Halogen-substituted (C-β-d-glucopyranosyl)-hydroquinone regioisomers: Synthesis, enzymatic evaluation and their binding to glycogen phosphorylase

Article abstract of DOI:10.1016/j.bmc.2011.07.024

Electrophilic halogenation of C-(2,3,4,6-tetra-O-acetyl-β-d- glucopyranosyl) 1,4-dimethoxybenzene (1) afforded regioselectively products halogenated at the para position to the d-glucosyl moiety (8, 9) that were deacetylated to 3 (chloride) and 16 (bromid

Full text of DOI:10.1016/j.bmc.2011.07.024

Bioorganic & Medicinal Chemistry 19 (2011) 5125–5136
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Halogen-substituted (C-b-D-glucopyranosyl)-hydroquinone regioisomers:
Synthesis, enzymatic evaluation and their binding to glycogen phosphorylase
Kyra-Melinda Alexacou a,b, Yun Zhi Zhang c,d,e,f, Jean-Pierre Praly c,d,e,f, , Spyros E. Zographos a,
⇑
Evangelia D. Chrysina a, Nikos G. Oikonomakos a,z, Demetres D. Leonidas g,
⇑
a Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece
b Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany
c Université de Lyon, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires associé au CNRS, UMR 5246, Laboratoire de Chimie Organique 2-Glycochimie, Bâtiment
Curien, 43 boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
d Université Lyon 1, F-69622 Villeurbanne, France
e CNRS, UMR5246, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Laboratoire de Chimie Organique 2-Glycochimie, Bâtiment Curien, 43 boulevard du 11
Novembre 1918, F-69622 Villeurbanne, France
f CPE-Lyon, F-69616 Villeurbanne, France
g Department of Biochemistry and Biotechnology, University of Thessaly, 26 Ploutonos Str. 41221 Larissa, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Electrophilic halogenation of C-(2,3,4,6-tetra-O-acetyl-b-
afforded regioselectively products halogenated at the para position to the
D
-glucopyranosyl) 1,4-dimethoxybenzene (1)
-glucosyl moiety (8, 9) that
were deacetylated to 3 (chloride) and 16 (bromide). For preparing meta regioisomers, 1 was efficiently
oxidized with CAN to afford C-(2,3,4,6-tetra-O-acetyl-b- -glucopyranosyl) 1,4-benzoquinone 2 which,
Received 18 May 2011
Revised 12 July 2011
Accepted 13 July 2011
Available online 21 July 2011
D
D
in either MeOH or H2O–THF containing few equivalents of AcCl, added hydrochloric acid to produce pre-
dominantly meta (with respect to the sugar moiety) chlorinated hydroquinone derivatives 5 and 18, this
latter being deacetylated to 4. The deacetylated meta (4, 5) or para (3, 16) halohydroquinones were eval-
uated as inhibitors of glycogen phosphorylase (GP, a molecular target for inhibition of hepatic glycogen-
olysis under high glucose concentrations) by kinetics and X-ray crystallography. These compounds are
Keywords:
Type 2 diabetes
Glycogen phosphorylase
C-glucosyl aryl
C-glucopyranosyl hydroquinone
Inhibition
competitive inhibitors of GPb with respect to a-D-glucose-1-phosphate. The measured IC50 values (lM)
[169.9 10.0 (3), 95 (4), 39.8 0.3 (5) 136.4 4.9 (16)] showed that the meta halogenated inhibitors
(4, 5) are more potent than their para analogs (3, 16). The crystal structures of GPb in complex with these
compounds at high resolution (1.97–2.05 Å) revealed that the inhibitors are accommodated at the cata-
lytic site and stabilize the T conformation of the enzyme. The differences in their inhibitory potency can
be interpreted in terms of variations in the interactions with protein residues of the different substituents
on the aromatic part of the inhibitors.
X-ray crystallography
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
cases.1,2 It is characterized by abnormal insulin secretion and/or
insulin resistance. The combination of pancreatic b-cell dysfunc-
The most common type of diabetes is type 2 diabetes mellitus
(T2DM) and it accounts for approximately 90–95% of diabetes
tion and insulin resistance by namely skeletal muscle, the liver
and fat tissue, gives rise to increased blood glucose. The number
of diabetic cases has been increasing exponentially over the past
years and it is estimated that by the year 2030 the number of pre-
valent patients will reach 300 million.3 The commonly prescribed
hypoglycemic agents (biguanides, sulfonylureas, thiazolidinedi-
Abbreviations: GP, glycogen phosphorylase; GPb, rabbit muscle glycogen
phosphorylase b; PLP, pyridoxal 50-phosphate; Glc-1-P,
a-D-glucose 1-phosphate;
rmsd, root–mean–square deviation.
ones,
a-glucosidase inhibitors) for symptomatic treatment of
⇑
Corresponding authors. Address: Department of Biochemistry and Biotechnol-
ogy, University of Thessaly, 26 Ploutonos Str., 41221 Larissa, Greece. Tel.: +30 2410
565278; fax: +30 2410 565290 (D.D.L.); Université de Lyon, Institut de Chimie et
Biochimie Moléculaires et Supramoléculaires associé au CNRS, UMR 5246, Labora-
toire de Chimie Organique 2-Glycochimie, Bâtiment Curien, 43 boulevard du 11
Novembre 1918, F-69622 Villeurbanne, France. Tel.: +33 (0) 4 72 43 11 61; fax : +33
(0) 4 72 43 27 52 (J.-P.P.).
diabetes4,5 have undesirable side effects and are inadequate for
30–40% of patients.6 This inadequacy of the current pharmacolog-
ical agents for the treatment of T2DM is an incentive for research
efforts towards new therapies.
Glycogen phosphorylase (GP) is a regulatory enzyme that cata-
lyzes the first step of the intracellular degradation of glycogen, an
important source of glucose production. This enzyme is found in
E-mail addresses: jean-pierre.praly@univ-lyon1.fr (J.-P. Praly), ddleonidas@
bio.uth.gr (D.D. Leonidas).
z
Deceased on August 31st 2008
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.024

5126
K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
every species from unicellular organisms and bacteria to the com-
plex tissues of higher plants and mammals, where it plays a key
role in carbohydrate metabolism. The best studied example of gly-
cogen phosphorylase is the rabbit muscle enzyme, which was first
isolated and characterized by G.T. Cori and C.F. Cori in 1936.7 Rab-
bit muscle GP is a dimer composed of two identical subunits 842
amino acids each, and a co-factor, pyridoxal 50-phosphate (PLP).
It is regulated allosterically by phosphorylation and it exists in
two forms: the unphosphorylated T state GPb and the phosphory-
lated R state GPa.8 A large number of compounds have been re-
ported to bind at five distinct binding sites: the catalytic, the
allosteric, and the new allosteric, the inhibitor and the glycogen
storage site.9,10 The efficacy of such inhibitors on blood glucose
control and hepatic glycogen balance has been confirmed from ani-
mal studies and in vitro cell biology experiments9,11,12 validating
GP as an important target for structure based inhibitor design of
new hypoglycemic agents.
The catalytic site of the enzyme is a deep cavity 15 Å from the
protein surface accessible via a channel blocked by the inhibitor
site and the 280s loop (residues 282–287). From T state to R state
transition (activation of the enzyme), the 280s loop becomes disor-
dered and displaced, opening this channel, allowing the entrance of
the substrate and inducing the conformational change of a crucial
residue, Arg569, to enter the catalytic site in place of Asp283 in or-
der to create the phosphate recognition site.8 Inhibition of glyco-
genolysis towards antidiabetic and other therapies has been
reviewed recently, and updated information about glycogen phos-
phorylase is available, concerning in particular computations,13
inhibitors binding at the active site14,15 and the physiological con-
trol of liver glycogen metabolism studied in the light of novel gly-
cogen phosphorylase inhibitors (GPis) .16
1,4-benzoquinone, while the 1,4-dimethoxy analog was not an inhib-
itor of GPb.25 Annelation of dimethyl b-
-glucosyl-hydroquinones led
to C-b- -glucopyranosyl-chromanes and C-b- -glucopyranosyl-vita-
min derivatives which showed interesting anti-oxidant
D
D
D
E
properties.26 Related C-glycosyl amino-substituted hydro- and ben-
zoquinones were synthesized and evaluated for their antitumor
activity.27 Although weak inhibitors, the C-glucosyl hydro- and ben-
zoquinones represented a new type of GPis, with a C-glycosidic bond
resistant to hydrolysis and a six-membered ring amenable to struc-
tural modifications that might result in improved affinity for the
enzyme. Introduction of substituents at the C-5 and C-6 positions of
glucosyl hydroquinone (see Fig. 1 for numbering) was considered
promising due to the proximity of the Asn284 sidechain with the phe-
nyl ring and the interactions observed by crystallography for these
two carbon atoms with other residues of the enzyme (C-5 with N
Asn284, C-6 with OD1 Asp283, and CB Leu136; see a reported discus-
sion25 in which the C-5 and C-6 carbons of the hydro/benzoquinone
ring werenumbered C-10 and C-11, asfor the nextstructuralstudies).
With our experience on the regioselective nitration of 2-(b-
pyranosyl)-1,4-dimethoxy-benzene,27 we synthesized C-5 or C-6 hal-
ogenated C- -glucosyl hydroquinones and four of them were
D-gluco-
D
evaluated as GPis by kinetic experiments. Furthermore, to explore
the molecular basis of their inhibition we have also determined the
crystal structures of each of these four compounds in complex with
GPb and the results are disclosed herein.
2. Materials and methods
2.1. Organic synthesis
2.1.1. General methods
Within collaborative programs devoted to the design, synthesis,
and evaluation by enzymology and crystallography of glucose-based
molecules, we recently demonstrated that glucose-based motifs of
the spirobicyclic or heterocyclic type (Fig. 1) are promising GPis, as
when equipped with a 2-naphthyl substituent. The corresponding
Dichloromethane was washed three times with water, dried
(CaCl2), and distilled over CaH2 before use. Other organic solvents
were distilled. Thin-layer chromatography (TLC) was carried out
on aluminium sheets coated with silica gel 60 F254 (Merck, Darms-
tadt, Germany). TLC plates were inspected under UV light (254,
312 nm), and/or developed by treatment with a mixture of 5%
H2SO4 in EtOH followed by heating. Silica gel column chromatogra-
spiro-isoxazoline
(Ki = 0.16
M),19,20 3- (or 5)-C-b-
C21 and D,17 (Ki = 38 and 11.6
1-(3-deoxy-3-fluoro-b- -glucopyranosyl) cytosine
M),23 wereto dateamong thebestGPisknowntobindat theenzyme
catalytic site. We also reported on the synthesis, by multi-step
efficient routes, of C- -glucopyranosyl hydro-(and -1,4-benzo)qui-
A
(Ki = 0.63
l
M),17,18 spiro-oxathiazole
B
l
D
-glucopyranosyl-1,2,4-oxadiazoles,
22
phy was performed with GeduranÒ silica gel Si 60 (40–63
lm) pur-
lM, respectively)
and N-benzoyl
(Ki = 46.42
chased from Merck. 1H and 13C NMR spectra were recorded at 23 °C
using Bruker AC200, DRX300 or DRX500 spectrometers with the
residual solvent as the internal standard. The following abbrevia-
tions are used to indicate the observed multiplicities: s, singlet;
d, doublet; dd, doublet of doublet; t, triplet; q, quadruplet; m, mul-
tiplet; br, broad. NMR solvents were purchased from Euriso-Top
(Saint Aubin, France). HRMS (LSIMS) mass spectra were recorded
in the positive mode (unless stated otherwise) using a Thermo
Finnigan Mat 95 XL spectrometer. MS (ESI) mass spectra were
D
E
l
D
nones ultimately deacetylated to afford potentially active water-sol-
uble materials.24 Kinetic and crystallographic studies showed that 7
andF (Fig. 1)are competitive inhibitorsof GPdue tobindingatthecat-
alytic site, with Ki values found to be 0.9 and 1.3 mM for, respectively,
2-(b-D-glucopyranosyl)-hydroquinone and the corresponding
Figure 1. Recently reported inhibitors of GP, with their Ki values against RMGPb (lM) and new ones described herein.

K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
5127
recorded in the positive mode using a Thermo Finnigan LCQ spec-
trometer. Optical rotations were measured using a Perkin Elmer
241 polarimeter in a 1 dm cell. Compounds 1, 2, 5, 6, and 7 were
obtained as described previously.26
2.1.3.1. Compound 9. White solid; mp 118.5–120 °C (CH2Cl2/
petroleum ether/Et2O), ½a D18
ꢁ
ꢂ19.4 (c 0.69 CH2Cl2); lit.28
½ ꢁ
a 1D8
ꢂ18.7 (c 1.0 CH2Cl2); 13C NMR (50 MHz, CDCl3): d 170.7 (C@O, acet-
yl), 170.3 (C@O, acetyl), 169.6 (C@O, acetyl), 169.2 (C@O, acetyl),
151.7 (C –OMe), 150.5 (C –OMe), 124.5 (Carom.), 116.7 (CHarom.),
112.5 (Carom.), 111.9 (CHarom.), 76.3, 74.5, 73.3, 71.8, 68.8 (C1–C5),
62.4 (C6), 56.9 (OMe), 56.6 (OMe), 20.8, 20.7, 20.7, 20.5 (4 CH3,
acetyl).
2.1.2. 5-Chloro-2-(2,3,4,6-tetra-O-acetyl-b-
1,4-dimethoxy-benzene (8) and 6-chloro-2-(2,3,4,6-tetra-O-
acetyl-b- -glucopyranosyl)-1,4-dimethoxy-benzene (10)
D-glucopyranosyl)-
D
A mixture of compound 1 (637 mg, 1.36 mmol), N-chlorosuccin-
imide (254 mg, 1.91 mmol, 1.4 equiv) and NH4NO3 (21.8 mg,
0.272 mmol, 0.4 equiv) in dry CH3CN (10 mL) was stirred at 60–
65 °C for 3 h. As indicated by TLC, the starting material (Rf 0.29
petroleum ether/AcOEt 3:2) was totally converted into two more
mobile compounds (Rf 0.31 and 0.34, respectively, petroleum
ether/AcOEt 3:2, red-violet under UV 312 nm). The reaction mix-
ture was poured into water (20 mL) and extracted with CH2Cl2
(3 ꢀ 20 mL). The extracts were combined and washed with brine,
dried over MgSO4, and evaporated. The residue was purified by
chromatography (petroleum ether/AcOEt 4:1) on silica gel to afford
pure compound 8 (633 mg, yield 93%) and compound 10 (12 mg,
yield 1.5%).
2.1.3.2. Compound 11. Pale yellow oil; 1H NMR (300.14 MHz,
CDCl3): d 7.08 (d, 1H, J = 3.0 Hz, Ar), 6.89 (d, 1H, Ar), 5.39 (t, 1H,
J3,4 = 9.3 Hz, H3), 5.34 (t, 1H, J2,3 = 9.3 Hz, H2), 5.21 (m, 1H, H4),
4.84 (br d, 1H, J1,2 = ꢃ9.9 Hz, H1), 4.24 (dd, 1H, J5,6 = 5.1 Hz,
0
0
0
J6,6 = 12.3 Hz, H6), 4.12 (dd, 1H, J5,6 = 2.7 Hz, H6 ), 3.89 (m, 1H,
H5), 3.83 (s, 3H, OMe), 3.77 (s, 3H, OMe), 2.06, 2.06, 2.02, 1.82
(4s, 12H, OAc); 13C NMR (75.5 MHz, CDCl3): d 171.0, 170.7, 169.9,
169.5 (4 C@O, acetyl), 156.7 (C–OMe), 150.0 (C–OMe), 131.5 (Car-
om.), 120.4 (CHarom.), 117.9 (Carom.), 112.9 (CHarom.), 76.8 (C5), 74.9
(C1 and C2), 71.6 (C3), 69.1 (C4), 62.8 (C6), 62.5 (OMe), 56.5
(OMe), 21.1, 21.1, 21.1, 20.9 (4 CH3, acetyl); MS(CI) m/z (%): 547
(90%)
C
[ [
79Br+H]+, 549 (100%) 81Br+H]; HRMS-CI: Calcd for
22H28BrO11 [M+H]+: 547.0815. Found: 547.0815.
2.1.2.1. Compound 8. White solid mp 110–112 °C (CH2Cl2/petro-
leum ether), ½a 1D8
ꢁ
ꢂ16.2 (c 0.76 CH2Cl2); 1H NMR (300.13 MHz,
2.1.4. 5-Chloro-2-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-
1,4-benzoquinone (12)
CDCl3): d 6.96 (s, 1H, Ar), 6.90 (s, 1H, Ar), 5.40–5.17 (m, 3H, H2,
0
H3, H4), 4.91 (d, 1H, J1,2 = 9.5 Hz, H1), 4.26 (dd, 1H, J6,6 = 12.4 Hz,
2.1.4.1. Prepared from 8. Compound 8 (256 mg, 0.508 mmol)
dissolved in CH3CN (2 mL) was reacted with CAN (1.02 g,
2.03 mmol, 4 equiv) dissolved in water (5 mL). After stirring the
mixture for 2 h at rt, TLC showed the complete conversion of com-
pound 8, in favor of compound 12 only (Rf 0.48 petroleum ether/
AcOEt 5:4 red-violet under UV 312 nm, black under UV 254 nm).
Water (10 mL) was poured into the reaction mixture which was
extracted with CH2Cl2 (3 ꢀ 15 mL). The extracts were combined
and washed with brine, water, dried over MgSO4, and concen-
trated. Chromatography of residue on silica gel with petroleum
ether/AcOEt 3/2 led to pure 12 (172 mg, 95% yield).
0
0
J5,6 = 4.8 Hz, H6), 4.12 (dd, 1H, J5,6 = 2.3 Hz, H6 ), 3.86 (s, 3H,
OMe), 3.79 (s, 3H, OMe), 3.88–3.75 (m, 1H, H5), 2.06, 2.05, 2.00,
1.79 (4s, 12H, OAc); 13C NMR (50 MHz, CDCl3): d 170.7 (C@O, acet-
yl), 170.3 (C@O, acetyl), 169.6 (C@O, acetyl), 169.1 (C@O, acetyl),
151.5 (C–OMe), 149.5 (C–OMe), 123.7 (Carom.), 123.3 (Carom.),
113.7 (CHarom.), 112.2 (CHarom.), 76.3, 74.5, 73.2, 71.8, 68.7 (C1-
C5), 62.4 (C6), 56.7 (OMe), 56.5 (OMe), 20.8, 20.7, 20.7, 20.4 (4
CH3, acetyl); MS(CI) C22H27ClO11: 503 (100%, 35Cl), 505 (33%, 37Cl)
[M+H]+.
2.1.2.2. Compound 10. Pale yellow oil, ½a D20
ꢂ16.5 (c 3.2 CH2Cl2);
ꢁ
1H NMR (300.14 MHz, CDCl3): d 6.92 (d, 1H, J = 3.0 Hz, Ar), 6.84 (d,
1H, Ar), 5.38 (t, 1H, J2,3 = 6.9 Hz, H2), 5.35 (t, 1H, J3,4 = 6.9 Hz, H3),
5.22 (m, 1H, H4), 4.85 (br d, 1H, J1,2 = ꢃ9.9 Hz, H1), 4.25 (dd, 1H,
2.1.4.2. Prepared from 14. 5-Chloro-2-(2,3,4,6-tetra-O-acetyl-b-
D-glucopyranosyl)-hydroquinone (14) [prepared from 2 (285 mg,
0.6 mmol) and (CH3)3SiCl—vide infra] was dissolved in CHCl3.
Freshly prepared Ag2O (1.18 g, 5.1 mmol, 8.5 equiv) was added
by small portions while stirring the mixture at rt. TLC indicated
the complete conversion of the starting material into a more mo-
bile product after 3 h (Rf = 0.59 petroleum ether/AcOEt 3:2). After
removal of the solids by filtration, the solvent was evaporated un-
der reduced pressure. The residue was purified by chromatography
(petroleum ether/AcOEt 2:1) on silica gel to afford 12 (221 mg, 78%
yield).
0
0
0
J5,6 = 5.1 Hz, J6,6 = 12.3 Hz, H6), 4.11 (dd, 1H, J5,6 = 2.1 Hz, H6 ),
3.91–3.85 (m, 1H, H5), 3.84 (s, 3H, OMe), 3.77 (s, 3H, OMe), 2.06,
2.06, 2.02, 1.82 (4s, 12H, OAc); 13C NMR (50 MHz, CDCl3): d 170.7
(C@O, acetyl), 170.3 (C@O, acetyl), 169.6 (C@O, acetyl), 169.1
(C@O, acetyl), 156.1 (C–OMe), 148.5 (C–OMe), 131.2 (Carom.),
128.4 (Carom.), 117.1 (CHarom.), 111.8 (CHarom.), 76.4, 74.6, 74.3,
71.3, 68.7 (C1-C5), 62.4 (C6), 61.9 (OMe), 55.8 (OMe), 20.8, 20.7,
20.7, 20.5 (4 CH3, acetyl); HRMS-EI M+ꢀ calcd for C22H27ClO11
502.1242; found 502.1241.
:
2.1.4.3. Compound 12. Golden-yellow solid, mp 101–102 °C
(CH2Cl2/petroleum ether); Rf = 0.59 (petroleum ether/AcOEt 3:2
2.1.3. 5-Bromo-2-(2,3,4,6-tetra-O-acetyl-b-
1,4-dimethoxy-benzene (9) and 6-bromo-2-(2,3,4,6-tetra-O-
acetyl-b- -glucopyranosyl)-1,4-dimethoxy-benzene (11)
D-glucopyranosyl)-
red-violet under UV 312 nm, black under UV 254 nm); ½a D19
ꢁ
D
ꢂ40.7 (c 0.78 CH2Cl2); 1H NMR (300.13 MHz, CDCl3:) d 7.03 (s,
1H, Ar), 6.98 (s, 1H, Ar), 5.36 (t, 1H, J3,4 = 9.3 Hz, H3), 5.13 (t, 1H,
J4,5 = 9.6 Hz, H4), 4.94 (t, 1H, J2,3 = 9.6 Hz, H2), 4.61 (d, 1H,
A mixture of compound 1 (240 mg, 0.513 mmol), N-bromosuc-
cinimide (147 mg, 0.821 mmol, 1.6 equiv) and NH4NO3 (16.5 mg,
0.205 mmol, 0.4 equiv) in dry CH3CN (8 mL) was stirred at rt for
about 3 h. TLC showed the starting material (Rf 0.42 petroleum
ether/AcOEt 1:1) was completely converted into 2 more mobile
compounds (Rf 0.46, 0.48, petroleum ether/AcOEt 1:1, red-violet
under UV 312 nm). Water (15 mL) was added to the reaction mix-
ture which was extracted with CH2Cl2 (3 ꢀ 15 mL). The extracts
were combined, washed with brine, water, then dried over MgSO4,
and evaporated. The residue was purified by chromatography
(petroleum ether/AcOEt 4:1) on silica gel to afford pure com-
pounds 928 (261 mg, 93% yield) and 11 (4.7 mg, 2% yield).
0
J1,2 = 9.9 Hz, H1), 4.24 (dd, 1H, J5,6 = 4.8 Hz, J6,6 = 12.3 Hz, H6), 4.12
0
0
(dd, 1H, J5,6 = 2.1 Hz, H6 ), 3.80 (ddd, 1H, H5), 2.09, 2.05, 2.01,
1.91 (4 s, 12H, OAc); 13C NMR (50 MHz, CDCl3): d 183.5 (C@O,
benzoquinone), 179.2 (C@O, benzoquinone), 170.6 (C@O, acetyl),
170.0 (C@O, acetyl), 169.9 (C@O, acetyl), 169.5 (C@O, acetyl),
144.9 (Cquin.), 144.2 (Cquin.), 133.4 (CHquin.), 133.3 (CHquin.), 76.3,
73.5, 72.6, 72.0, 68.2 (C1–C5), 62.0 (C6), 20.8 (CH3, acetyl), 20.6
(CH3, acetyl), 20.6 (CH3, acetyl), 20.5 (CH3, acetyl); MS (ESI) 495.0
(100%), 497.0 (43%) [M+23]+; MS (CI) [M+H]+ 473; HRMS (CI):
Calcd for C20H22O11Cl [M+H]+ 473.0851. Found: 473.0852.

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K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
2.1.5. 5-Bromo-2-(2,3,4,6-tetra-O-acetyl-b-
D
-glucopyranosyl)-
2.1.7. 5-Bromo-2-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-
1,4-benzoquinone (13)
hydroquinone (15)
Compound 13 was prepared by CAN oxidation of 9 in 90% yield
as for synthesis of 12. Compound 13: golden-yellow solid, mp
A freshly prepared aqueous solution (6 mL) of Na2S2O4 (85%
tech., 349 mg, 2.01 mmol, 6 equiv) was introduced in a flask con-
taining compound 13 (173 mg, 0.335 mmol) dissolved in 5 mL
CHCl3. The reaction mixture was stirred vigorously at rt. After
80 min, TLC showed compound 13 (Rf 0.60, petroleum ether/THF
3:2 red-violet under UV 312 nm, dark spot under UV 254 nm)
was changed into a more polar compound (Rf 0.37, petroleum
ether/THF 3:2 red-violet under UV 312 nm). The reaction mixture
was extracted with CHCl3 (3 ꢀ 15 mL), washed with brine, water,
dried over MgSO4. After filtration and concentration, the residue
was purified by chromatography (petroleum ether/AcOEt 3:2) on
silica gel to afford pure compound 15 (162 mg, 93% yield).
141–142 °C (CH2Cl2/petroleum ether), ½a D20
ꢂ40.0 (c 0.66 CH2Cl2);
ꢁ
1H NMR (300.13 MHz, CDCl3): d 7.28 (s, 1H, Ar), 7.09 (s, 1H, Ar),
5.37 (t, 1H, J3,4 = 9.3 Hz, H3), 5.14 (t, 1H, J4,5 = 9.6 Hz, H4), 4.95 (t,
1H, J2,3 = 9.3 Hz, H2), 4.61 (d, 1H, J1,2 = 9.9 Hz, H1), 4.26 (dd, 1H,
0
0
0
J5,6 = 4.8 Hz, J6,6 = 12.6 Hz, H6), 4.14 (dd, 1H, J5,6 = 2.1 Hz, H6 ),
3.81 (ddd, 1H, H5), 2.11, 2.06, 2.03, 1.93 (4 s, 12H, OAc); 13C NMR
(50 MHz, CDCl3): d 183.1 (C@O, benzoquinone), 179.1 (C@O,
benzoquinone), 170.6 (C@O, acetyl), 170.0 (C@O, acetyl), 169.9
(C@O, acetyl), 169.5 (C@O, acetyl), 144.8 (Cquin.), 137.8 (CHquin.),
137.6 (Cquin.), 133.1 (CHquin.), 76.3, 73.5, 72.6, 72.0, 68.2 (C1–C5),
62.0 (C6), 20.8, 20.6, 20.6, 20.5 (4 CH3, acetyl); MS (+c ESI) for
C
20H21O11Br: 456.8 (79Br) and 458.8 (81Br) [M+HꢂCH3COOH]+,
2.1.7.1. Compound 15. White solid, mp 155–156 °C; ½a D23
ꢂ32.7
ꢁ
516.7 [M+H]+, 538.9 (79Br) and 541.0 (81Br) [M+Na]+.
(c 0.7 CH2Cl2); 1H NMR (300.13 MHz, CDCl3) d 7.03 (s, 1H, Ar),
6.68 (s, 1H, exchangeable with D2O, OH), 6.68 (s, 1H, Ar), 5.33 (t,
1H, J3,4 = 9.0 Hz, H3), 5.26 (t, 1H, J2,3 = 9.6 Hz, H2), 5.24 (t, 1H,
J4,5 = 9.6 Hz, H4), 5.17 (s, 1H, exchangeable with D2O, OH), 4.52
2.1.6. 5-Chloro-2-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-
hydroquinone (14)
2.1.6.1. Prepared from 12. A freshly prepared aqueous solution
(4 mL) of Na2S2O4 (85% tech., 222 mg, 1.27 mmol, 6 equiv) was
introduced in a flask containing a solution of compound 12
(100 mg, 0.212 mmol) in 5 mL CHCl3. After the mixture was stirred
vigorously for 40 min at rt, TLC showed compound 12 (Rf 0.60,
petroleum ether/AcOEt 1:1 red-violet under UV 312 nm, dark spot
under UV 254 nm) was completely converted into a more polar
compound (Rf 0.37, petroleum ether/AcOEt 1:1 red-violet under
UV 312 nm). After extracting the reaction mixture with CHCl3
(3 ꢀ 15 mL), the combined organic phase was washed with brine,
water, and dried over MgSO4. After filtration and concentration,
the residue was purified by chromatography (petroleum ether/
AcOEt 3:2) on silica gel to afford pure compound 14 (88 mg, 88%
yield).
0
(d, 1H, J1,2 = 9.3 Hz, H1), 4.32 (dd, 1H, J5,6 = 3.6 Hz, J6,6 = 12.6 Hz,
0
0
H6), 4.16 (dd, 1H, J5,6 = 2.1 Hz, H6 ), 3.87 (dq, 1H, H5), 2.12, 2.06,
2.01, 1.88 (4s, 12H, OAc); 13C NMR (50 MHz, CDCl3): d 170.7,
170.4, 169.5, 168.9 (4 C@O, acetyl), 149.0 (C–OH), 145.9 (C-OH),
121.7 (Carom.), 120.9 (CHarom.), 115.2 (CHarom.), 111.0 (Carom.), 79.2,
76.2, 73.6, 70.6, 67.9 (C1–C5), 61.6 (C6), 20.7, 20.7, 20.6, 20.5
(4 CH3, acetyl); MS-CI: m/z: 519, 521 [M+H]+; HRMS-CI: Calcd for
C20H24BrO11 [M+H] 519.0502. Found: 519.05045.
2.1.8. 6-Chloro-2-(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-
hydroquinone (18)
2.1.8.1. Prepared from 2. Compound 2 (169 mg, 0.386 mmol)
was dissolved in 15 mL dry THF. Then freshly distilled AcCl
(110 lL, 1.54 mmol, 4 equiv) and water (0.3 mL) were successively
added to the above solution. After 6 h, TLC showed that compound
2 (Rf 0.60, petroleum ether/AcOEt 1:1 red-violet under UV 312 nm,
dark spot under UV 254 nm) almost disappeared while four more
polar spots became visible on the plates. After adding more water
(5 mL), the mixture was extracted with CH2Cl2 (3 ꢀ 15 mL) and the
combined organic phase was washed with brine, water, and dried
over MgSO4. After filtration and concentration, the residue was
purified by chromatography (petroleum ether/AcOEt 5:2) on silica
gel to afford 14 (45 mg, 24% yield) and 18 (109 mg, 60% yield).
2.1.6.2. Prepared from 2. Neat chlorotrimethylsilane (CH3)3 SiCl
(0.15 mL, 1.2 mmol, 1.5 equiv) and BF3ꢄEt2O (0.01 mL, 0.08 mmol,
0.1 equiv) were successively added to a stirred solution of 2-
(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-1,4-benzoquinone (2,
350 mg, 0.8 mmol) in 10 mL CH3CN. TLC showed that the starting
material was completely transformed into a more polar product
within 14 h. After quenching by addition of water (15 mL), the reac-
tion mixture was extracted with CH2Cl2 (3 ꢀ 15 mL). The extracts
were combined, washed with brine, dried (MgSO4), and evaporated.
The residue was purified by chromatography (petroleum ether/
AcOEt 7:3) on silica gel to afford pure 14 (318 mg, 84% yield). See be-
low for another access from 2 to 14 and 18 as, respectively, the minor
and major products.
2.1.8.2. Prepared from 10. In another route, as compound 10 was
only partially converted (ꢃ10%) to 17 upon oxidation with CAN
(10 equiv, two days), this mixture was treated with aqueous
Na2S2O4 to afford, after separation on column of unreacted 10, a
low amount of 18 (7 mg).
2.1.6.3. Compound 14. White solid, mp 165–166 °C (not recrys-
tallized); Rf = 0.20 (petroleum ether/AcOEt 2:1); ½a D21
ꢂ33.1 (c
ꢁ
0.66 CH2Cl2); 1H NMR (300.14 MHz, CDCl3): d 6.89 (s, 1H, Ar),
6.74 (br s, 1H, OH exchangeable with D2O), 6.69 (s, 1H, Ar),
5.35–5.23 (m, 3H, H2, H3, H4), 5.17 (br s, OH exchangeable with
D2O), 4.53 (d, 1H, J1,2 = 9.2 Hz, H1), 4.33 (dd, 1H, J5,6 = 3.6 Hz,
2.1.8.3. Compound 18. Colorless oil, Rf = 0.46 (petroleum ether/
AcOEt 1:1 red-violet under UV 312 nm); ½a D20
ꢂ15.7 (c 1.10, CHCl3);
ꢁ
1H NMR (500 MHz, CDCl3): d 6.83 (d, 1H, J = 2.8 Hz, Ar), 6.69 (d, 1H,
J = 2.8 Hz, Ar), 5.90 (br s, 1H, OH), 5.37 (t, 1H, J = 9.4 Hz), 5.23 (q,
2H, J = 9.7 Hz), 4.81 (d, 1H, J1,2 = 9.9 Hz, H1), 4.30 (dd, 1H,
0
0
0
J6,6 = 12.6 Hz, H6), 4.17 (dd, 1H, J5,6 = 2.2 Hz, H6 ), 3.88 (ddd, 1H,
J4,5 = 9.3 Hz, H5), 2.12, 2.07, 2.01, 1.89 (4 s, 12H, OAc); 13C NMR
(50.32 MHz, CDCl3): d 170.7, 170.4, 169.4, 168.9 (4 C@O, acetyl),
148.9 (C-OH), 144.8 (C-OH), 121.0 (Carom.), 120.9 (Carom.), 118.1
(CHarom.), 115.5 (CHarom.), 79.4, 76.2, 73.6, 70.6, 67.8 (C1-C5), 61.5
(C6), 20.7, 20.7, 20.6, 20.5 (4 CH3, acetyl); MS (+c ESI): 474.8
[M+H]+; 491.9 [M+NH4]+; 497.0 [M+Na]+; 970.5, 972.6 [2 M+Na]+;
MS (ꢂc ESI): 472.8: [MꢂH]ꢂ; 508.9 [M+Cl]ꢂ; 946.7 [2MꢂH]ꢂ;
0
0
J5,6 = 4.5 Hz, J6,6 = 12.5 Hz, H6), 4.15 (m, 2H, OH, H6 ), 3.88 (m, 1H,
0
J4,5 = 10 Hz, J5,6 = 4.5 Hz, J5,6 = 2 Hz, H5), 2.09, 2.06, 2.01, 1.86 (4s,
12H, OAc); 13C NMR (75 MHz, CDCl3) d 170.9, 170.5, 169.7, 169.3
(4 C@O, acetyl), 149.4, 143.6 (2 C–OH), 123.5, 120.9, 116.9, 113.8
(4 C, Ar), 76.1, 75.6, 74.0, 71.6, 68.3 (C1–C5), 62.0 (C6), 20.7, 20.6,
20.6, 20.4 (4 CH3, acetyl); MS(ESI+) m/z (%):475.1 (25) [M]+,
497.1 (100) and 499.1 (32) [M+Na]+, 1116.6 [2 M+Na]+; HRMS(E-
SI+) m/z: Calcd for [M+Na]+ C20H23Na1Cl1O11: 497.0827. Found:
497.0821.
HRMS-CI: Calcd for
473.1211.
C20H24ClO11 [M+H] 473.1214. Found:

K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
5129
2.1.9. 5-Chloro-2-(b-
2.1.9.1. Prepared by acid-catalyzed deacetylation of 14.
2-(2,3,4,6-Tetra-O-acetyl-b- -glucopyranosyl)-5-chloro-1,4-hydro-
quinone (14) (142 mg, 0.30 mmol) was dissolved in 10 mL dry
CH3OH. AcCl (61 L, 0.86 mmol, 2.9 equiv) was added to the solu-
tion. The mixture protected from light was stirred at rt under argon
for six days, whereupon TLC showed that the starting material (Rf
0.37 petroleum ether/AcOEt 1:1 red-violet under UV 312 nm)
was converted into a new very polar product (Rf 0.24 CH2Cl2/
CH3OH 4:1, red-violet under UV 312 nm). After concentration un-
der vacuum, the residue was purified by chromatography
(CH2Cl2/CH3OH 10:1) on silica gel to afford 3 (85 mg, 93% yield).
D
-glucopyranosyl)-hydroquinone (3)
4H, H2, H3, H4, H5); 13C NMR (100.6 MHz, CD3OD) d 150.5, 143.6
(2 C–OH), 128.9, 121.7, 115.3, 113.4 (4 C, Ar), 80.9, 78.4, 77.0,
74.8, 70.2 (C1–C5), 61.5 (C6); 13C NMR (50.32 MHz, D2O) d 150.0,
143.9 (2 C–OH), 128.1, 123.0, 117.2, 114.4 (4 C, Ar), 80.6, 77.8,
76.9, 73.9, 70.1 (C1–C5), 61.2 (C6); MS(ESI+) m/z (%): [M+Na]+
329.0 (100), [M+Na]+ 331.0 (30); HRMS (ESI+) m/z: Calcd for
[M+Na]+ C12H15Na1Cl1O7: 329.0404. Found: 329.0408.
D
l
2.1.11. 5-Bromo-2-(b-
After compound 15 (127 mg, 0.245 mmol) was dissolved in
10 mL dry CH3OH, AcCl (50 L, 0.49 mmol, 2 equiv) was added to
D-glucopyranosyl)-1,4-hydroquinone (16)
l
the solution. The mixture protected from light was stirred at rt un-
der argon for six days. TLC showed that the starting material (Rf
0.34 petroleum ether/AcOEt 1:1 red-violet under UV 312 nm)
was converted into a new polar product (Rf 0.24 CH2Cl2/CH3OH
4:1, red-violet under UV 312 nm). After concentration under vac-
uum, the residue was purified by chromatography (CH2Cl2/CH3OH
10:1) on silica gel to afford compound 16 (82 mg, 95% yield).
2.1.9.2. Prepared by base-catalyzed deacetylation of 14.
2-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-5-chloro-1,4-hydro-
quinone 14 (100 mg, 0.21 mmol) was dissolved in 8 mL dry CH3OH.
A solution of CH3ONa in methanol (0.1 M, 4.2 mL) was added under
argon. After the reaction mixture was stirred at rt for 2 h, TLC
showed compound 14 changed into a more polar compound (Rf
0.24 CH2Cl2/CH3OH 4:1, red-violet under UV 312 nm). Then Dowex
50wX2 (H+) ion-exchange resin was cautiously added to the reac-
tion mixture, so as to reach pH ꢃ6 (litmus paper). Without delay,
the resin was removed by filtration and after concentration, the
residue was applied to a short column (CH2Cl2/CH3OH 10:1) to af-
ford compound 3 (62 mg, 96% yield).
2.1.11.1. Compound 16. White foam. ½a D20
ꢁ
+3.4 (c 1 CH3OH); 1H
NMR (300.13 MHz, D2O) d 7.09 (s, 1H, Ar), 6.95 (s, 1H, Ar), 4.55
0
(d, 1H, J1,2 = 9.3 Hz, H1), 3.82 (dd, 1H, J5,6 = 1.2 Hz, J6,6 = 12 Hz,
0
0
H6), 3.72 (dd, 1H, J5,6 = 4.2 Hz, H6 ), 3.64–3.50 (m, 4H, H2, H3, H4,
H5); 13C NMR (50.32 MHz, D2O) d 148.8 (C–OH), 146.6 (C–OH),
125.4 (Carom.), 120.9 (CHarom.), 116.4 (CHarom.), 110.5 (Carom.), 80.6,
77.9, 76.3, 73.7, 70.1 (C1–C5), 61.2 (C6); MS ESI +c: 373, 375 1:1
ratio [M+Na]+; 722.7, 724.6, 726.8 1:2:1 ratio [2M+Na]+; MS ESI
ꢂc: 349.0, 351 1:1 ratio [MꢂH]ꢂ; 386.8 [M+Cl]ꢂ; 734,6, 736.6,
738.6 [2M+Cl]ꢂ; HRMS-LSIMS: Calcd for C12H14BrO7 [MꢂH]ꢂ
348.9923. Found: 348.9920.
2.1.9.3. Prepared by acid-catalyzed deacetylation of 2. After
compound 2 (252 mg, 0.575 mmol) was dissolved in 10 mL MeOH,
freshly distilled AcCl (1 mL, 25 equiv) was added via a syringe into
the above mixture (9% v/v AcCl in MeOH). At once, the color of the
solution changed from orange to almost colorless. After the mix-
ture was stirred at rt for 30 h, TLC showed the starting material
was converted to more polar deacetylated compounds giving two
spots (Rf 0.55 and 0.18, MeOH/CH2Cl2 1:4). The volatiles were
evaporated under reduced pressure. The residue was applied to a
column of chromatography on silica gel (MeOH/CH2Cl2 1:10) to af-
ford compound 5 (18 mg, 0.06 mmol, 10.4% yield) ,25 and the
inseparable mixture of 3 and 4 (82 mg, 46.7% yield, 3/4 ratio:
ꢃ65:35), as a glassy yellow solid, Rf 0.18, MeOH/CH2Cl2 1:4. A sim-
ilar experiment with 100 mg of 2 confirmed these results. In an-
other experiment, compound 2 (90 mg, 0.205 mmol) dissolved in
MeOH (10 mL) was deacetylated with AcCl (1.5 mL, 100 equiv)
upon stirring for 30 h at rt to afford 41 mg (65% yield) of 3 and 4,
with no detectable amount of 5.
2.2. Enzyme isolation and kinetic experiments
Glycogen phosphorylase (GPb) was isolated from rabbit skeletal
muscle and purified as described previously.29 Kinetic studies were
performed in the direction of glycogen synthesis with 5 lg/ml en-
zyme, constant concentrations of glycogen (0.2% w/v), Glc-1-P
(2 mM), AMP (1 mM) and various concentrations of inhibitors.
They were carried out in the presence of 30 mM imidazole/HCl buf-
fer 60 mM KCl, 0.6 mM EDTA, and 0.6 mM dithiothreitol (pH 6.8).
Experiments were performed in the presence of 1% or 2% DMSO
and the Km of the enzyme was 2.1–2.6 mM. Enzyme activity was
measured at pH 6.8 by the release of inorganic phosphate as de-
scribed previously.30
2.1.9.4. Compound 3½a. ꢁD22 +14.4 (c 0.8 CH3OH); 1H NMR (200 MHz,
D2O) d 7.04 (s, 1H, Ar), 7.02 (s, 1H, Ar), 4.64 (d, 1H, J1,2 = 8.8 Hz,H1),
2.3. X-ray crystallography
3.95–3.60 (m, 6H, H2, H3, H4, H5, H6, H6 ); 13C NMR (50.32 MHz,
GPb crystals, grown in the tetragonal lattice, space group
P43212, as previously described31 were soaked with 100 mM of a
mixture of 3 and 4 (21 h), 10 mM of 5 (2.5 h), 20 mM of 16 (6 h),
or 70.1 mM of 3 (20 h) in a solution of the crystallization media
prior to data collection. Diffraction data were collected at the
Daresbury Synchrotron Radiation Source, UK and at the EMBL
Hamburg Outstation, Germany. Data reduction and integration fol-
lowed by scalling and merging of the intensities obtained was per-
formed with the HKL program suite.32 Crystallographic refinement
of the complexes was performed by maximum-likelihood methods
using REFMAC.33 The starting model employed for the refinement
of the complexes was the structure of the native T state GPb com-
plex determined at 1.9 Å resolution (Leonidas et al., unpublished
results). Ligand models of the compounds were fitted to the
electron density maps after adjustment of their torsion angles.
Alternate cycles of manual rebuilding with COOT34 and refinement
with REFMAC improved the quality of the models. The stereochem-
istry of the protein residues was validated by PROCHECK.35 Hydro-
gen bonds and van der Waals interactions were calculated with the
0
D2O) d 148.7 (C-OH), 145.5 (C-OH), 124.7 (Carom.), 121.5 (Carom.),
118.0 (CHarom.), 116.8 (CHarom.), 80.7, 77.9, 76.2, 73.7, 70.2 (C1-C5),
61.3 (C6); MS FAB negative mode: 305 [MꢂH]ꢂ; HRMS (LSIMS):
Calcd for C12H14ClO7 [MꢂH]ꢂ: 305.0428. Found: 305.0435.
2.1.10. 6-Chloro-2-(b-D-glucopyranosyl)-hydroquinone (4)
Compound 18 (96 mg, 0.202 mmol) was dissolved in 6 mL dry
CH3OH. AcCl (0.805 mmol, 4 equiv) was added to the solution.
The mixture protected from light was stirred at rt under argon
for four days. TLC showed that the starting material (Rf 0.46 petro-
leum ether/AcOEt 1:1 red-violet under UV 312 nm) was converted
into a new polar product (Rf 0.20 CH2Cl2/CH3OH 4:1, red-violet un-
der UV 312 nm). After concentration under vacuum, the residue
was purified by chromatography (CH2Cl2/CH3OH 15:1) on silica
gel to afford pure compound 4 (57 mg, 92% yield). ½a D20
ꢁ
+27.2 (c
0.8, CH3OH); 1H NMR (500 MHz, D2O): d 6.93 (d, 1H, J = 2.9 Hz,
Ar), 6.81 (d, 1H, Ar), 4.64 (d, 1H, J = 9.3 Hz, H1), 3.84 (d, 1H,
0
J = 12.2 Hz, H6), 3.73 (dd, 1H, J = 3.2, 12.4 Hz, H6 ), 3.65–3.52 (m,

5130
K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
program CONTACT as implemented in CCP436 applying a distance
cut off of 3.3 and 4.0 Å, respectively. Protein structures were super-
imposed using LSQKAB as implemented in CCP4.36 Figures were
prepared with the programs MolScript37 and PyMol38 rendered
with Raster3D.39 The coordinates of the new structures have been
deposited with the RCSB Protein Data Bank (http://www.rcsb.org/
pdb) with codes 3NP7 (mixture of 3 and 4), 3NP9 (5), 3NPA (16)
and 3S0J (3).
was carried out with lower amounts of AcCl in MeOH, 2 was con-
verted cleanly within one week to 5 (80% yield) which displayed a
methoxy group at the C-4 position.25 As reduction of 2 to hydroqui-
none 6 was straightforward as was its deacetylation to 7 under
either acid or base-catalyzed conditions,25 another route to the tar-
get compounds could be followed. Compound 1 was halogenated
41
by either N-chlorosuccinimide (NCS)
(NBS)
or N-bromosuccinimide
with NH4NO3 as catalyst41,43 affording 827 and 9 (C-5
42
chloro and bromo isomers) both in 93% yield, and trace amount
of the C-6 halogenated regioisomers 10 (chloro) and 11 (bromo).
Upon treatment with CAN in aqueous CH3CN, compounds 8 and
9 were oxidized to the corresponding 1,4-benzoquinones 12 and
13 obtained in high yield. Equally high yielding was their
Na2S2O4-mediated reduction to the corresponding hydroquinones
14 and 15. These were deacetylated in good yields under acid-cat-
alyzed conditions (cat. AcCl in MeOH), while deacetylation under
Zemplén conditions (MeONa in MeOH) proved less reproducible,
with formation of unidentified byproducts. In contrast, the minor
C-6 halogenated isomers 10 and 11 could not be transformed effi-
ciently into benzoquinones because of scarcity and also due to
their higher stability: treatment of 10 with CAN resulted in a
ꢃ10% conversion only, to produce low amounts of 17. As 10 and
17 had a similar mobility on TLC plates, separation of 17 was not
attempted, and the mixture from oxidation was subjected to
reduction with Na2S2O4. The more polar 6-chloro-hydroquinone
18 was easily separated from the starting material 10. However,
the obtained amount (7 mg) was so low that synthesizing 18 by
another route was needed. In agreement with earlier data,44 we
observed that treatment of the glycosyl benzoquinone 2 with
3. Results and discussion
3.1. Organic synthesis
Tetra-O-acetyl-b-D-glucopyranosyl-1,4-dimethoxybenzene 1
(the numbering shown in Scheme 1 is used for discussing the syn-
theses and for the compounds’ names in the Experimental section),
prepared in good yield from penta-O-acetyl-b-
1,4-dimethoxybenzene28 was oxidized with ceric ammonium ni-
trate (CAN) in aqueous acetonitrile to afford b- -glucopyranosyl-
1,4-benzoquinone 2, isolated as moderately stable yellow needles
in almost quantitative yield.24,25 Acid-catalyzed deacetylation of
2 (MeOH containing acetyl chloride, 25/100 equiv) led by addition
of hydrochloric acid and complete deacetylation, to 3 and 4 insep-
arable in our hand (46–65% total yield, ratio: ꢃ65:35), and to 5
formed in less than 10–12% yield. The major isomer 3 displayed
a chlorine atom at the C-5 position (Scheme 1), para related to
the sugar substituent, showing an addition regioselectivity already
D-glucopyranose and
D
observed when 2-(b-
D-glucopyranosylthio)-1,4-benzoquinone was
treated with HCl in CHCl3.40 To our surprise, when deacetylation
Scheme 1. Synthetic routes to halogenated b-D-glucopyranosyl hydroquinone regioisomers. Reagents conditions: (a) CAN (3 equiv), CH3CN/H2O, 1:1 or 2:3; (b) MeOH, AcCl
(25–100 equiv), 30 h; (c) MeOH, AcCl (ꢃ3.3 equiv), eight days; (d) NaBH4 (2 equiv), EtOAc, rt, 30 min; (e) CH3CN, NCS (1.4 equiv), NH4NO3 (0.4 equiv), 60–65 °C, 3 h; (f) CH3CN,
NBS (1.6 equiv), NH4NO3 (0.4 equiv), rt, 3 h; (g) CAN (4 equiv), CH3CN/H2O 2:5, rt, 2 h; (h) Na2S2O4 (6 equiv), CHCl3/H2O, rt; (i) MeOH, AcCl (2.0–2.9 equiv), rt, six days; (j)
catal. MeONa in MeOH, rt, 2 h; (k) (a) CAN (10 equiv), CH3CN/H2O, two days, 10% conversion of 10; b) Na2S2O4 (6 equiv), CHCl3/H2O, 18 (7 mg); (l) CH3CN, Me3SiCl (1.5 equiv),
BF3ꢄOEt2 (0.1 equiv), rt, 14 h; (m) THF/H2O, AcCl (4 equiv), 6 h; (n) MeOH, AcCl (4 equiv), rt, four days; (o) CHCl3, Ag2O (8.5 equiv), rt, 3 h.

K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
5131
Table 1
chlorotrimethylsilane Me3SiCl in the presence of BF3ꢄEt2O in CH3CN
afforded cleanly the corresponding C-5 chlorinated hydroquinone
14 already obtained by another route. We reasoned that the C-6
halogenated hydroquinone could be produced under conditions
similar to those applied for the synthesis of the C-6 chlorinated
compound 5. To our delight, the glycosyl benzoquinone 2, when
treated with AcCl in THF/H2O, gave easily separable C-5 and C-6
chlorinated hydroquinones 14 and 18 in 24 and 60% isolated yield,
respectively. Finally, acid-catalyzed deacetylation (MeOH contain-
ing 0.5% AcCl, rt) proved to be efficient, so the desired compounds 3
and 4 were obtained in high yield and sufficient quantity for fur-
ther tests. The halogenation pattern of the prepared compounds
was established easily by 1H NMR spectroscopy. In case of C-5 sub-
stitution, the two para related protons of the phenyl or 1,4-benzo-
quinone ring appeared both as singlet (Jpara = ꢃ0 Hz), while the
meta related counterparts associated to the C-6 halogenation gave
two doublets (Jmeta = ꢃ3 Hz). In the 1H NMR spectra of 8 and 9 re-
corded at 300 MHz, signals for H-1 appeared as doublet as ex-
pected, but for 10 and 11, the H-1 signals were more complex,
suggesting the existence of conformers due to a restricted rotation
of the C-glycosidic bond, as already observed for related dimethyl
meta-substituted analogs.26
Inhibitory efficiency of the hydroquinone derivatives and the atom numbering
scheme used in crystal structures
Compounds
Chemical structure
IC50 (lM)
O7
OH
O6
OH
C9
O5
O
C6
C8
C7
C4
C10
C11
O4
C5
7
977a
HO
HO
C2
C1
C12
C3
O3
OH
O2
OH
O8
O7
OH
C9
O6
OH
C6
Br
O5
C4
C8
C7
C10
O4
C5
O
16
136.4 4.9
169.9 10.0
140.0 10.0
HO
C11
HO
C2 OH
C1
C12
C3
O3
O2
OH
O8
O7
OH
C9
O6
OH
C6
Cl
O5
C4
C8
C7
C10
O4
C5
O
3
HO
C11
HO
C2 OH C1
C12
C3
O3
O2
OH
O8
O7
OH
C9
3.2. Enzyme kinetics
O6
OH
O5
O
C6
C10
C4
C8
C7
The inhibitory efficiency of three hydroquinone derivatives (3,
5, 16) was tested in kinetic experiments with rabbit muscle glyco-
gen phosphorylase b (GPb) while it was estimated for one (4) and
their inhibitory constants (IC50, Table 1) are compared to that of 7,
as a reference. All compounds displayed competitive inhibition
with respect to Glc-1-P, at constant concentrations of glycogen
(0.2% w/v), AMP (1 mM) and Glc-1-P (2 mM). Kinetic experiments
were carried out in the direction of glycogen synthesis and IC50 val-
O4
C5
Cl
Mixture of 3 and 4
HO
C1 1
HO
C2 OH
C1
C12
C3
O3
O2
OH
O8
O7
OH
C9
O6
OH
O5
O
C6
C10
C11
Cl
C8
C7
C1
C4
O4
C5
4
5
95b
HO
HO
C2
ues obtained are in the lM range. The lead compound 7 displays a
C12
C3
O3
OH
Ki of 0.9 mM25 and it is a slightly better inhibitor than
a
-D
-glucose
OH
O2
O8
45
(Ki = 1.7 mM)
possibly because of the additional interactions of
C13
O7OMe
the 1,4-hydroquinone group with the protein residues.25 Com-
O6
OH
C9
pared to 7, the halogen substituted hydroquinones studied were
O5
O
C6
C5
C4
C8
C7
C2 OH C1
C10
O4
found more potent inhibitors, with IC50 values (lM) comprised be-
39.8 0.3
HO
C11
HO
Cl
tween 169.9 10 (3) and 39.8 0.3 (5).
C12
C3
O3
O2
OH
Comparing compounds 16 and 3 reveals that the inhibitory po-
tency does not change significantly by the replacement of the bro-
mide group by a chloride. However, replacing the hydroxyl with a
methoxy group and having a chloride on C-11of the aromatic ring
(compound 5), has a profound effect on potency, lowering the IC50
O8
a
25
Value calculated from the Ki reported in.
Value calculated using the IC50 value of the mixture of 3 and 4, the IC50 of pure 3
b
and a ratio of 3:4 in the mixture of 0.6:0.4.
to 39.8 0.3
mixture of compounds 3 (60%) and 4 (40%). From the IC50 value
of pure 3 (169.9 M) and using the compound composition in
the mixture the IC50 of 4 was estimated to 95 M. With respect
lM (Table 1). An IC50 of 140 lM was measured for a
l
l
to the lead compound 7 it seems that the addition of a chloride
or a bromide atom to the aromatic ring has improved the potency
of the inhibitors since all four compounds presented in this study
have IC50 values 5–25 times lower than that of 7 for GPb (Fig. 2).
Furthermore, bromine is more favorable than chlorine at C-10 (Ta-
ble 1) while chlorine has a positive influence in potency at the C-11
(Table 1) position.
3.3. Structural studies
Figure 2. A schematic representation of the IC50 values of the four inhibitors
studied.
In order to elucidate the structural basis of inhibition we have
determined the crystal structure of GPb in complex with each of
the compounds 3, 5, 16, and with the mixture of 3 and 4. A sum-
mary of the data processing and refinement statistics for the inhib-
itor complex structures is given in Table 2. The 2Fo–Fc and Fo–Fc
electron density maps clearly defined the position of each atom
of the inhibitors and specifically showed that all the studied com-
pounds were bound at the catalytic site of GPb (Fig. 3).

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K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
Additional electron density was found in the GPb complex with
the mixture of compounds 3 and 4 at a site distinct from the five
known GPb binding sites (Fig. 4).
The superimposition of the structures of native GPb and the
GPb–inhibitor complexes over well defined residues (18–249,
262–312, 326–829) gave rmsd values of 0.1 Å (mixture of 3
and 4), 0.16 Å (5), 0.09 Å (16) and 0.1 Å (3) (Ca positions) indi-
cating that the inhibitors were accommodated at the catalytic
site without any major conformational change of the protein
structure. The mode of binding and the hydrogen bonding net-
work of interactions of the glucopyranose moiety are analogous
to those observed for the glucopyranose moiety of the lead com-
pound 725 and other glucopyranose derivatives.8–10,23 Thus, the
hydroxyl groups of glucopyranose engage in hydrogen bond
interactions with side chain atoms of Asn284, His377, Asn484,
Tyr573 and Glu672, and with main chain atoms of Ala673,
Ser674 and Gly675. Furthermore, three conserved water mole-
cules mediate hydrogen bond interactions between the glucopyr-
anose moiety of each ligand and main chain atoms of His377,
Thr671, Ala673, Asp283 and Lys574, and the main and side chain
atoms of Thr676 and the phosphate group of the co-factor PLP
(Fig. 5).
Upon binding of the lead compound 7 to GPb the hydroquinone
group is accommodated at the b-pocket of the catalytic site. There
O8 makes direct polar interactions with Leu136N and Asp283 OD1,
and water-mediated hydrogen bond interactions with Gly134N,
Gly135N, Glu88 OE2, Asp283 OD1 and Asp283 OD2. Atom O7 also
forms a hydrogen bond with Asp339 OD1 of the b-pocket.25 In
addition it participates in an extended network of van der Waals
interactions with residues of the 280s loop.25
Figure 3. 2Fo-Fc electron density maps of hydroquinone compounds bound at the
catalytic site. The maps are contoured at 1
molecule in the refinement process.
r level before the inclusion of the ligand
3.3.1. The binding of 2,5-dihydroxy-4-(b-D-glucopyranosyl)-
bromo-benzene (16) and 2,5-dihydroxy-4-(b-D-glucopyranosyl)-
chlorobenzene (3)
Upon binding to the catalytic site of GPb compound 16 forms 12
hydrogen bonds and 84 van der Waals interactions, while com-
pound 3 forms 16 hydrogen bonds and 84 van der Waals interac-
tions with protein residues (Table 2). The hydrogen bond pattern
of the glycopyranose ring is the same for both compounds (Table
1), and compound 3 forms two additional hydrogen bonds; atom
Table 2
Summary of the diffraction data processing, refinement and protein ligand interactions statistics for GPb–inhibitor complexes
GPb complex
16
3
Mixture of 3 and 4
5
Data collection and processing statistics
Resolution (Å)
No. of observations
30.0–1.97
368,477
64,845 (3109)
30.0–2.00
634,277
65,452 (3213)
0.040 (0.398)
30.0–2.05
380,693
60,600 (2641)
30.0–2.00
460,082
63,987 (3140)
0.050 (0.295)
No. of unique reflections
a
Rsymm
0.065 (0.298)
93.9 (90.9)
2.00–1.97
11.3 (4.2)
3.7 (3.8)
0.061 (0.339)
Completeness (%)
Outermost shell (Å)
<I/r (I)>
98.7 (99.0)
2.03–2.00
21.2 (5.2)
5.4 (5.4)
31.1
86.8 (97.8)
2.09–2.05
14.8 (3.9)
3.9 (3.1)
29.8
98.0 (97.3)
2.03–2.00
16.1 (4.7)
4.0 (3.7)
22.3
Redundancy
B-values (Å2)
26.5
Refinement statistics
b
Rcryst
Rfree
0.19 (0.21)
0.23 (0.26)
0.19 (0.21)
0.24 (0.27)
0.19 (0.22)
0.25 (0.29)
0.19 (0.21)
0.22 (0.249)
c
No. of solvent molecules
335
265
250
326
rms deviation from ideality
In bond lengths (Å)
In bond angles (°)
0.007
1.022
0.007
1.028
0.007
1.039
0.007
0.014
Average B factor (A2)
Protein atoms
27.5
35.0
32.3
24.9
Ligand atoms
Solvent molecules
20.1
35.2
25.0
39.8
19.7 (1a), 20.3 (1b), 51.7 (novel site)
35.8
14.3
31.9
No. of hydrogen/halogen bond interactions
Glucose ring
Aromatic moiety
9
3
0
11
5
0
8d
3d
1d
10
2
1
Halogen atom
No. of van der Waals interactions
Glucose ring
Aromatic moiety
Halogen atom
47
34
3
45
34
5
42d
42
33
3
31d
5d
a
b
c
Rsymm
Rcryst
=
RhRi|I(h) ꢂ Ii(h)/RhRiIi(h) where Ii(h) and I(h) are the ith and the mean measurements of the intensity of reflection h.
=
Rh|Fo ꢂ Fc|/RhFo, where Fo and Fc are the observed and calculated structure factors amplitudes of reflection h, respectively.
Rfree is equal to Rcryst for a randomly selected 5% subset of reflections not used in the refinement. Values in parentheses are for the outermost shell.
Values are for compound 4.
d

K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
5133
that all three inhibitors bind similarly within the active site of GPb
(Fig. 6). The hydroquinone groups are accommodated at the b-
pocket of the catalytic site and stabilize the closed conformation
of the 280s loop. Thus, the hydroxyl group O7 makes direct polar
interactions with Asp283 OD1 while O8 is involved in hydrogen
bond interactions with Leu136N and Asp283 OD1, and water-med-
iated interactions with Glu88 OE2, Gly134N, Gly135N, Gly137N
and Asp283 OD1 and OD2 (Fig. 5). The chloride at the C-11 position
is involved in a direct halogen bond with OD1 of Asp283 and two
water-mediated interactions with the main chain nitrogen of
Gly134 and OE2 of Glu88 (Fig. 5). These interactions are possibly
the main reason for the sevenfold increase in the inhibitory po-
tency of the mixture of compounds 4 and 3 with respect to that
of compound 7. This is supported by the fact that compound 3
alone displays an IC50 of 169.9 lM (5.8 times smaller compared
to that of 7). Furthermore, the direct halogen interactions of the
chloride group at position C-11 (compound 4) with GPb residues
along with its water-mediated interactions which do not exist
when chloride is in C-10 position (compound 3) seem to form
the structural basis of the increased potency of compound 4 with
respect to that of 3.
In the GPb complex structure with the mixture of 3 and 4 com-
pound 3 was also found bound to a new site of GPb. This novel
binding site is located ꢃ27 Å from the catalytic site, ꢃ38 Å from
the allosteric site and ꢃ45 Å from the new allosteric site (Fig. 4).
Figure 4. A schematic diagram of the GPb dimeric molecule viewed down the
molecular dyad. One subunit is colored in dark purple and the other in light purple.
The catalytic and the novel binding sites are marked by bound compound 3 (in
yellow and cyan, respectively).
This binding pocket is formed by residues 122–124 (of the a5-helix
residues 118–124), residues 124–129 (of the b3 strand-residues
129–131), residues 494–508 (of the 16 helix-residues 496–508),
residues 527–553 (of the 17 helix-residues 527–553) and resi-
dues 649–657 (of the 20 helix-residues 649–657). The inhibitor
a
a
a
is surrounded by a hydrophobic environment formed by the aro-
matic amino acids Tyr548 and Phe545. It is also surrounded by
Lys545 and Lys544. Lysine contains four methylene groups capped
by an ammonium ion at neutral pH. Although sometimes lysine is
considered as a hydrophylic amino acid, the apolar methylene
groups of the residue often participate in favorable hydrophobic
interactions within folded peptides and proteins. In addition, when
found in a hydrophobic environment, lysine stabilizes this environ-
ment by forming salt bridges. Therefore, it is likely to assume that
these two lysines surrounding the binding pocket possibly play a
role in stabilizing the hydrophobic environment surrounding the
inhibitor. It is noteworthy that O2, O3 and O4 of glucose are ex-
posed to the solvent. O2 forms a hydrogen bond with Lys544, while
O4 forms a hydrogen bond with Leu494 and Gln96 (Fig. 7) and O7
forms a hydrogen bond with Lys655 via a water molecule. Chloride
forms a halogen bond to the main chain carbonyl oxygen of Lys544.
Overall, upon binding to the enzyme, the inhibitor forms 6 hydro-
gen bonds and 61 van der Waals interactions (Fig. 7). The number
of hydrogen bonds is reduced as compared to the number observed
for the catalytic site. Compounds 5 or 16 were not found bound at
this site. It is interesting that compound 3 was found bound at this
site only when GPb crystals were soaked with a mixture of com-
pounds 3 and 4 and not when soaked with a solution of compound
3 alone. Presumably, this can be attributed to differences between
the experimental conditions for forming these protein inhibitor
complexes and those for forming the GPb-mixture of 3 and 4 or
GPb-3 complexes (if lower inhibitor concentration of the soaking
solution and shorter soaking time were applied). However, exper-
imental conditions were similar when soaking GPb native crystals
with either a solution of a mixture of compounds 3 and 4 (100 mM,
21 h) or a solution of 3 (70 mM, 20 h). Therefore it is not clear
whether this new site represents a genuine new binding site with
a regulatory function or it is an artifact of the experiment condi-
tions. However, it has to be noted that since from the mixture of
compounds 3 and 4 only 3 binds to this site, the site displays some
sort of specificity.
O2 with Asn284 OD1, and O3 with Ala673N. In both GPb-inhibitor
complexes the hydroquinone group is accommodated at the
b-pocket and O8 of the inhibitor is hydrogen bonded to residues
of the glycine helix (134–150) Leu136N and Gly135N, Gly134N
and Gly137N via Wat53 and Wat74. This atom is also hydrogen
bonded to residues of the 280s loop (282–287) directly and via
two water molecules (Fig. 5). Atom O7 takes part in hydrogen bond
interactions with either one (compound 16) or the two carboxyl
oxygen atoms of the side chain of Asp339 (compound 3). The bro-
mide or the chloride group is not involved in any halogen bond
interactions but both participate in 3 van der Waals interactions
with His341 CE1, NE2 and Asn284N while the chloride group forms
2 additional van der Waals contacts with the side chain atoms of
Asp339. Structural comparison of the GPb-7, GPb-16 and GPb-3
complexes revealed that all three inhibitors bind similarly to the
active site and the existence of the halogen group triggered the dis-
placement of one the conserved water molecules from the active
site and the shift by 2.0 Å of another. Therefore the sevenfold
and the 5.7-fold increase in the inhibitory potency of 16 and 3,
respectively, with respect to 7 may be attributed to the van der
Waals interactions of the halogen group.
3.3.2. Binding of the mixture of structural isomers 2,5-dihydr
oxy-4-(b-
D-glucopyranosyl)-chlorobenzene (3) and 2,5-dihyd
roxy-3-(b-
D
-glucopyranosyl)-chlorobenzene (4)
A solution of a mixture of the two structural isomers 3 and 4
(60% and 40%, respectively) was used for soaking GPb native crys-
tals to obtain the enzyme–inhibitor complex. Thus, in the crystal
structure both compounds 3 and 4 were found bound at the cata-
lytic site of GPb with occupancies of 0.6 and 0.4, respectively, in a
similar structural mode. Compound 3 is bound in an identical man-
ner with the one found in the GPb-3 complex described above.
Upon binding to the catalytic site of GPb, compound 4 forms 11
hydrogen bonds and 78 van der Waals interactions (Table 2,
Fig. 5). A structural comparison with the lead compound 7 reveals

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K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
Figure 5. LIGPLOT46 diagram of the network of interactions of 3 (a), 4 (b) 5 (c) and 16 (d) with GPb residues in the vicinity of the catalytic site. Hydrogen bonds are
represented by green dashed lines and hydrophobic contacts by arcs with radiating spokes. Corresponding atoms, involved in hydrophobic contacts, are represented by black
circles
3.3.3. Binding of 3-(b-
chlorobenzene (5)
Upon binding to the catalytic site of GPb the inhibitor 5 forms
12 hydrogen bonds (one more than 4) and 79 van der Waals
interactions with protein residues. It also participates in an exten-
D
-glucopyranosyl)-2-hydroxy-5-methoxy-
sive network of water-mediated interactions, stabilizing the 280s
loop and hence the T state of the enzyme (Fig. 5). A structural com-
parison between the GPb-7 and the GPb-5 complexes revealed that
the two inhibitors bind similarly to the enzyme’s active site
forming a similar hydrogen bond and van der Waals interactions

K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
5135
Figure 6. A structural comparison between inhibitors 5 (purple) and 7 (yellow) at the catalytic site of GPb in stereo.
Figure 7. Stereo diagram showing the network of interactions formed between inhibitor 3 and residues at the novel binding site of GPb. Inhibitor molecules are shown in
cyan and GPb residues in purple.
network with GPb residues. The chloride group of 5, like that of 4,
is involved in a halogen interaction with Asp283 OD1 (albeit weak
since the distance is 3.36 Å) and water-mediated halogen interac-
tions with Leu136N and Gly137N. Comparative structural analysis
of the GPb-4 and the GPb-5 complexes has shown that the two
compounds bind similarly to the active site of the enzyme. How-
ever, the hydrogen bond interactions of hydroxyl O7 of compound
4 to Asp283 OD1 does not exist in the complex with compound 5
but it seems that the 8 additional van der Waals interactions of the
methoxy group counteract this loss. Furthermore, these interac-
tions are possibly responsible for the 2.4 times increase in the
inhibitory potency of compound since 5 with respect to 4.
and Asp339. These interactions serve to stabilize a conformation
of the 280s loop that blocks access of the substrate glycogen to
the catalytic site. Furthermore, the halogen interactions provide a
rationale for the increased potency of these halogen derivatives
glucosyl(hydro)quinones to inhibit GPb activity with respect to
the lead compound, and should assist in the design of more effec-
tive compounds. Thus, it seems, that bromine substitution is more
favorable than chlorine at C-10 position, chlorine has more favor-
able interactions at C-11 position, and a methoxy group at C-9 po-
sition is better than a hydroxyl group.
This study is part of an ongoing project to discover new GP
inhibitors, as potential drugs against type 2 diabetes and although
GP is a well documented enzyme, the findings presented here of-
fer new possibilities for structure based drug design of type 2 dia-
betes, and proves that new pathways are yet to be exploited.
4. Conclusions
Thus, this study shows that C-b-D-glucopyranosyl hydroquinones,
In summary, the kinetic and structural studies of the binding of
derivatives of C-glucosylhydroquinone showed that these com-
pounds are potent competitive inhibitors of GPb with respect to
Glc-1-P and bind at the catalytic site of the enzyme. One of the
inhibitors was also found bound at a novel site of GPb. On binding
to the enzyme, the inhibitors stabilize the less active T state of the
enzyme through an extensive network of direct and water-medi-
ated hydrogen and halogen bonds, as well as a network of van
der Waals interaction with residues of the 280s loop (Asn283
and Asn284), the glycine helix (Gly134, and Gly137) and Glu88
in which the glucose moiety is linked by a highly stable C-gluco-
sidic bond to a hydroquinone ring are well suited for regioselec-
tive modifications of the aglycon that modulate the binding to GP
and inhibitory properties. More work in this series, possibly with
the help of calculations and predictive methods should result in
the design and synthesis of accessible, hydrolytically stable and
more potent GPis, which will be subjected to further tests with
cells and animals to better evaluate their potential as anti-hyper-
glycemic molecules.

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K.-M. Alexacou et al. / Bioorg. Med. Chem. 19 (2011) 5125–5136
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This work was supported by the EU Marie Curie Early Stage Train-
ing (EST) contract no. MEST-CT-020575 and by the Commission of
the European Communities—under the FP7 ‘SP4-Capacities Coordi-
nation and Support Action, Support Actions’ EUROSTRUCT project
(CSA-SA_FP7-REGPOT-2008-1 Grant Agreement N° 230146). This
work was also supported by grants from European Community —
Research Infrastructure Action under the FP6 ‘Structuring the Euro-
pean Research Area’ Program (through the Integrated Infrastructure
Initiative ‘Integrating Activity on Synchrotron and Free Electron
Laser Science’) for work at the Synchrotron Radiation Source MAX-
lab, Lund, Sweden, and EMBL Hamburg Outstation, Germany. Région
Rhône-Alpes is warmly thanked for a stipend (Z.Y.Z.) and generous
financial support allocated in the frame of the MIRA collaborative
programme with Shanghai City (MIRA Recherche 2005). Other
financial supports from CNRS (France), University Claude-Bernard
Lyon 1 (for co-tutored PhD), ARCUS-Chine 2005 (joint support from
Région Rhône-Alpes and French Ministry of Foreign Affairs), and the
French Agence Nationale de Recherche (support of the ANR project
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