C, C-40 0 0 0 ), 70.7 (s, 3C, C-20 0 0 0 ), 72.3 (s, 3C, C-10 0 0 0 ), 74.1
(s, 3C, C-30 0 0 0 ), 77.0 (s, 3C, C-50 0 0 0 ), 122.2 (s, 3C, C-50), 127.0
(s, 3C, C-2, C-4, C-6), 128.39, 128.43, 128.5 (3s, 24C, CH-ar),
128.69, 128.71, 128.8, 129.5 (4s, 12C, CIV-ar), 129.7, 129.78,
129.82, 129.9 (4s, 24C, CH-ar), 133.2, 133.4, 133.6 (3s, 12C,
CH-ar), 137.0 (s, 3C, C-1, C-3, C-5), 145.6 (s, 3C, C-40), 164.9,
165.2, 165.8, 166.2 (4s, 12C, COPh), 166.4 (s, 3C, C-30 0 0),
180.0 (s, 3C, C-500 0). ESI-MS (positive mode) m/z: 1173.5
3,5-Bis[30-C-(b-D-glucopyranosyl)-10,20,40-oxadiazol-50-yl]-
benzoic acid (12). A solution of tris-O-acylamidoxime 10
(532 mg, 256 mmol) in 1,4-dioxane (12 mL) was stirred at
100 1C for 4 days. The reaction was then cooled to room
temperature and the solvent evaporated off. The crude pro-
duct 11 was used without further purification. A solution of
crude 11 (239 mg) and NaOMe (10 mg) in CH2Cl2/MeOH
(5 mL, 1:1) was stirred at room temperature for 5 hours. The
solvent was evaporated off and the crude mixture was purified
by flash reverse-phase silica gel chromatography (H2O then
H2O/MeOH 7:3) to afford the benzoic acid derivative 12
(95 mg, 58% over two steps) as a white foam. Rf = 0.20
(EtOAc/MeOH 1:1). [a]D = +7.1 (c = 0.41/MeOH). 1H
NMR (300 MHz, CDCl3) d 3.62–3.76 (m, 3H, H-300, H-400,
H-500), 3.81–3.89 (m, 2H, H-200, H-600a), 3.99 (m, 1H, H-600b),
4.75 (m, 1H, H-100), 8.77 (s, 2H, H-ar), 8.87 (s, 1H, H-ar). 13C
NMR (75 MHz, CDCl3) d 61.2 (C-600), 69.7, 77.1, 80.8 (3s, 3C,
C-300, C-400, C-500), 72.1 (C-200), 73.4 (C-100), 124.7 (s, 2C,
CIV-ar), 126.9 (CIV-ar), 129.8 (CH-ar), 133.0 (s, 2C, CH-ar),
[M + 2H]2+
.
1,3,5-Tris-40-200-[30 00-C-(b-D-glucopyranosyl)-10 00,20 00,40 00-oxa-
diazol-50 0 0-yl]ethyl-10,20,30-triazol-10-ylmethylbenzene (9).
solution of benzoylated tris-cycloadduct 8 (114 mg, 49 mmol)
and NaOMe (5 mg, 92 mmol) in CH2Cl2/MeOH (5.5 mL, 10:1)
was stirred at room temperature for 6 hours. The solution was
neutralized with a cation exchange resin (Amberlite IR-120,
H+ form) and resin washed with MeOH (3 ꢄ 5 mL). The
filtrate was evaporated off and the residue was dissolved in
MeOH then precipitated with PE. The resulting solid was
washed with PE (5 ꢄ 5 mL), dissolved into pure water and
freeze-dried to afford the hydroxylated tris-cycloadduct 9
A
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154 | New J. Chem., 2009, 33, 148–156

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139.5 (CO2H), 168.4 (C-30), 175.8 (C-50). ESI-MS (negative
mode) m/z: 581 [M ꢀ H]ꢀ. HR-ESI-MS (negative mode) m/z:
calcd for C23H25N4O14 [M ꢀ H]ꢀ 581.1367, found 581.1369.
glycogen phosphorylase were collected using different concen-
trations of a-D-glucose-1-phosphate (2–20 mM), constant
concentrations of glycogen (1% w/v) and AMP (1 mM), and
various concentrations of inhibitors. Inhibitors were dissolved
in dimethyl sulfoxide (DMSO) and diluted in the assay buffer
(50 mM triethanolamine, 1 mM EDTA and 1 mM dithio-
threitol) so that the DMSO concentration in the assay should
be lower than 5%. The enzymatic activities were presented in
the form of double-reciprocal plots (Lineweaver–Burk) apply-
ing a nonlinear data analysis program. The inhibitor constants
(Ki) were determined by Dixon plots, by replotting the
slopes from the Lineweaver–Burk plots against the inhibitor
concentrations.26,27 The means of standard errors for all
calculated kinetic parameters averaged to less than 10%.
Ki values for compounds 9 and 14 were also estimated and
found to be 490 ꢃ 45 mM and 535 ꢃ 50 mM, respectively.
The poor solubility of inhibitors 5, 7 and 15 limited the
concentrations used in the kinetic studies. The inhibition of
glycogen phosphorylase was therefore determined at 625 mM
and 2.5 mM concentrations of these inhibitors and given in
Table 2.
1,3,5-Tris[30-C-(200,300,400,600-tetra-O-benzoyl-b-D-glucopyra-
nosyl)-10,20,40-oxadiazol-50-yl]benzene (13). In a CEM Discover
5 mL vial was introduced a solution of O-acylamidoxime
10 (454 mg, 0.22 mmol) and TBAF (70 mL, 70 mmol, 1 M in
THF) in toluene (5 mL). The reaction vial was heated
at 150 1C for 40 min upon microwave irradiation (200 W).
The solvent was evaporated and the residue purified by
flash silica gel column chromatography (PE then PE/EtOAc,
1:1) to afford the trivalent oxadiazole 13 (317 mg, 72%) as a
white foam. Rf = 0.79 (PE/EtOAc, 1:1). [a]D = ꢀ35.1 (c =
1
1.00/CH2Cl2). H NMR (300 MHz, CDCl3) d 4.41 (ddd, 3H,
J = 3.1 Hz, J = 5.1 Hz, J = 9.5 Hz, H-500), 4.59 (dd, 3H,
J = 5.2 Hz, J = 12.5 Hz, H-600a), 4.71 (dd, 3H, J = 2.9 Hz,
J = 12.5 Hz, H-600b), 5.22 (d, 3H, J = 9.5 Hz, H-100), 5.91
(t, 3H, J = 9.7 Hz, H-400), 6.04–6.15 (m, 6H, H-200 H-300),
7.26–7.52 (m, 36H, H-ar), 7.82–8.02 (m, 24H, H-ar), 8.96
(s, 3H, H-2). 13C NMR (75 MHz, CDCl3) d 63.3 (s, 3C,
C-600), 69.4 (s, 3C, C-400), 70.8 (s, 3C, C-200), 72.6 (s, 3C, C-100),
74.1 (s, 3C, C-300), 77.4 (s, 3C, C-500), 126.2 (s, 3C, C-1 C-3
C-5), 128.5, 128.5, 128.6 (3s, 24C, CH-ar), 129.9, 130.0
(2s, 24C, CH-ar), 131.3 (s, 3C, C-2 C-4 C-6), 133.3, 133.4,
133.6, 133.6 (4s, 12C, CH-ar), 164.9, 165.2, 165.9, 166.3
(4s, 12C, COPh), 167.6 (s, 3C, C-30), 174.1 (s, 3C, C-50).
ESI-MS (positive mode) m/z: 2039.6 [M + Na]+.
Acknowledgements
The authors wish to thank Universite
and CNRS for financial support. A stipend to S. C. and
financial support from Region Rhone-Alpes )Cluster de
´
Claude Bernard Lyon 1
´
Recherche Chimie* are gratefully acknowledged. CNRS is
also thanked for additional funding through )Programme
1,3,5-Tris[30-C-b-D-glucopyranosyl)-10,20,40-oxadiazol-50-yl]-
benzene (14). A solution of benzoylated tris-oxadiazole 13 (296
mg, 0.15 mmol) and NaOMe (15 mg, 0.3 mmol) in CH2Cl2/
MeOH (8 mL, 3:5) was stirred at room temperature for 3
hours. The solution was neutralized with a cation exchange
resin (Amberlite IR-120, H+ form) and the resin washed with
MeOH (3 ꢄ 5 mL). The filtrate was evaporated off and the
residue was dissolved in MeOH then precipitated with
CH2Cl2. The resulting solid was washed with CH2Cl2 (2 ꢄ
5 mL) and dried under vacuum to afford the hydroxylated tris-
oxadiazole 14 (114 mg, 99%) as a white foam. Rf = 0.63
(CH2Cl2/MeOH, 9:1). [a]D = +10.6 (c = 1.00/H2O). 1H
NMR (300 MHz, D2O) d 3.61–3.84 (m, 15H, H-200 H-300
H-400 H-500 H-600a), 3.97 (d, 3H, J = 11.9 Hz, H-600b), 4.67
(d, 3H, J = 9.4 Hz, H-100), 8.74 (s, 3H, H-2 H-4 H-6). 13C
NMR (75 MHz, D2O) d 59.0 (s, 3C, C-600), 71.0 (s, 3C, C-100),
67.4, 69.9, 74.9, 78.6 (4s, 12C, C-200 C-300 C-400 C-500), 123.5
(s, 3C, C-1 C-3 C-5), 129.2 (s, 3C, C-2 C-4 C-6), 166.5 (s, 3C,
C-30), 172.0 (s, 3C, C-50). ESI-MS (negative mode) m/z:
812.9 [M + HCO2ꢀ]ꢀ. ESI-MS (positive mode) m/z: 791.7
[M + Na]+. HR-ESI-MS (positive mode) m/z: calcd. for
C30H36N6NaO18 [M + Na]+ 791.1984, found 791.1981.
Interdisciplinaire: Chimie pour le Developpement Durable*.
´
This work was also supported by grants from the Hungarian
Science Research Fund (OTKA K60620) and the Hungarian
Ministry of Health (ETT 083/2006).
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