In vitro studies of 3-hydroxy-4-pyridinones and their glycosylated derivatives as potential agents for Alzheimer's disease

Article abstract of DOI:10.1039/b918439b

Glycosides of 3-hydroxy-4-pyridinones were synthesized and characterized by mass spectrometry, elemental analysis, ¹H and ¹³C NMR spectroscopy, and in one case by X-ray crystallography. The Cu²⁺ complex of a novel 3-hydroxy-4-pyridinone was synthesized and characterized by IR and X-ray crystallography, showing the ability of these compounds to chelate potentially toxic metal ions. An MTT cytotoxicity assay of a selected glycosylated compound showed a relatively low toxicity of IC₅₀ = 570 ± 90 μM in a human breast cancer cell line. The pyridinone glycosides could be cleaved by a broad specificity beta-glycosidase, Agrobacterium sp.β-glucosidase, and for one compound k_cat and K_m were determined to be 19.8 s^-1 and 1.52 mM, respectively. Trolox Equivalent Antioxidant Capacity (TEAC) values were determined for the free pyridinones, indicating the good antioxidant properties of these compounds. Metal-Aβ_1-40 aggregates with zinc and copper were resolubilized by the non-glycosylated pyridinone ligands. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b918439b

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PAPER
www.rsc.org/dalton | Dalton Transactions
In vitro studies of 3-hydroxy-4-pyridinones and their glycosylated derivatives
as potential agents for Alzheimer’s disease†
David E. Green,a Meryn L. Bowen,a Lauren E. Scott,a Tim Storr,a Michael Merkel,a Karin Bo¨hmerle,a
Katherine H. Thompson,a Brian O. Patrick,a Harvey J. Schugarb and Chris Orvig*a
Received 7th September 2009, Accepted 10th November 2009
First published as an Advance Article on the web 11th December 2009
DOI: 10.1039/b918439b
Glycosides of 3-hydroxy-4-pyridinones were synthesized and characterized by mass spectrometry,
elemental analysis, 1H and 13C NMR spectroscopy, and in one case by X-ray crystallography. The Cu2+
complex of a novel 3-hydroxy-4-pyridinone was synthesized and characterized by IR and X-ray
crystallography, showing the ability of these compounds to chelate potentially toxic metal ions. An
MTT cytotoxicity assay of a selected glycosylated compound showed a relatively low toxicity of IC50
=
570 90 mM in a human breast cancer cell line. The pyridinone glycosides could be cleaved by a broad
specificity beta-glycosidase, Agrobacterium sp. b-glucosidase, and for one compound kcat and Km were
determined to be 19.8 s-1 and 1.52 mM, respectively. Trolox Equivalent Antioxidant Capacity (TEAC)
values were determined for the free pyridinones, indicating the good antioxidant properties of these
compounds. Metal-Ab1-40 aggregates with zinc and copper were resolubilized by the non-glycosylated
pyridinone ligands.
In an effort to reduce the harmful effects of excess metal
Introduction
ions and oxidative stress in AD, therapies involving metal ion
chelation and/or removal are being investigated.12–19 Proponents
of the MPAC (metal protein attenuating compound) approach
to treating AD have previously used an FDA-approved agent
Clioquinol (CQ)(Fig. 1) as a proof of principle drug.20 CQ, and a
similar compound, known as PBT2, are 8-hydroxyquinolines, and
bind in a bidentate fashion with high affinity to a range of metal
ions, serving as gravimetric agents for many.21 These compounds
are able to slow the progression of dementia in Tg2576, AD type
transgenic mice.20,22 They are also able to affect the levels of Ab
found in the plasma of human subjects with AD,15,23 though it is
not yet known if they will be effective at alleviating AD symptoms
over the long term. As compounds of these types only treat one
specific point in the complex cascade that leads to the effects of
AD, current research often tries to build on the basic promise
seen in approaches like this by adding multiple functionalities
into one molecule to better treat the many factors occurring
simultaneously in AD. In addition, specific, rather than systemic,
chelation of excess metal ions in the brain of AD patients could
reduce side effects of treatments. Metal chelators with additional
antioxidant properties may also help to reduce the oxidative stress
that accompanies AD.24
The amyloid cascade hypothesis was first postulated by Hardy
and Higgins to explain the progression of Alzheimer’s disease
(AD) in 1992.1 This hypothesis proposed that the deposition of
b-amyloid (Ab) was the cause and first step in the progression of
AD, and that all other histologies and symptoms are downstream
effects.1 The key evolution to this theory since this time is that it
is not the characteristic amyloid deposits themselves that are the
major problematic species, but soluble forms of amyloid in close
proximity with metal ions that are the key toxic species.2
Ab plaques found in AD brains contain elevated levels of the
metal ions copper, zinc and iron; up to 400 mM for copper, 1 mM
for zinc, and 1 mM for iron.3,4 Cu and Zn ions are thought to
play key roles in binding to the Ab protein and in causing some of
the subsequent aggregation, redox activity and downstream effects
that lead to the symptoms of AD.5,6 The strong metal binding sites
in the Ab proteins are known to rely largely on histidine side chains,
though these interactions are still the subject of controversy and
current research.7–9 It has been postulated that the natural roles
of endogenous Ab and its precursor, amyloid precursor protein
(APP), are as copper binding and regulating proteins.10,11
aMedicinal Inorganic Chemistry Group, Department of Chemistry, Univer-
sity of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T
1Z1. E-mail: orvig@chem.ubc.ca; Tel: 1 604 822 4449
bDepartment of Chemistry and Chemical Biology, Rutgers, The State
University of New Jersey, 610 Taylor Rd, Piscataway, New Jersey, 08854-
8087, USA
† Electronic supplementary information (ESI) available: TLCs of Abg
reaction, initial rate graph for 2d and Abg, concentration vs. cell viability
for 5d and cisplatin, selected crystallographic data for 2d and Cu(5b)2,
TEAC values for 2d–6d and turbidity assay resolubilization data for
2b and 4b. CCDC reference numbers 746849 and 746850. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/b918439b
Fig. 1 Metal chelators of interest in removing excess metal ions in vivo;
Clioquinol (CQ) and Deferiprone (L1 or 2b).
Deferiprone (in this work, 2b)(Fig. 1) is a 3-hydroxy-4-
pyridinone used in the clinic to chelate and help excrete excess
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iron in thalassemia patients.25 Iron is sequestered by 2b to form
its iron complex Fe(2b)3, which is thermodynamically stable.26,27
3-Hydroxy-4-pyridinones such as 2b also have a relatively high
affinity for Cu(II) ions27–29 moderate affinity for Zn(II),27 and
very little affinity for common biological electrolyte ions such as
sodium, potassium, calcium and magnesium.30 It has been shown
recently that the chelation of iron with pyridinones is able to reduce
oxidative stress.31–33
to act as antioxidants and to redissolve metal-aggregated Ab
peptide species. Some of the work presented here was briefly
communicated previously, along with proof that these glycosylated
pyridinones are capable of crossing the BBB.16 Herein, we present
the complete report including full characterization and a detailed
analysis of results.
The pyridinones may be able to bind copper, zinc and iron in
the brains of AD sufferers and thereby help to break apart the
Ab aggregates (soluble or precipitated) already present, as well as
prevent further Ab aggregation from occurring. This would reduce
oxidative stress as the redox active metal ions would no longer be
free to react.
Results and discussion
Synthesis and characterization
Four of the 3-hydroxy-4-pyridinones were synthesized by con-
densation of maltol (1) with the appropriate amine (2a–5a)
(Scheme 1) in modest yields (23–51%), similar to those previously
reported.41,42 The pyridinones, 2b–5b, were then coupled to 2,3,4,6-
tetra-O-benzyl-D-glucopyranose, 7, under Mitsunobu conditions43
(Scheme 1) to give moderate yields (47–61%) of the b anomers
of compounds 2c–5c following column chromatography. Depro-
tection using H2 and Pd gave 2d–5d in 65–73% yield following
recrystallization.
Compound 6d was made via a different route (Scheme 2), as the
hydroxyl group on the N-phenyl ring required selective protection
prior to the coupling of the sugar. Pyridinone 8 was synthesized
as previously reported.44 The phenolic oxygen was then acetylated
to give 9, which was debenzylated to yield 10. The free hydroxyl
group was coupled to 2,3,4,6-tetra-O-benzyl-D-glucopyranose via
a Mitsunobu reaction to give the fully protected glycoconjugate 11.
This was deacetylated with NaOMe in MeOH to give 6c, which
was debenzylated via a Pd catalysed hydrogenation reaction to
yield 6d.
The Mitsunobu reaction was used to produce the pyridinone
glycosides (2c–6c). The choice of ADDP instead of diethyl
azodicarboxylate (DEAD) as a Mitsunobu reagent was because
the former gives a more basic intermediate43 and would likely
be more effective in deprotonation of the relatively high pKa
pyridinones (pKa ~9.4–9.9). Tributylphosphine was used because
it is more nucleophilic than triphenylphosphine, which is typically
used with DEAD. The Mitsunobu reaction produced mainly the
b-anomers of the protected pyridinone glycosides (2c–6c). This
was because the carbohydrate starting material 7, 2,3,4,6-tetra-O-
benzyl-D-glucopyranose, was 80–90% a-anomer, and the inversion
in the SN2 displacement that occurs during the reaction resulted in
80–90% b anomer of the pyridinone glycosides being formed. The
a and b anomers were separable with column chromatography,
We were interested in functionalizing these molecules further
in an effort to prevent premature systemic metal chelation prior
to passage across the blood brain barrier (BBB). We therefore
investigated using a prodrug that is targeted to the brain and then
deprotected after crossing the BBB. Protection of the 3-hydroxy-
4-pyridinones at the 3-hydroxy position forms a simple, function-
alized prodrug as this prevents metal chelation by blocking one of
the ligating atoms from binding metal ions. Glucose was chosen
for use in the prodrugs, as this would protect the pyridinone and
impart water solubility to the resulting compounds. In addition,
a high density of GLUT-1 transporters are present at the BBB34
and this may allow glucose conjugates to utilize GLUT-1 and gain
access to the brain. Glycoconjugates have been used to deliver
many types of drugs across the BBB, including anticonvulsants,35
analgesics,36 dopamine derivatives,37 anticancer compounds38 and
HIV therapeutics.39 The glycosidic link has the potential to be
cleaved in vivo by a number of b-glucosidases, some of which are
found naturally in the brain.40 If the pyridinone glycosides were
able to penetrate the BBB then be cleaved to produce the free
pyridinonate anions, metal complexes could form in the brain in a
manner that breaks apart and/or prevents Ab-metal aggregates
from forming. The compounds formed would be neutral and
relatively lipophilic, and may passively diffuse across the BBB,
and be redistributed or excreted.
Herein, we report the synthesis and characterization of pyridi-
none glycosides that may be suitable for passivating and/or
removing excess metal ions from the brain of AD patients using
a multifunctional approach. The pyridinone glycosides presented
here were assessed for their toxicity and for their ability to be
enzymatically cleaved to the free pyridinones. The pyridinones
produced by this cleavage were then tested for their abilities
Scheme
1
Synthesis of pyridinones and pyridinone glycosides 2–5; (a) H+, H2O–MeOH, reflux, yields for 2b–5b range from 23–51%;
(b) 2,3,4,6-tetra-O-benzyl-D-glucopyranose (7), ADDP, Bu3P, CH2Cl2, yields for 2c–5c range from 47–61%; (c) 10% Pd/C, 1 atm. H2, MeOH–H2O,
yields for 2d–5d range from 65–73%.
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Scheme 2 Synthesis of 6d: (a) Ac2O, pyridine, 1.5 h, 91%; (b) 10% Pd/C, H2, AcOEt–EtOH, 12 h, 82%; (c) 2,3,4,6-tetra-O-benzyl-D-glucopyranose (7),
ADDP, Bu3P, CH2Cl2, 72 h, 38%; (d) NaOMe, MeOH, 82%; (e) 10% Pd/C, H2, CH2Cl2–MeOH, 12 h, 72%. 6d shows the assignment used in NMR
spectra.
1
◦
˚
and the b anomer was isolated in each case, as verified by H
NMR spectroscopy.
Table 1 Selected bond lengths (A) and angles ( ) in 2d
O(1)–C(1)
C(4)–C(5)
N(1)–C(7)
O(2)–C(2)
C(7)–C(8)
N(1)–C(13)
O(4)–C(4)
C(8)–C(9)
1.412(3)
1.530(3)
1.378(4)
1.419(3)
1.370(4)
1.474(4)
1.414(3)
1.439(4)
1.520(4)
1.407(3)
1.352(5)
112.9(2)
107.1(2)
109.2
119.6(2)
113.1(2)
110.9(2)
119.7(2)
124.2(2)
118.4(2)
109.9(2)
121.4(2)
122.9(2)
111.4(2)
109.6(2)
109.5(2)
111.3(2)
O(7)–C(9)
O(1)–C(5)
C(5)–C(6)
N(1)–C(11)
O(3)–C(3)
C(7)–C(12)
C(1)–C(2)
O(5)–C(6)
1.256(4)
1.428(3)
1.518(3)
1.354(4)
1.426(3)
1.490(4)
1.525(3)
1.410(4)
1.427(4)
1.520(3)
1.388(3)
109.5(2)
115.0(2)
110.4(2)
106.7(2)
120.7(3)
106.7(2)
112.0(2)
106.0(1)
113.5(2)
118.8(2)
108.3(2)
122.9(2)
119.3(2)
116.4(2)
124.1(2)
122.4(2)
Debenzylation of 2c–6c was accomplished using H2 and 10%
Pd/C; yields following recrystallization ranged from 65–73% for
2d–6d. As the anomeric oxygen is bound to the pyridinone,
mutarotation does not occur, and the anomers of the starting
material in this reaction are the same as the anomers obtained in
the products. Thus the isolated b anomers of compounds 2c–6c
gave the corresponding b anomers of the deprotected compounds
2d–6d. The b anomers were the compounds of interest in this study,
which is why the a starting material was used. The b anomers were
desirable as the glucosidase available to us was a b-glucosidase,
and there is evidence that there are glucosidases of this type in the
human brain.40,45
All novel compounds in these reaction schemes were character-
ized by 1H NMR spectroscopy as well as mass spectrometry and
elemental analysis. The final compounds 2d–6d, were additionally
characterized by 13C NMR spectroscopy, with 2D-NMR spec-
troscopy used to help assign peaks as necessary (data not shown).
All compounds showed the expected spectral characteristics for
the predicted products of the given experimental conditions.
X-Ray quality crystals of one of the pyridinone glycosides,
2d, were grown by slow evaporation of a MeOH solution; the
structure is shown in Fig. 2, and selected bond lengths and angles
in given in Table 1. The bond lengths and angles are typical for
pyridinone structures.41,46 The torsional angles indicate the near-
planarity of the pyridinone ring, and the N-CH3 carbon atom
(C13) lies approximately in the same plane as the pyridinone ring,
as seen in other pyridinone structures.41,46 The shortest hydrogen
C(2)–C(3)
O(6)–C(1)
C(10)–C(11)
C(9)–C(10)
C(3)–C(4)
O(6)–C(8)
C(1)–O(1)–C(5)
O(3)–C(3)–C(4)
O(1)–C(5)–C(4)
C(7)–N(1)–C(11)
O(4)–C(4)–C(3)
C(4)–C(5)–C(6)
C(11)–N(1)–C(13)
O(7)–C(9)–C(10)
N(1)–C(7)–C(8)
O(1)–C(1)–C(2)
C(9)–C(10)–C(11)
C(8)–C(7)–C(12)
O(2)–C(2)–C(1)
O(2)–C(2)–C(3)
C(1)–C(2)–C(3)
O(3)–C(3)–C(2)
C(3)–C(4)–C(5)
C(1)–O(6)–C(8)
C(2)–C(3)–C(4)
O(1)–C(5)–C(6)
C(7)–N(1)–C(13)
O(4)–C(4)–C(5)
O(5)–C(6)–C(5)
O(1)–C(1)–O(6)
C(8)–C(9)–C(10
N(1)–C(7)–C(12)
O(6)–C(1)–C(2)
N(1)–C(11)–C(10)
O(6)–C(8)–C(7)
O(6)–C(8)–C(9)
C(7)–C(8)–C(9)
O(7)–C(9)–C(8)
bond in this structure is between H14 and O7 (keto-oxygen) with
a distance of 1.76(3) A. The b-anomer of glucose is displayed in
˚
this solid state structure, in agreement with the coupling constant
and chemical shift in the 1H NMR solution spectrum for purified
2d, the latter data indicating the presence of only one isomer.
Much data in the literature shows the utility of pyridinone
compounds as chelators for a range of metal ions.29,42,44,47 To show
the potential utility of the pyridinones in AD we verified the
binding of novel pyridinone, 5b, with the AD relevant metal ion
Cu2+. Slow addition of deprotonated ligand to a stirred solution
of copper perchlorate gave a bright green solution from which a
fine green precipitate was produced. This precipitate was filtered,
washed and dried, and examined by IR spectroscopy to help
verify the nature of the compound present. The IR showed the
four strong bands of the pyridinone ring had shifted significantly
between the ligand where they appear from 1488–1625 cm-1, and
the metal complex where they appear between 1464–1588 cm-1.
This bathochromic shift of 24–38 cm-1 for each of these four peaks
is characteristic of the binding of a pyridinone to a metal ion.42
The broad –OH stretching band at around 3170 cm-1 present in
Fig. 2 ORTEP diagram of 2d showing 50% thermal probability ellipsoids.
Hydrogen atoms have been omitted for clarity.
1606 | Dalton Trans., 2010, 39, 1604–1615
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◦
˚
pyridinone and phenyl rings are very nearly planar, and that they
are offset from each other by about 76◦. We published the crystal
structure of Cu(4b)2 recently,18 and the orientation between the
pyridinone and the phenyl rings in that complex was slightly less
than that observed here, at 63◦.18 It is expected that in the solution
state any differences in orientation would become negligible due
to rotation around the inter-ring C–N bond. There is disorder in
the isobutyl group (about C13 and C29) on the phenyl ring, and
this was modelled in two orientations.
Table 2 Selected bond lengths (A) and angles ( ) in Cu(5b)2
O(1)–Cu(1)
O(3)–Cu(1)
C(1)–N(1)
C(1)–C(6)
C(2)–C(3)
C(3)–C(4)
C(5)–N(1)
C(13)–C(14)
1.9118(15)
1.9165(14)
1.382(3)
1.502(4)
1.429(3)
1.399(4)
1.349(4)
1.542(17)
1.387(3)
86.11(7)
94.41(7)
179.39(7)
109.52(12)
110.06(15)
119.9(2)
123.1(2)
119.7(2)
116.9(2)
120.4(3)
121.8(2)
120.6(2)
119.3(2)
125.8(4)
100.6(6)
114.5(9)
104.2(7)
115.0(10)
O(2)–Cu(1)
O(4)–Cu(1)
C(1)–C(2)
C(2)–O(1)
C(3)–O(2)
C(4)–C(5)
C(7)–N(1)
C(15)–C(16)
1.9304(17)
1.9379(16)
1.389(3)
1.326(3)
1.300(3)
1.366(4)
1.457(3)
1.49(2)
C(17)–C(18)
O(1)–Cu(1)–O(2)
O(1)–Cu(1)–O(4)
O(2)–Cu(1)–O(4)
C(18)–O(3)–Cu(1)
C(3)–O(2)–Cu(1)
N(1)–C(1)–C(6)
O(1)–C(2)–C(1)
C(1)–C(2)–C(3)
O(2)–C(3)–C(2)
C(5)–C(4)–C(3)
C(5)–N(1)–C(1)
C(1)–N(1)–C(7)
C(12)–C(7)–N(1)
C(9)–C(10)–C(13)
C(9)–C(10)–C(13B)
C(15)–C(13)–C(14)
C(14)–C(13)–C(10)
C(16)–C(15)–C(13)
O(3)–Cu(1)–O(2)
O(3)–Cu(1)–O(4)
C(2)–O(1)–Cu(1)
C(19)–O(4)–Cu(1)
O(1)–Cu(1)–O(3)
C(2)–C(1)–C(6)
O(1)–C(2)–C(3)
O(2)–C(3)–C(4)
C(4)–C(3)–C(2)
N(1)–C(5)–C(4)
C(5)–N(1)–C(7)
C(8)–C(7)–N(1)
N(1)–C(1)–C(2)
C(11)–C(10)–C(13B)
C(11)–C(10)–C(13)
C(15)–C(13)–C(10)
C(13)–C(10)–C(13B)
93.18(6)
86.30(6)
109.54(13)
109.56(13)
177.95(7)
121.1(2)
117.18(19)
124.9(2)
118.2(2)
120.9(3)
117.6(2)
120.0(2)
119.0(2)
140.7(6)
116.4(4)
111.8(5)
25.8(4)
MTT cytotoxicity assay
Human breast cancer cells (MDA-MB-435S)48 were used to
study the toxicity of one of the pyridinone glycosides (5d). The
MTT assay used is described in detail elsewhere.49 This assay
is a colorimetric determination of cell viability during in vitro
treatment of various concentrations of test compound.50 The assay,
developed as an early stage drug screen, measures the amount
of MTT reduction by mitochondrial dehydrogenase and assumes
that the reductive activity, as measured by the production of purple
formazan that is assayed spectrophotometrically is proportional
to the number of viable cells.51 A low IC50 implies cytotoxicity
or antiproliferation at low drug concentrations. For example,
cisplatin was used as a positive control in this experiment, and was
found to have IC50 = 35 5 mM (see ESI),† which is consistent with
other values in the literature.49 The MTT plot (see ESI)† reveals a
relatively high IC50 value for 5d of 570 90 mM. This bodes well
for administering relatively high concentrations of these glycosyl-
protected pyridinones if necessary, without damage to healthy
tissue.
the IR spectrum of 5b, is no longer present in Cu(5b)2, suggesting
that this O is now bound to the metal ion rather than a proton.
Cu(5b)2 was dissolved in DMSO and the solvent was allowed to
slowly evaporate, producing fine green crystals suitable for analysis
by X-ray crystallography. The solid state structure is shown in
Fig. 3, and confirms the coordination pattern predicted from the
IR spectroscopic data. Table 2 gives selected bond lengths and
angles.
Enzymatic cleavage of pyridinone glycosides
The pyridinone glycosides (2d–6d) require the glucose moiety
to be cleaved before metal chelation by the pyridinone oxygen
atoms is possible. There are a number of enzymes that cleave b-
glycosides in humans.52 Some of these include glucocerebrosidase
(EC 3.2.1.45), cytosolic b-glucosidase (EC 3.2.1.21), and lactase
phlorizin hydrolase (EC 3.2.1.62, EC 3.2.1.108).40,53 Other enzymes
such as b-galactosidases may have some activity toward b-
glycosides. For instance, cytosolic b-glucosidase has catalytic
activity toward b-D-glucosides and b-D-galactosides.54
An initial screen using a broad specificity b-glucosidase,
Agrobacterium sp. b-glucosidase (Abg) was performed to assess
the potential of the pyridinone glycosides to act as substrates for
glucosidases (Scheme 3). TLC monitoring of the Abg reactions
was used to determine whether the pyridinone glycosides showed
any sign of being cleaved by Abg. With an Abg concentration
of 3.9 nM and initial substrate concentrations of 7.5 (3d–5d)or
15 mM (2d), some cleavage of the glucose moiety was observed
for each pyridinone glycoside studied (see TLCs in ESI).† In
all cases, significant cleavage was observed after 20–30 min at
ambient temperature. The free pyridinones (highest Rf spots)
turned purple with FeCl3 spray and did not char, unlike the
pyridinone glycosides and glucose, which both char due to the
presence of the carbohydrates.
Fig. 3 ORTEP diagram of Cu(5b)2 showing 50% thermal probability
ellipsoids. Hydrogen atoms have been omitted for clarity.
This compound crystallized with two DMSO molecules in
the unit cell (not shown), in a monoclinic crystal lattice. The
coordination mode of the two pyridinones is as seen in other
copper-pyridinato complexes with the two keto oxygen atoms and
the two phenolic oxygen atoms being trans to each other.18,29 The
distances between the oxygen atoms and the copper centre are
also within the normal limits of these types of compounds, with
˚
the phenolic oxygen atoms being 1.912 and 1.9167 A from the
˚
metal while the keto oxygen atoms were 1.930 and 1.938 A. The
geometry about the copper centre is very close to square planar,
with the angles between the adjacent oxygen atoms ranging from
86.1–94.4◦, and the angles between the two pyridinones being
178.0–179.4◦. This is very similar to what has been seen in previous
copper-pyridinone structures.18 The torsional angles show that the
To obtain more detailed information about Abg and pyridinone
glycoside substrates, some kinetic parameters were obtained for
one of the pyridinone glycosides, 2d. If Michaelis–Menten kinetics
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Table 3 Parameters for the glycosidic cleavage of 2d by Abg at 37 ◦C (from this work), compared to other Abg data56 for phenolic substrates with similar
pKa values
Substrate
pKa of aglycone
k
cat/s-1
Km/mM
k
cat/Km/s-1mM-1
4-Bromophenyl-b-D-glucopyranoside
4-Chlorophenyl-b-D-glucopyranoside
2-Naphthyl-b-D-glucopyranoside
2d
9.34
9.38
9.51
9.86
9.99
28.8
29.6
25.3
19.8
5.44
0.56
0.64
0.16
1.52
2.12
52
46
160
13
2.6
Phenyl-b-D-glucopyranoside
Scheme 3 Abg enzymatic cleavage of the pyridinone glycosides to
produce free pyridinone and glucose. TLC was used to follow the reactions
by assessing for formation of the free pyridinone (see ESI).†
are observed, a plot of initial rates versus substrate concentrations
generates a hyperbolic curve and fitting of this curve can determine
the Km and Vmax values. If the total enzyme concentration is
known, then kcat can be determined from Vmax
.
For Abg and 2d, the initial rates (no) were determined for
substrate concentrations ranging from 20–3000 mM. From a plot
of the initial rates versus substrate concentrations (Fig. 4), it
appeared that Michaelis–Menten kinetics were being observed at
substrate concentrations from 20–1000 mM. At higher substrate
concentrations (above ~1000 mM), there were noticeable decreases
in initial rates, indicating that the substrate inhibited the enzyme
activity; these points were not used in determining kinetic pa-
rameters. Assuming no substrate inhibition was taking place at
substrate concentrations from 20–1000 mM, a number of kinetic
parameters were determined for 2d as shown in Table 3.
There does not appear to be substrate inhibition of enzyme
at the lower substrate concentrations for 2d, as evidenced by the
linear nature of the early part of the main graph in Fig. 4. Analysis
of the initial rates only, as shown in the ESI, resulted in a kcat/Km
value very similar to that obtained above: kcat/Km was calculated
to be 12.2 0.8 s-1mM-1 using the initial rates method, and 13.0
0.8 s-1mM-1 via the hyperbolic curve fitting method. The similarity
between these numbers helps verify that relatively little substrate
inhibition occurs at concentrations below 1000 mM.
Fig. 4 Saturation curve for initial rates (DA.U./s) versus substrate
concentrations (mM) for 2d and Abg (3.9 nM) in sodium phosphate buffer
at 37 ◦C. The graph was fitted and Michaelis–Menten kinetic parameters
Vmax = 0.0102 0.0004 DA.U./s and Km = 1.5213 0.0944 mM were
determined using GraFit.55 Substrate concentrations above 1 mM were not
used in determining these kinetic parameters. The units for Vmax convert
to 1.72 0.07 ¥ 10-6 M s-1. Inset is a Lineweaver–Burk reciprocal plot.
substrate for Abg. However, the pyridinone glycosides may behave
differently with other glucosidases in vivo, and may provide enough
free pyridinone for therapeutic metal chelation. The slow cleavage
may provide a long-lived continuous release for chronic delivery
of pyridinone.
TEAC antioxidant assay
The Trolox Equivalent Antioxidant Capacity (TEAC) assay is
a simple method to quantify the total antioxidant activity of a
compound.57 This assay examines the quenching of the ABTS∑+
radical cation by monitoring the disappearance of the ABTS∑+
signal. Trolox, a more water soluble analogue of a-tocopherol,
is used as a standard, with its quenching value normalized to 1.
TEAC values for 2b–6b are plotted in Fig. 5 and listed in a table in
the ESI.† All of the pyridinones showed equal or enhanced values
compared to those of known antioxidants a-tocopherol and BHT,
demonstrating the radical quenching ability of these compounds.
In this assay, 6b showed the highest activity after 1, 3, and 6 min,
likely due to its phenol substituent, which is generally associated
with a more protective function against oxidative stress. After
Previous work with Abg has shown that substrates with
relatively poor leaving groups (pKa > 8) exhibit a significant
dependence of kcat on the leaving group ability.56 A value of kcat
=
~20 s-1 for 2d does not seem out of place compared to phenolic
substrates with similar pKa values (Table 3), suggesting that the
kcat values would likely increase for pyridinones with lower pKa
values, such as 4d and 5d whose aglycones, 4b and 5b, have pKa
values of ~9.4.
With a high Km value and relatively low kcat and kcat/Km values,
this pyridinone glycoside 2d does not appear to be the perfect
1608 | Dalton Trans., 2010, 39, 1604–1615
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Fig. 6 Relative resolubilization efficiency towards Cu(II) or Zn(II) medi-
ated Ab1-40 aggregates of 2b and 4b as compared to DTPA (normalized to
100).
Fig. 5 TEAC values at 1, 3, and 6 min for known antioxidants
a-Toc (a–tocopherol) and BHT (butylated hydroxytoluene), and various
3-hydroxy-4-pyridinones. Means are the average of three independent
trials, and error bars show standard deviations.
of both metal ions, but particularly for Zn2+. Log K1 for 2b is 9.76,
◦
and log K2 is 3.62 at 25 C,63 while for 4b log K1 is 9.40 and log
K2 is 3.03 under the same conditions.42 The higher log K values of
2b are consistent with the increased resolubilization seen, as this
resolubilization is a result of 2b binding more of the metal ions
that were previously bound to Ab than 4b is able to bind.
3 and 6 min, all aromatic pyridinones (4b–6b) displayed higher
TEAC values than those of the alkyl pyridinones (2b and 3b).
Ab aggregate resolubilization assay
Two representative pyridinones, 2b and 4b, with an alkyl and an
aromatic N-substituent, respectively, were tested for their ability
to resolubilize metal-aggregated Ab1-40 species. Because activity in
this assay is dependent upon metal ion chelation, the free ligands
were used, as opposed to their glycosylated prodrug forms.
Zinc(II) and copper(II) are known to cause Ab aggregation in
solution.58,59 The Ab1-40 peptide was dissolved and then incubated
with either Cu2+ or Zn2+, in different trials, and aggregates were
allowed to form. Copper and zinc were used in this assay as
they are found in elevated concentrations in Alzheimer’s amyloid
plaques.60,61 Ligand effect on metal-induced Ab1-40 aggregation was
assessed at physiological pH, 7.4, for Zn2+ and at a lower pH, 6.6,
for Cu2+. The interaction of the Ab peptide with Cu2+ is known to
be pH-dependent with no aggregation apparent at physiological
pH.58
The formation of aggregates leads to increased light scattering,
which was verified by increased solution turbidity as measured
at 405 nm prior to addition of test compound. Addition of
metal binding ligands reduces this apparent absorption and thus
a comparison of Ab disaggregating ability can be made; lower
absorbance indicates greater compound efficacy. Diethylenetri-
aminepentaacetic acid (DTPA) was used as a positive control
in this experiment as it is known to bind strongly to a range
of transition metal ions and to dissolve metal-containing Ab
aggregates.62 DTPA, 2b or 4b was added to the metal-peptide
aggregates and incubated for 45 min before the solution turbidity
was again measured via its absorbance at 405 nm. Changes in
absorbance value over this time were recorded, with the values
for DTPA being normalized to a resolubilization efficiency of
100%. The results of this assay are presented in Fig. 6, and
numerically in a table in the ESI.† Both 2b and 4b provide
significant resolubilization in the timeframe of this assay. 2b,
however, shows significantly more resolubilization for aggregates
Conclusions
Several pyridinones and pyridinone glycosides were synthesized
and characterized by mass spectrometry, 1H and 13C NMR
spectroscopies, elemental analysis and for one compound (2d), X-
ray crystallography. Studies of one of the pyridinone glycosides,
5d, with human breast cancer cells demonstrated a relatively high
IC50 value of 570 mM, indicating that high concentrations of 5d
could be used without expectation of toxicity to other human
cells. The pyridinone glycosides are cleaved by a broad specificity
b-glucosidase (Abg), indicating they may be useful prodrugs for
the pyridinones. The free pyridinones are able to act as competent
antioxidants, as indicated by their radical quenching abilities
in the TEAC assay. The free pyridinones are also able to re-
dissolve metal-mediated Ab aggregates, as exhibited in a turbidity
assay. Overall these compounds have shown great promise for
developing a multifunctional treatment for AD, and work on
further derivatives and expanded assays is ongoing in our lab.
Experimental
Materials and instrumentation
Reagents and atomic absorption standards were from Aldrich
(Madison, WI) unless otherwise stated, and were used without
further purification. All solvents were purchased from Fisher
(Ottawa, ON), and anhydrous solvents that were required were
dried according to standard procedures.64 3-Hydroxy-2-methyl-
4-pyranone (maltol, 1) was obtained from Pfizer (New York,
NY). H2 and N2 gases were from Praxair. Agrobacterium sp. b-
glucosidase56 (Abg) was generously provided by Prof. S. G. Withers
(UBC). Synthetic Ab1-40 peptide was purchased from Bachem
(Torrance, CA). 2b was synthesized as previously reported,41 or
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purchased from Aldrich. Compound 3b (1-hexyl-3-hydroxy-2-
methyl-4(1H)-pyridinone),42 compound 4b (1-phenyl-3-hydroxy-
2-methyl-4(1H)-pyridinone)42 and compound 8 (3-benzyloxy-
1-(4-hydroxyphenyl)-2-methyl-4(1H)-pyridinone)44 were synthe-
sized as previously reported.
solid was washed with cold MeOH, and dried in vacuo for 24 h
producing 2.94 g (23% yield) of an off-white solid that was
presumably a mixture of enantiomers. Anal. calcd (found) for
C22H19NO2: C 71.63 (71.41), H 5.51 (5.44), N 6.96 (6.91). MS
(+LSIMS): m/z 258 [M + H]+. 1H NMR (300 MHz, CD3OD): d
0.85 (t, 3 H, 2-butyl CH3, J = 7.1 Hz), 1.28 (d, 3 H, 2-butyl CH3,
J = 7.1 Hz), 1.66 (m, 2 H, 2-butyl CH2), 2.11 (s, 3 H, pyrid. CH3),
2.72 (m, 1 H, 2-butyl CH), 6.46 (d, 1 H, pyrid. H5, J = 7.3 Hz),
7.35 (m, 4 H, arom. C6H5), 7.58 (d, 1 H, J = 7.3 Hz, pyrid. H6).
Infrared spectrum (cm-1, total reflectance): 3169 (w, br), 1625 (s),
1575 (m), 1532 (s), 1488 (s), 1300 (s), 1231 (s), 823 (s).
Water was deionized and organics were removed (Barnstead
D9802 and D9804 cartridges) and this was followed by distillation
with a Corning MP-1 Mega-Pure Still. Sephadex G10 was allowed
to swell in the solvent system being used for at least 2 h
before column packing. Silica for column chromatography was
from Silicycle (Quebec City, PQ). Thin layer chromatography
(TLC) was performed using silica TLC plates with aluminium
backing (Merck). TLC plates were developed with UV light or
other selected reagents such as ninhydrin spray (~0.2% wt/wt
in EtOH) followed by heating (heat gun), a solution of FeCl3
(FeCl3·6H2O appropriately diluted for TLC visualization with
water and adjusted to ~pH 2 with 1 M HCl), and a sulfuric acid
spray (5% vol/vol H2SO4 in EtOH) followed by charring (heat
gun). All 10% Pd/C was wetted first with water before contact with
other solvents. MeOH and EtOH can ignite on contact with dry
Pd/C. Latex balloons (Aldrich) used for hydrogenation reactions
were ~11 L when filled to 80% capacity and were discarded after
each reaction. Nylon filters were from Gelman (25 mm, 0.2 mm).
The pH was measured using an Accumet 950 pH/ion Meter
with a Metrohm Porotrode micro-combination glass electrode.
Infrared spectra were recorded between 400 and 4000 cm-1 in
transmission mode on a Nicolet 6700 FT-IR spectrophotometer
at a resolution of 0.09 cm-1. NMR spectra were recorded on
a Bruker Avance 300, Avance 400, or AMX 500 instrument
at ambient temperature. NMR spectra were calibrated with
the residual solvent peak in the deuterated solvent. 2D-NMR
techniques such as COSY, HMQC and HMBC were used to aid
in the characterization of all final products (2d–6d). The a and
b anomers of the glucose derivatives were confirmed by analysis
3-(2¢,3¢,4¢,6¢-Tetra-O-benzyl-D-glucopyranosyloxy)-1,2-dimethyl-
4(1H)-pyridinone (2c). This Mitsunobu reaction43 was carried
out under typical conditions. Dry CH2Cl2 (~220 mL) was added
to 2,3,4,6-tetra-O-benzyl-D-glucopyranose (5.407 g, 10.0 mmol)
and 2b (1.670 g, 12.0 mmol). After addition of tributylphosphine
(3.74 mL, 15.0 mmol) and azodicarboxylic acid dipiperidide
(ADDP) (3.785 g, 15 mmol) some material remained undissolved
but eventually went into solution after ~1 h. The flask contents
were stirred for 12 h at RT, the CH2Cl2 volume was reduced to
~30 mL using a rotary evaporator and crystallization occurred
upon cooling (ADDP byproduct). The byproduct was removed
on a coarse frit, and rinsed with 2 ¥ 10 mL cold CH2Cl2. After
CH2Cl2 removal using a rotary evaporator, a second crop of
ADDP byproduct was recrystallized from a minimum of hot
EtOAc, followed by filtering and rinsing with 2 ¥ 10 mL cold
EtOAc. Evaporation using a rotary evaporator produced an
orange-brown oil that was taken up in a minimum amount of
EtOAc and applied to a silica column (5 ¥ 45 cm) run with
EtOAc. The product was collected, rotoevaporated, and dried on
the vacuum line for 48 h producing a viscous yellow oil (3.083 g,
47% yield). Anal. calcd (found) for C41H43NO7: C 74.41 (74.23),
H 6.55 (6.60), N 2.12 (2.13). MS (+LSIMS): m/z 662 [M + H]+.
1H NMR (300 MHz, CDCl3): d 2.29 (s, 3 H, pyrid. CH3), 3.44
(m, 1 H), 3.44 (s, 3 H, N–CH3), 3.71 (m, 5 H), 4.47 (m, 2 H, Bn
CH2), 4.57 (d, 1 H, Bn CH2, J = 11.2 Hz), 4.79 (m, 3 H, Bn CH2),
4.99 (d, 1 H, Bn CH2, J = 10.9 Hz), 5.25 (d, 1 H, Bn CH2, J =
11.2 Hz), 5.49 (d, 1 H, anomeric H, J = 7.3 Hz), 6.42 (d, 1 H,
pyrid. H, J = 7.3 Hz), 7.25 (m, 19 H, Bn C6H5), 7.46 (m, 2 H).
1
of the anomeric proton doublets in the H NMR spectra. Mass
spectra were obtained with either a Kratos Concept II H32Q
instrument (Cs+-LSIMS) or a Macromass LCT (electrospray
ionization, ESI).† Microanalyses for C, H and N were performed
in the Department of Chemistry at UBC (Mr P. Borda or
Mr M. Lakha) or by Delta Microanalytical Services (Delta, BC).
X-Ray data were collected and processed64 using a Rigaku/ADSC
CCD diffractometer. The solid state structures were solved using
direct methods65 and expanded using Fourier techniques.66 The
non-hydrogen atoms were refined anisotropically. Some hydrogen
atoms were refined isotropically, the rest were included in fixed
positions. All refinements were performed using SHELXL-97.67
3-(2¢,3¢,4¢,6¢-Tetra-O-benzyl-D-glucopyranosyloxy)-1-hexyl-2-
methyl-4(1H)-pyridinone (3c). This procedure was similar to that
used for the synthesis of 2c with the following substitutions:
dry CH2Cl2 (~80 mL) was added to 2,3,4,6-tetra-O-benzyl-D-
glucopyranose (2.703 g, 5.0 mmol) and 3b (1.256 g, 6.0 mmol) to
dissolve these materials. Tributylphosphine (2.49 mL, 10.0 mmol)
and ADDP (2.523 g, 10.0 mmol) were added to the reaction
mixture. Columnpurification using EtOAc withsilicagel produced
1.946 g (53% yield) of a viscous yellow oil. Anal. calcd (found) for
C46H53NO7: C 75.49 (75.09), H 7.30 (7.35), N 1.91 (2.10). MS
Synthesis
1-[4-(2-Butyl)phenyl]-3-hydroxy-2-methyl-4(1H)-pyridinone (5b).
This pyridinone was prepared in a similar manner to previously
published procedures for other pyridinones.42 To a 500 mL round
bottom flask (RBF) was added maltol (1) (6.306 g, 50.0 mmol), ( )-
4-(2-butyl)aniline (5a) (14.924 g, 100 mmol), a dilute HCl solution
(3 mL conc. HCl and 100 mL water), and 40 mL MeOH. The
mixture was refluxed under Ar for 72 h. The solvents were removed
on the rotary evaporator and the product was recrystallized from
a minimum amount of refluxing MeOH. The solution containing
recrystallized product was filtered using a coarse frit, the collected
1
(+LSIMS): m/z 732 [M + H]+. H NMR (500 MHz, CDCl3): d
0.88 (m, 3 H, hexyl CH3), 1.27 (m, 6 H, hexyl CH2), 1.61 (m, 2 H,
hexyl CH2), 2.33 (s, 3 H, pyrid. CH3), 3.41 (m, 1 H), 3.70 (m, 7 H),
4.45 (m, 2 H, Bn CH2), 4.56 (d, 1 H, Bn CH2, J = 11.0 Hz), 4.76
(m, 2 H, Bn CH2), 4.82 (d, 1 H, Bn CH2, J = 11.0 Hz), 4.95 (d,
1 H, Bn CH2 J = 11.0 Hz), 5.25 (d, 1 H, Bn CH2, J = 11.2 Hz),
5.50 (d, 1 H, anomeric H, J = 7.6 Hz), 6.41 (d, 1 H, pyrid. H, J =
7.6 Hz), 7.24 (m, 19 H, Bn C6H5), 7.46 (m, 2 H).
1610 | Dalton Trans., 2010, 39, 1604–1615
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3-(2¢,3¢,4¢,6¢-Tetra-O-benzyl-D-glucopyranosyloxy)-2-methyl-1-
phenyl-4(1H)-pyridinone (4c). This procedure was similar to that
used for the synthesis of 2c with the following substitutions:
dry CH2Cl2 (~120 mL) was added to 2,3,4,6-tetra-O-benzyl-D-
glucopyranose (4.325 g, 8.0 mmol) and 4b (1.932 g, 9.6 mmol) to
dissolve these materials. Tributylphosphine (3.0 mL, 12.0 mmol)
and ADDP (3.028 g, 12.0 mmol) were added to the reaction
mixture. Column purification as for 2c produced 2.028 g (56%
yield) of a viscous yellow oil. Anal. calcd (found) for C46H45NO7:
C 76.33 (76.18), H 6.27 (6.41), N 1.93 (2.02). MS (+LSIMS): m/z
724 [M + H]+. 1H NMR (500 MHz, CDCl3): d 2.07 (s, 3 H, pyrid.
CH3), 3.47 (m, 1 H), 3.66 (m, 4 H), 3.79 (dd, 1 H, J = 9.0 Hz, J =
9.0 Hz), 4.46 (s, 2 H, Bn CH2), 4.56 (d, 1 H, Bn CH2, J = 11.0 Hz),
4.80 (m, 2 H, Bn CH2), 4.96 (d, 1 H, Bn CH2, J = 11.0 Hz), 5.28
(d, 1 H, Bn CH2, J = 11.1 Hz), 5.56 (d, 1 H, anomeric H, J =
7.6 Hz), 6.48 (d, 1 H, pyrid. H, J = 7.6 Hz), 7.21 (m, 24 H, arom.
C6H5), 7.47 (m, 2 H).
material was used to saturate a solution of MeOH; X-ray quality
crystals were grown by slow evaporation at RT from a slightly
opened vial. Anal. calcd (found) for C13H19NO7: C 51.82 (51.76),
H 6.36 (6.06), N 4.65 (4.66). MS (+LSIMS): m/z 324 [M + Na]+,
302 [M + H]+. 1H NMR (500 MHz, CD3OD): d 2.54 (s, 3 H, C7-
CH3), 3.23 (ddd, 1 H, H5, 3J5,4 = 9.5 Hz, 3J5,6a = 2.3 Hz, 3J5,6b
=
5.5 Hz), 3.33 (dd, 1 H, H4, 3J4,5 = 9.5 Hz, 3J4,3 = 9.5 Hz), 3.39 (dd,
partially overlapped with H2, H3), 3.42 (dd, partially overlapped
with H3, H2), 3.67 (dd, 1 H, H6b, 2J6b,6a = 11.9 Hz, 3J6b,5 = 5.5 Hz),
3.75 (s, 3 H, N–CH3), 3.84 (dd, 1 H, H6a, 2J6a,6b = 11.9 Hz, 3J6a,5
=
3
2.3 Hz), 4.62 (d, 1 H, H1, J1,2 = 7.4 Hz), 6.44 (d, 1 H, H10,
3
1
3J10,11 = 7.4 Hz), 7.72 (d, 1 H, H11, J11,10 = 7.4 Hz). 13C{ H}
NMR (125 MHz, CD3OD): d 14.04 (C7-CH3), 42.60 (N-CH3),
62.64 (C6), 71.07 (C4), 75.50 (C3), 78.49 (C2), 78.70 (C5), 107.19
(C1), 116.78 (C10), 142.63 (C11), 146.12 (C8), 147.40 (C7), 174.06
(C9).
3-(D-Glucopyranosyloxy)-1-hexyl-2-methyl-4(1H)-pyridinone
(3d). This hydrogenolysis reaction was carried out in a similar
fashion as the synthesis of 2d with the following changes: 3c
(1.934 g, 2.64 mmol) was dissolved in MeOH (60 mL) and 10%
Pd/C (1.124 g, 1.06 mmol Pd) was added. The off-white solid
obtained from this reaction (873 mg) was recrystallized from EtOH
to give 635 mg (65% yield) of a white solid. Anal. calcd (found)
for C18H29NO7: C 58.21 (58.21), H 7.87 (7.96), N 3.77 (3.87). MS
(+LSIMS): m/z 394 [M + Na]+, 372 [M + H]+. 1H NMR (400 MHz,
CD3OD): d 0.91 (m, 3 H, hexyl CH3), 1.35 (m, 6 H, hexyl CH2),
1.76 (m, 2 H, hexyl CH2), 2.57 (s, 3 H, C7-CH3), 3.24 (ddd, 1 H,
H5, 3J5,4 = 9.5 Hz, 3J5,6a = 2.2 Hz, 3J5,6b = 5.6 Hz), 3.33 (dd, 1 H,
H4, 3J4,5 = 9.5 Hz, 3J4,3 = 9.5 Hz), 3.39 (dd, partially overlapped
with H2, H3), 3.42 (dd, partially overlapped with H3, H2), 3.67
3-(2¢,3¢,4¢,6¢-Tetra-O-benzyl-D-glucopyranosyloxy)-1-[4-(2-butyl)-
phenyl)]-2-methyl-4(1H)-pyridinone (5c). This procedure was
similar to that used for the synthesis of 2c with the following
substitutions: dry CH2Cl2 (~100 mL) was added to 2,3,4,6-tetra-
O-benzyl-D-glucopyranose (4.866 g, 9.0 mmol) and 5b (2.779 g,
10.8 mmol) to dissolve these materials. Tributylphosphine
(3.36 mL, 13.5 mmol) and ADDP (3.406 g, 13.5 mmol) were
added to the reaction mixture. Column purification produced
4.248 g (61% yield) of a viscous yellow oil. A presumed mixture
of the 2-butyl stereogenic carbon atom results in the pure
b-anomer being a diastereomeric mixture. Anal. calcd (found)
for C50H53NO7: C 77.00 (76.83), H 6.85 (6.82), N 1.80 (2.00). MS
1
(+LSIMS): m/z 780 [M + H]+. H NMR (500 MHz, CDCl3): d
2
3
0.84 (t, 3 H, 2-butyl CH3, J = 7.3 Hz), 1.26 (d, 3 H, 2-butyl CH3,
J = 6.0 Hz), 1.62 (m, 2 H, 2-butyl CH2), 2.07 (s, 3 H, pyrid. CH3),
2.67 (m, 1 H, 2-butyl CH), 3.45 (m, 1 H), 3.65 (m, 4 H), 3.78 (dd,
1 H, J = 9.0 Hz, J = 9.0 Hz), 4.46 (s, 2 H, Bn CH2), 4.56 (d, 1 H,
Bn CH2, J = 11.0 Hz), 4.79 (m, 3 H, Bn CH2), 4.96 (d, 1 H, Bn
CH2, J = 11.0 Hz), 5.28 (d, 1 H, Bn CH2, J = 11.2 Hz), 5.55
(d, 1 H, anomeric H, J = 7.6 Hz), 6.41 (d, 1 H, pyrid. H, J =
7.6 Hz), 7.25 (m, 23 H, arom. C6H5), 7.47 (m, 2 H).
(dd, 1 H, H6b, J6b,6a = 11.9 Hz, J6b,5 = 5.6 Hz), 3.84 (dd, 1 H,
H6a, 2J6a,6b = 11.9 Hz, 3J6a,5 = 2.2 Hz), 4.04 (t, 2 H, N–CH2,3J =
3
7.7 Hz), 4.63 (d, 1 H, H1, J1,2 = 7.5 Hz), 6.48 (d, 1 H, H10,
3
1
3J10,11 = 7.4 Hz), 7.76 (d, 1 H, H11, J11,10 = 7.4 Hz). 13C{ H}
NMR (100 MHz, CD3OD): d 13.61 (C7-CH3), 14.27 (hexyl CH3),
23.55 (hexyl CH2), 27.06 (hexyl CH2), 31.53 (hexyl CH2), 32.44
(hexyl CH2), 55.56 (N-CH2), 62.67 (C6), 71.10 (C4), 75.54 (C3),
78.50 (C2), 78.67 (C5), 107.19 (C1), 117.05 (C10), 141.95 (C11),
146.35 (C7), 146.48 (C8), 173.96 (C9).
3-(b-D-Glucopyranosyloxy)-1,2-dimethyl-4(1H)-pyridinone (2d).
To a 150 mL RBF was added 2c (1.078 g, 1.63 mmol) which was
dissolved in MeOH (50 mL). The 10% Pd/C (694 mg, 0.65 mmol
Pd) was wetted with water (10 mL) before being added to the
reaction vessel, and the vial was rinsed with MeOH (10 mL).
An 11 L latex balloon was filled with H2 gas and fitted to a
stopcock adaptor, which was opened after placement in the RBF
ground glass joint. The flask contents were stirred vigorously at RT
for 24 h, the reaction mixture was suction-filtered (medium frit),
Pd/C was stirred with an additional 10 mL of water in the frit,
suction-filtered, and the combined filtrates were syringed through
a Nylon filter (25 mm, 0.2 mm) to remove fine Pd/C. The filtrate
was evaporated using a rotary evaporator (45 ◦C), and the residue
was dried in a vacuum desiccator for 24 h to give an off-white
product (468 mg). TLC (1 : 1 EtOAc–MeOH) indicated no starting
material was present, and FeCl3 spray indicated no deprotected
a-hydroxyketone present. This solid was recrystallized from a
minimum of hot MeOH (~20 mL). After filtering, drying in a
vacuum desiccator for 48 h, 335 mg (68% yield) of a crystalline
solid was isolated and characterized. A small amount of this
3-(D-Glucopyranosyloxy)-2-methyl-1-phenyl-4(1H)-pyridinone
(4d). This hydrogenolysis reaction was carried out in a similar
fashion as the synthesis of 2d with the following changes: 4c
(1.998 g, 2.76 mmol) and 10% Pd/C (1.175 g, 1.10 mmol Pd)
were used. The off-white solid obtained from this reaction (930
mg) was recrystallized from MeOH to give 704 mg (70% yield) of
a white solid. Anal. calcd (found) for C18H21NO7: C 59.50 (59.71),
H 5.82 (6.18), N 3.85 (3.73). MS (+LSIMS): m/z 386 [M + Na]+,
364 [M + H]+. 1H NMR (400 MHz, CD3OD): d 2.25 (s, 3 H, C7-
CH3), 3.27 (ddd, 1 H, H5, 3J5,4 = 9.5 Hz, 3J5,6a = 2.2 Hz, 3J5,6b
=
5.5 Hz), 3.33 (dd, 1 H, H4, 3J4,5 = 9.6 Hz, 3J4,3 = 9.6 Hz), 3.42 (dd,
partially overlapped with H2, H3), 3.45 (dd, partially overlapped
2
3
with H3, H2), 3.66 (dd, 1 H, H6b, J6b,6a = 11.9 Hz, J6b,5
5.5 Hz), 3.84 (dd, 1 H, H6a, J6a,6b = 11.9 Hz, J6a,5 = 2.2 Hz),
4.74 (d, 1 H, H1, J1,2 = 7.6 Hz), 6.56 (d, 1 H, H10, J10,11
=
2
3
3
3
=
7.4 Hz), 7.43 (m, 2 H, arom. C6H5), 7.59 (m, 3 H, arom. C6H5),
7.74 (d, 1 H, H11, J11,10 = 7.4 Hz). 13C{ H} NMR (100 MHz,
CD3OD): d 15.68 (C7-CH3), 62.67 (C6), 71.13 (C4), 75.58 (C3),
78.50 (C2), 78.72 (C5), 107.05 (C1), 116.85 (C10), 127.97 (C14),
3
1
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Dalton Trans., 2010, 39, 1604–1615 | 1611
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View Article Online
131.11 (C15), 131.18 (C13), 142.22 (C11), 143.06 (C12), 145.90
(C8), 146.69 (C7), 174.89 (C9).
H13, J = 12.8 Hz), 7.54 (d, 2 H, H14, J = 12.8 Hz), 7.56 (d, 1 H,
H11, J = 11.0 Hz).
1-[4-(2-Butyl)phenyl)]-3-(b-D-glucopyranosyloxy)-2-methyl-4(1H)-
pyridinone (5d). This was obtained as a mixture of diastereomers
resulting from a mixture of the 2-butyl stereogenic carbon atom.
This hydrogenolysis reaction was carried out in a similar fashion
as the synthesis of 2d with the following changes: 5c (2.535 g,
3.25 mmol) was dissolved with MeOH (90 mL) and 10% Pd/C
(1.383 g, 1.30 mmol Pd) was added. The off-white solid obtained
from this reaction (1.335 g) was recrystallized from MeOH to
give 991 mg (73% yield) of a white solid. Anal. calcd (found)
for C22H29NO7: C 62.99 (62.95), H 6.97 (6.89), N 3.34 (3.23).
1-(4-Acetoxyphenyl)-3-(2¢,3¢,4¢,6¢-tetra-O-benzyl-D-glucopyran-
osyloxy)-2-methyl-4(1H)-pyridinone (11). To a suspension of 10
(4.16 g, 16 mmol) and 2,3,4,6-tetra-O-benzyl-D-glucopyranose
(7.23 g, 13 mmol) in dry CH2Cl2 (150 mL) under argon was added
first tributylphospine (6.7 mL, 27 mmol) and then a solution of
ADDP (6.53 g, 26 mmol) in dry CH2Cl2 (50 mL), yielding a dark
orange solution. After stirring for 72 h, the colourless precipitate
was filtered off, the filtrate was concentrated and chromatographed
on silica gel using EtOAc as the eluent to afford 4.02 g (38%) of a
pale orange solid. Anal. calcd (found) for C48H47NO9·0.3 H2O: C
73.35 (73.23), H 6.18 (6.09), N 2.20 (1.78). MS (ESI): m/z 782 [M +
H+]. 1H NMR (300 MHz, CD3OD): d 2.06 (s, 3 H, OCOCH3), 2.29
(s, 3 H, C7-CH3), 3.44 (m, 1 H, H5), 3.48-3.71 (m, 5 H, H2,3,4,6),
4.42 (s, 2 H, BnCH2), 4.58 (d, 1 H, BnCH2, J = 11.0 Hz), 4.80 (m,
3 H, BnCH2), 4.98 (d, 1 H, BnCH2, J = 11.0 Hz), 5.30 (d, 1 H,
BnCH2, J = 11.2 Hz), 5.57 (d, 1 H, H1, J = 7.4 Hz), 6.48 (d, 1 H,
H10, J = 7.6 Hz), 7.20 (m, 23 H, H11, H14, BnH), 7.46 (d, 2 H,
H13, J = 8.4 Hz).
1
MS (+LSIMS): m/z 442 [M + Na]+, 420 [M + H]+. H NMR
(400 MHz, CD3OD): d 0.85 (t, 3 H, 2-butyl CH3, J = 7.4 Hz),
1.28 (d, 3 H, 2-butyl CH3, J = 6.9 Hz), 1.66 (m, 2 H, 2-butyl
CH2), 2.25 (s, 3 H, C7-CH3), 2.73 (m, 1 H, 2-butyl CH), 3.27
(ddd, 1 H, H5, 3J5,4 = 9.5 Hz, 3J5,6a = 2.2 Hz, 3J5,6b = 5.5 Hz), 3.33
3
3
(dd, 1 H, H4, J4,5 = 9.6 Hz, J4,3 = 9.6 Hz), 3.41 (dd, partially
overlapped with H2, H3), 3.44 (dd, partially overlapped with H3,
2
3
H2), 3.67 (dd, 1 H, H6b, J6b,6a = 11.9 Hz, J6b,5 = 5.5 Hz), 3.84
(dd, 1 H, H6a, J6a,6b = 11.9 Hz, J6a,5 = 2.2 Hz), 4.73 (d, 1 H,
H1, J1,2 = 7.5 Hz), 6.55 (d, 1 H, H10, J10,11 = 7.4 Hz), 7.34 (d,
2
3
3
3
3-(2¢,3¢,4¢,6¢-Tetra-O-benzyl-D-glucopyranosyloxy)-1-(4-hydroxy-
phenyl)-2-methyl-4(1H)-pyridinone (6c). To a suspension of 11
(4.02 g, 5.2 mmol) in dry MeOH (50 mL) under argon was added
a suspension of sodium methoxide (0.57 g, 10.5 mmol) in dry
MeOH (10 mL). The reaction mixture was stirred for 2 h. To this
3
3
2 H, H14, J14,13 = 7.6 Hz), 7.42 (d, 2 H, H13, J13,14 = 7.6 Hz),
7.73 (d, 1 H, H11, J11,10 = 7.4 Hz). 13C{ H} NMR (100 MHz,
CD3OD): d 12.48 (2-butyl CH2CH3), 15.68 (C7-CH3), 22.17
(2-butyl CHCH3), 32.08 (2-butyl CH2), 42.72 (2-butyl CH), 62.65
(C6), 71.12 (C4), 75.57 (C3), 78.48 (C2), 78.70 (C5), 107.07 (C1),
116.80 (C10), 127.74 (C14), 129.72 (C13), 140.86, (C12), 142.35
(C11), 145.88 (C8), 146.85 (C7), 151.20 (C15) 174.81 (C9).
3
1
R
clear, slightly pink solution activated Rexynꢀ (8 spatulas) and
CH2Cl2 (50 mL) was added and the reaction mixture was stirred
for 10 min. This mixture was filtered, the filtrate evaporated and
recrystallized from EtOAc to afford 3.11 g (82% yield) of pale
pink crystals. Anal. calcd (found) for C46H45NO8·0.2 H2O: C
74.30 (74.31), H 6.14 (6.15), N 2.03 (1.88). MS (EI): m/z 740 [M
+ H+]. 1H NMR (300 MHz, CDCl3): d 2.13 (s, 3 H, C7-CH3), 3.36
(m, 1 H, H5), 3.40–3.76 (m, 5 H, H2,3,4,6), 4.53 (d, 1 H, BnCH2,
J = 11.0 Hz), 4.76 (m, 3 H, BnCH2), 4.91 (d, 1 H, BnCH2, J =
11.0 Hz), 5.18 (d, 1 H, BnCH2, J = 11.2 Hz), 5.41 (d, 1 H, H1,
J = 7.4 Hz), 6.81 (d, 1 H, H10, J = 7.3 Hz), 7.20 (m, 23 H, H11,
H14, BnH), 7.38 (d, 2 H, H13, J = 6.4 Hz).
1-(4-Acetoxyphenyl)-3-benzyloxy-2-methyl-4(1H)-pyridinone
(9). To a suspension of 8 (5.96 g, 19 mmol) in dry pyridine
(60 mL) under argon that was cooled in an ice bath, was added
acetic anhydride (14.7 mL, 155 mmol). After the addition was
complete, the ice bath was removed and the mixture stirred at
room temperature for 1.5 h. The clear solution was concentrated
to an orange oil and taken up in EtOAc (100 mL), then extracted
with aqueous NaCl solution (3 ¥ 200 mL). The organic layer was
dried over Na2SO4, concentrated, and the product recrystallized
from EtOAc–MeOH (25 : 2) to give 6.14 g (91% yield) of colourless
crystals. Anal. calcd. (found) for C21H19NO4·0.2H2O: C 71.57
(71.46), H 5.47 (5.54), N 4.03 (3.97). MS (EI): m/z 372 [M +
3-(b-D-Glucopyranosyloxy)-1-(4-hydroxyphenyl)-2-methyl-4(1H)-
pyridinone (6d). To a solution of 6c (3.05 g, 4.1 mmol) in a
mixture of MeOH and CH2Cl2 (1 : 1, 100 mL) was added 10%
Pd/C (1.75 g, 1.6 mmol Pd), wetted with water and suspended
in EtOH (20 mL). This suspension was set under hydrogen using
a 30 cm diameter balloon and stirred for 12 h. The catalyst was
filtered off and the filtrate concentrated and recrystallized from
MeOH/EtOAc to afford 1.12 g (72% yield) of a colourless solid.
Anal. calcd (found) for C18H21NO8: C 56.76 (56.99), H 5.69 (5.58),
N 3.87 (3.69). MS (EI): m/z 380 [M + H+]. 1H NMR (300 MHz,
CD3OD): d 2.26 (s, 3 H, C7-CH3), 3.36 (m, 2 H, H4, H5), 3.44
(m, 2 H, H2, H3), 3.63 (dd, 1 H, H6b, J = 8.9, 4.1 Hz), 3.85 (dd,
1 H, H6a, J = 11.8, 4.1 Hz), 4.73 (d, 1 H, H1, J = 7.4 Hz), 6.57
(d, 1 H, H10, J = 7.4 Hz), 6.93 (d, 2 H, H13, J = 6.7 Hz), 7.22 (d,
1
H+]. H NMR (200 MHz, CD3OD): d 1.89 (s, 3 H, OCOCH3),
2.31 (s, 3 H, C7-CH3), 5.14 (s, 2 H, BnCH2), 6.54 (d, 1 H, H10,
J = 11.0 Hz), 7.34 (m, 9 H, H13, H14, BnH), 7.68 (d, 1 H, H11,
J = 11.0 Hz).
1-(4-Acetoxyphenyl)-3-hydroxy-2-methyl-4(1H)-pyridinone
(10). To a solution of 9 (6.14 g, 18 mmol) in EtOAc (230 mL)
was added a suspension of 10% Pd/C (0.96 g, 0.9 mmol Pd) in
EtOH (40 mL) and set under hydrogen using a 30 cm diameter
balloon. After stirring for 12 h the catalyst was filtered off and
residual product extracted by stirring in MeOH–EtOH (4 : 1) for
2 h before filtering again. The filtrates were combined, evaporated
and recrystallized from EtOH to afford 3.75 g (82% yield) of
pale orange crystals. Anal. calcd (found) for C14H13NO4: C 64.90
(64.86), H 5.01 (5.05), N 5.38 (5.40). MS (EI): m/z 282 [M + H+].
1H NMR (300 MHz, DMSO-d6): d 1.99 (s, 3 H, OCOCH3), 2.33
(s, 3 H, C7-CH3), 6.22 (d, 1 H, H10, J = 10.2 Hz), 7.34 (d, 2 H,
1
2 H, H14, J = 5.9 Hz), 7.71 (d, 1 H, H11, J = 7.4 Hz). 13C{ H}
NMR (75 MHz, CD3OD): d 15.67 (C7-CH3), 62.65 (C6), 71.12
(C4), 75.58 (C3), 78.48 (C2), 78.72 (C5), 107.07 (C1), 116.58
(C10), 117.32 (C12), 128.96 (C14), 134.70 (C13), 142.81 (C11),
145.74 (C8), 147.61 (C7), 160.12 (C15), 174.70 (C9).
1612 | Dalton Trans., 2010, 39, 1604–1615
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Bis(1-[4-(2-Butyl)phenyl]-2-methyl-3-oxy-4(1H)-pyridinato)-
copper(II) (Cu(5b)2). 5b (64 mg, 0.25 mmol) was dissolved in
MeOH (4 mL), and triethylamine (34 mL, 2.4 mmol) was added.
Copper(II) perchlorate hexahydrate (46 mg, 0.12 mmol) was
dissolved in MeOH (4 mL) to give a pale blue solution. The
solution of deprotonated 5b was added dropwise to the copper
solution with stirring at RT. A fine green precipitate formed, and
this was left overnight in the fridge to allow full precipitation.
The product was filtered and washed with cold MeOH (2 ¥ 5 mL)
to give 38 mg (54% yield) of (Cu(5b)2). A small amount of this
material was used to saturate a solution of DMSO; X-ray quality
crystals were grown by slow evaporation at RT from a slightly
opened vial. Anal. calcd (found) for C32H36CuN2O4: C 66.71
(66.88), H 6.30 (6.23), N 4.86 (4.94). Infrared spectrum (cm-1,
total reflectance): 1588 (m), 1537 (s), 1503 (s), 1464 (s), 1350 (s),
1310 (s), 727 (m), 563 (s).
as Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid), a-tocopherol (a-Toc) and BHT (butylhydroxytoluene).
An improved ABTS∑+ (2,2¢-azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid) diammoniuim salt) radical cation decolourization
assay57 was used to determine relative TEAC values. ABTS was
dissolved in water (7 mM), then reacted with aqueous potassium
persulfate (2.45 mM), and placed in the dark for 16 h before
use. The ABTS∑+ product solution, after equilibrating to 30 ◦C
(Fisher Isotemp circulating water bath), was diluted with EtOH
to an absorbance of 0.70 ( 0.02) at 734 nm. Stock solutions of
the pyridinones in EtOH were diluted so that addition of 20 mL
to 2 mL of ABTS∑+ solution caused a reduction of 20–80% in
the absorbance as a result of the reduction to ABTS. To achieve
this, final concentrations for the pyridinones ranged from 2.5–
15 mM. Once the ABTS∑+ and the test compound were mixed, the
solutions were incubated at 30 ◦C, and the A734 readings were taken
after 1, 3, and 6 min. These measurements were done in triplicate.
The percentage inhibition of absorbance at 734 nm was calculated
and plotted as a function of pyridinone concentration. The slopes
were then compared to the standard Trolox, with its TEAC value
normalized to 100%.
Abg catalysed glycosidic cleavage
Preliminary reactivities of substrates (2d–5d) with Abg were tested
and monitored by TLC (see ESI). The reactions took place in 1 mL
Eppendorf tubes at RT with an Agrobacterium sp. b-glucosidase
(Abg) concentration of 3.9 nM. Initial substrate concentrations
were 7.5 mM for 3d–5d and 15 mM for 2d. TLC plates were
spotted 20–30 min after Abg initiated the reactions and were run
with 7 : 3 EtOAc–MeOH for 3d–5d, and 1 : 1 EtOAc–MeOH for
2d. A UV lamp, FeCl3 spray, and charring of TLC plates were
used in the characterization of compounds, as they show aromatic
compounds, free pyridinones,68 and carbohydrates, respectively.
MTT assay
Cells were received that had previously been cultured and prepared
with the experimental details described elsewhere.49 For this assay,
human breast cancer cells (MDA-MB-435S)48 were transferred to
a 96-well plate by addition of 100 mL of the cell solution (1 ¥ 104
cells) to 54 of the wells, and the plate was incubated at 37 ◦C
for 24 h. Phosphate buffered saline (PBS) solutions (100 mL)
of 5d at eight different concentrations (10-5000 mM) were then
added to the wells, and the plate was incubated at 37 ◦C for
3 d. A PBS solution of MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) (50 mL, 2.5 mg mL-1) was added to
each well of the plate which was then incubated for 3 h, by which
time a purple precipitate of formazan formed at the bottom of
certain wells.69 The contents of each well were carefully aspirated
off to leave the formazan that was then dissolved in DMSO (150
mL). The plate was shaken and analyzed by a plate reader (Spectra
Max 190 from Molecular Devices) to determine the absorbance of
each well at 570 nm. The percentage cell viability was calculated
by dividing the average absorbance of the cells treated with 5d by
that of the control.
Enzyme kinetics
A Hewlett-Packard(HP)model8453diodearrayspectrophotome-
ter, equipped with kinetics software package, was used to monitor
enzyme reactions with Agrobacterium sp. b-glucosidase (Abg).
The spectrophotometer cuvette holder was connected to a Fisher
Isotemp 1050 circulating water bath so that the temperature in the
cuvette was 37.0 0.2 ◦C after equilibration for 15 min. Enzyme re-
actions tookplaceinquartzcuvettes (semi-micro cells, 1.4mL total
volume, 10 mm path length), with each one having its background
measured while empty. The cuvette was then filled with 600 mL
of 50 mM sodium phosphate buffer (pH 6.8), 80 mL of a 50 mM
sodium phosphate buffered solution containing 1% bovine serum
albumin, and 100 mL of 2d in water and covered with parafilm to
prevent evaporation. The cuvette was equilibrated for 15 min in
the cuvette holder before 20 mL of Abg was syringed through the
parafilm to initiate the reaction (final [Abg] ~ 89 nM). The cuvette
was quickly inverted and replaced in the cuvette holder.
Turbidity assay for M-Ab aggregate dissolution
Lyophilized synthetic human Ab1-40 amyloid peptide was prepared
as a ~200 mM solution in distilled, deionized water. To achieve
full peptide dissolution while avoiding frothing, intermittent
sonication (1 min on, 30 s off) was repeated three times; the
solution was then filtered through a 0.2 mm syringe filter (What-
man) to remove any microparticulate protein matter. A bicin-
choninic acid assay was performed to quantify (relatively, versus
bovine serum albumin) the concentration of the Ab peptide.70
Two chelex-treated 4-(2-hydroxyethyl)-1- piperazineethanesul-
fonic acid (HEPES, 20 mM) buffer solutions containing 150 mM
NaCl were prepared with distilled, deionized water to pH values of
6.6 and 7.4. These solutions were filtered through 0.22 mm acetate
filters (Millipore) to remove any particulate material and were used
From 2d, the release of 3-hydroxy-1,2-dimethyl-4(1H)-
pyridinone (2b) (Scheme 3) was followed at 290 nm (lmax
=
285 nm). The initial rates were typically followed up to 2 min
after addition of Abg with data points being measured every 0.5 s.
For each concentration of substrate (2d at 0–3.0 mM), the initial
rates were determined in triplicate. The slopes for initial rates were
calculated using the on board HP software. Mathematical analyses
for kcat or Km values were done using GraFit software.55
Trolox equivalent antioxidant capacity (TEAC) assay
The 3-hydroxy-4-pyridinones were tested for their ability to
scavenge free radicals compared to antioxidant standards such
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Dalton Trans., 2010, 39, 1604–1615 | 1613
©

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in subsequent preparations of metal, ligand and reaction mixture
solutions. Stock solutions of CuII and ZnII were prepared from
atomic absorption standards to final concentrations of 200 mM
using pH 6.6 and 7.4 buffers respectively. Stock solutions of the
free ligands were prepared in HEPES buffer. The pyridinones were
used at 150 mM and diethylenetriaminepentaacetic acid (DTPA),
a known strong metal ion chelator, was used at 50 mM as a positive
control ligand.
Turbidity assays were performed in flat-bottomed 200 mL 96-
well microtiter plates (Falcon). The Ab1-40 peptide (~25 mM in
HEPES buffer) was exposed to either ZnII (25 mM, pH 7.4) or
CuII (25 mM, pH 6.6). After incubation for 3 min at RT, the test
compounds were added and the mixture incubated for a further
45 min at room temperature before absorbance measurements
were taken. A405 measurements were carried out with a Molecular
Devices Thermomax microplate reader, programmed to agitate the
plate for 30 s to evenly suspend any aggregates before all readings.
Immediately prior to ligand addition, aggregation appeared to be
complete (A405 0.03 for the ZnII samples and A405 0.04 for the
CuII samples). After a 45 min incubation with each test compound,
absorbance measurements showed lower turbidity, indicative of
disaggregation.
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Wells containing HEPES buffer only at the appropriate pH were
used as blanks. For each combination of metal ion, ligand and pH,
the analogous well was prepared containing metal ion, ligand and
HEPES buffer; in all cases where there was no Ab1–40 present,
A405 was negligible. MALDI spectroscopic analysis of selected
well solutions, performed after the turbidity assay, exhibited
peaks consistent with intact Ab1–40 peptide, confirming that no
degradation of the peptide occurred during the assay.
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Acknowledgements
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This work was supported by a Proof of Principle grant from the
Canadian Institutes of Health Research (CIHR) and a Strategic
grant from the Natural Sciences and Engineering Research
Council (NSERC). We also thank the Alexander von Humboldt
Foundation (M. M.), NSERC (T. S.), and the Alzheimer’s Society
of Canada (L. E. S.) for scholarship funding. The authors wish
to thank Prof. C. Overall and lab (Centre for Blood Research,
UBC) for the use of their plate reader for turbidometric assays,
and Prof. S. Withers (Chemistry, UBC) for a gift of Agrobacterium
sp. b-glucosidase.
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