'Click' assembly of glycoclusters and discovery of a trehalose analogue that retards Aβ40 aggregation and inhibits Aβ40-induced neurotoxicity

Article abstract of DOI:10.1016/j.bmcl.2014.07.077

Osmolytes have been proposed as treatments for neurodegenerative proteinopathies including Alzheimer's disease. However, for osmolytes to reach the clinic their efficacy must be improved. In this work, copper(I)-catalyzed azide-alkyne cycloaddition chemistry was used to synthesize glycoclusters bearing six copies of trehalose, lactose, galactose or glucose, with the aim of improving the potency of these osmolytes via multivalency. A trehalose glycocluster was found to be superior to monomeric trehalose in its ability to retard the formation of amyloid-beta peptide 40 (Aβ40) fibrils and protect neurons from Aβ40-induced cell death.

Full text of DOI:10.1016/j.bmcl.2014.07.077

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
‘Click’ assembly of glycoclusters and discovery of a trehalose
analogue that retards Ab40 aggregation and inhibits Ab40-induced
neurotoxicity
Hemalatha Rajaram a, Manoj Kumar Palanivelu a, Thiruma V. Arumugam b, Venkatesan M. Rao a,
P. Nicholas Shaw a, Ross P. McGeary c, Benjamin P. Ross a,
⇑
a The University of Queensland, School of Pharmacy, Brisbane, Queensland 4072, Australia
b Department of Physiology, Yong Loo Lin School Medicine, National University of Singapore, Singapore 117597, Singapore
c The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, Queensland 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Osmolytes have been proposed as treatments for neurodegenerative proteinopathies including Alzhei-
mer’s disease. However, for osmolytes to reach the clinic their efficacy must be improved. In this work,
copper(I)-catalyzed azide–alkyne cycloaddition chemistry was used to synthesize glycoclusters bearing
six copies of trehalose, lactose, galactose or glucose, with the aim of improving the potency of these
osmolytes via multivalency. A trehalose glycocluster was found to be superior to monomeric trehalose
in its ability to retard the formation of amyloid-beta peptide 40 (Ab40) fibrils and protect neurons from
Ab40-induced cell death.
Received 13 June 2014
Revised 25 July 2014
Accepted 29 July 2014
Available online xxxx
Keywords:
Alzheimer’s disease
Amyloid beta-peptides
Click chemistry
Glycoclusters
Ó 2014 Elsevier Ltd. All rights reserved.
Osmolytes
Alzheimer’s disease (AD) is the most common neurodegenera-
tive disorder and the leading cause of dementia.1 Currently, only
symptomatic treatments are available and there is an urgent need
for a disease-modifying drug.2 According to the modified amyloid
cascade hypothesis,3 AD is caused by small, soluble aggregates of
amyloid-beta peptide (Ab) which are highly neurotoxic.4 It follows
that one strategy to combat AD is to minimize the exposure of neu-
rons to these aggregates and approaches to achieve this include:
(a) inhibiting the production of Ab;5,6 (b) inhibiting Ab aggrega-
tion;7 (c) promoting off-pathway aggregation that produces non-
toxic Ab aggregates;8 and (d) improving clearance of Ab.9,10
Recently, osmolytes have been proposed as treatments for neu-
rodegenerative proteinopathies including AD.11 Osmolytes are
small organic molecules that protect intracellular macromolecules
from denaturation caused by environmental stresses such as per-
turbing solutes, dehydration, desiccation, extreme temperature,
and high hydrostatic pressure.12–14 There are three major catego-
ries of stabilizing osmolytes: carbohydrates; amino acids; and
methylammonium/methylsulfonium compounds.12,13 Many carbo-
hydrate osmolytes have been found both to inhibit Ab aggregation
and to attenuate Ab-induced neurotoxicity. Trehalose inhibits
Ab40/42 aggregation, dissociates pre-formed Ab40/42 aggregates
and decreases the toxicity of Ab40.15,16 Similarly, polymers carry-
ing trehalose suppress Ab40/42 fibril formation and reduce Ab40/
42-induced cytotoxicity.17,18 Myo-, scyllo-, and epi-inositol stabilize
a non-fibrillar Ab42 structure and attenuate Ab42-induced neuro-
toxicity.19–21 Fructose inhibits fibrillogenesis of Ab40/4222 and
attenuates Ab42-induced neurotoxicity.23 In many of these cases,
the carbohydrate osmolytes were active at millimolar concentra-
tions and for them to become viable treatments for AD their effec-
tiveness must be improved. It is well established that weak
interactions between proteins and carbohydrates can be amplified
by constructing multivalent carbohydrate ligands and this phe-
nomenon is known as the ‘cluster glycoside effect’.24,25 However,
very few studies have investigated whether this phenomenon
applies to carbohydrate osmolyte–Ab interactions and then only
for high MW glycopolymers.17,18 Herein we describe the use of
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) chemistry
to synthesize glycoclusters bearing six copies of trehalose, lactose,
galactose or glucose, with the aim of improving the potency of
the osmolytes’ effect on Ab secondary structure, aggregation, and
Ab-induced neurotoxicity.
Scheme 1 describes the synthesis of the glycoclusters. Dip-
entaerythritol (1) was chosen as the core branching unit onto
which six mono- or disaccharides were grafted. Hexa-alkyne 2
⇑
Corresponding author. Tel.: +61 7 334 61900.
E-mail address: b.ross1@uq.edu.au (B.P. Ross).
http://dx.doi.org/10.1016/j.bmcl.2014.07.077
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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2
H. Rajaram et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
azide 5.30 Peracetylated 6-azido-6-deoxy trehalose 6 was prepared
from trehalose which was iodinated with Ph3P/I2 followed by
immediate acetylation with Ac2O/pyridine to form 1-O-(2,3,4,6-
tetra-O-acetyl-a-D-glucopyranosyl)-2,3,4-tri-O-acetyl-6-deoxy-6-iodo-
a-D
-glucopyranoside.18 This compound was then reacted with
NaN3 in DMF to obtain azide 6.18 Azides 3-6 were ‘clicked’ onto
hexa-alkyne 2 by a CuAAC reaction31,32 followed by Zemplén
deacetylation33 to afford the target glycoclusters 7–10.
The effect of glycoclusters 7–10 on Ab40 aggregation kinetics,
secondary structure, fibril morphology and neurotoxicity was eval-
uated using the thioflavin T (ThT) fluorescence assay, circular
dichroism (CD) spectroscopy, transmission electron microscopy
(TEM), and the cell-based trypan blue dye exclusion assay. Control
glucose, galactose, lactose, and trehalose were tested at concentra-
tions six times greater than the glycoclusters (7–10) to ensure an
equivalent concentration of mono- and disaccharide units. To
obtain accurate and precise data from biophysical and biochemical
experiments using Ab, it is important to remove aggregates and
prepare monomer-rich starting solutions of Ab, commonly referred
to as low molecular weight (LMW) Ab.34,35 However, the high ten-
dency of Ab to aggregate makes this a challenging task.35 In this
study, LMW Ab40 was prepared by a facile procedure involving
dissolution of Ab40 in dilute aqueous NaOH solution (pH 10.5–
12)35 followed by dilution in phosphate buffer (pH 7.4), sonication
for 60 s, and ultracentrifugation (80,000 rpm for 90 min at 4 °C). As
described below, the supernatant obtained from this procedure
exhibited properties consistent with LMW Ab including a sigmoi-
dal kinetic growth curve with a long lag phase.
Glycoclusters 7–10 were screened for their effect on Ab40
aggregation using a continuous ThT assay under quiescent condi-
tions at pH 7.4 and 37 °C. ThT undergoes a large enhancement of
its fluorescence emission upon binding to amyloid fibrils hence
ThT fluorescence can be used to monitor Ab aggregation.36,37
A
concentration range of 10 nM to 100 lM of glycocluster was cho-
sen for screening because these concentrations might enable iden-
tification of potent compounds that are amenable to drug
development. Ab40 (35 lM) exhibited a sigmoidal kinetic growth
curve (Fig. 1) consistent with an autocatalytic process in which
new oligomers initially form only via slow primary nucleation,
and after elongation produces fibrils, fast fibril-catalyzed second-
ary nucleation commences and combines with elongation to
generate new fibrils.38,39 This autocatalytic positive feedback loop
results in exponential growth and secondary nucleation
Scheme 1. Synthesis of the glycoclusters. (a) Propargyl bromide, NaOH, TBAB,
toluene/water, 80 °C, 16 h, 33% yield; (b) (i) azides 3–6, CuSO4, sodium ascorbate,
THF/water, 40 °C, 6–18 h, (ii) 10 mM NaOMe in MeOH, 3 h, (iii) Amberlite IR-
120(H+), yields 56%⁄ (7), 63%⁄ (8), 28%⁄⁄ (9), 20%⁄⁄ (10), ⁄purification by silica flash
column chromatography, ⁄⁄purification by RP-HPLC.
was obtained by alkylating dipentaerythritol (1) with propargyl
bromide under phase-transfer conditions similar to those used by
Nouguier and McHich to alkylate pentraerythritol.26 An alternative
synthesis of 2 adapted from the method used by Touaibia et al. to
alkylate pentraerythritol,27 involving reaction of dipentaerythritol
(1) with propargyl bromide in DMF/KOH, was inferior (6% yield)
to the phase-transfer method (33% yield). Peracetylated b-glucosyl
azide 3 and peracetylated b-galactosyl azide 4 were prepared from
the corresponding 1,2,3,4,6-penta-O-acetyl-b-
treatment with with 33% HBr in AcOH to form the 2,3,4,6-tetra-
O-acetyl- -glycopyranosyl bromides, followed by immediate
D-glycopyranoses by
a-D
nucleophilic displacement with NaN3 in a phase-transfer reaction
according to the method of Tropper et al.28 Peracetylated b-lactosyl
azide 5 was synthesized by first acetylating lactose with Ac2O/
NaOAc and recrystallizing the crude product from hot MeOH to
give 1,2,3,6-tetra-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-
pyranosyl)-b-
-glucopyranose.29 This compound was converted to
2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b- -galactopyran-
osyl)- -glucopyranosyl bromide by treatment with 33% HBr in
AcOH, followed by immediate reaction with NaN3 in DMF to afford
D-galacto-
Figure 1. Aggregation of Ab40 monitored by in situ ThT fluorescence. Ab40 (35
was incubated with or without trehalose glycocluster 10 in phosphate buffer
(20 mM, pH 7.4, I 0.15 M, containing 20 M ThT and 0.007% NaN3) at 37 °C under
quiescent conditions. Fluorescence readings (kex 440 nm, kem 480 nm) were taken
every 5 min. Four representative kinetic traces are shown for each group.
lM)
D
D
l
a-D
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H. Rajaram et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
dominating primary nucleation as the main source of new oligo-
mers.38,39 An empirical sigmoidal model was fitted to each of the
fluorescence intensity versus time data sets and the amplitude
and lag time were estimated.40 Figure 2 shows the average results
of four independent experiments. All of the compounds extended
the lag phase of Ab40 aggregation with a concentration-dependent
increase in lag time observed for most compounds. In general, gly-
coclusters (7–10) at the two highest concentrations (10
100 M) extended the lag phase more than the corresponding
mono- and disaccharides (60 M and 600 M), and all of the gly-
coclusters (7–10) exhibited statistically significant activity at
100 M with lag times increased by between 28% and 53%. Three
lM and
l
l
l
l
instances of statistically significant changes in the amplitude of
Ab40 aggregation were observed, however the differences were
only small (ꢀ10%). Generally, the compounds did not effect the
amplitude (Figs. 1 and 2), indicating that ultimately the same
amount of fibrils is present at equilibrium.41
The extension of the lag phase of Ab40 aggregation caused by
trehalose glycocluster 10 was clearly concentration-dependent,
and 100
lM
of 10 resulted in an extremely significant
(p <0.0001) 45% increase in lag time (Figs. 1 and 2), consequently
trehalose glycocluster 10 was chosen for further investigation.
Figure 1 demonstrates that the lag phase of Ab40 aggregation is
extended as the concentration of 10 increases from 1 to 100 lM;
the slope of the curve, however, remains constant. Hence, once
the concentration of fibrils reaches a threshold level, the rate of
fibril formation is the same and independent of the lag time.42
Although the lag time and slope are each determined by both
nucleation and growth processes,41 the combination of an increase
in lag time with no change in slope suggests that trehalose glyco-
cluster 10 inhibits the rate of primary nucleation, with no or only a
minor effect on secondary nucleation and elongation.42
CD spectroscopy was used to confirm the results of the ThT
assay and to determine the secondary structure of the aggregates.
This technique relies on the well-established principle that LMW
Ab with predominantly unordered structure43 transitions to
b-sheet rich aggregates,34,44 and it has previously been used to
study inhibition of Ab aggregation.45 Trehalose glycocluster 10
was incubated with Ab40 at molar ratios of 1:5, 1:1 and 5:1 under
quiescent conditions at pH 7.4 and 37 °C. The CD spectra were
recorded over the wavelength range 190–250 nm and deconvo-
luted using the CONTIN algorithm46,47 on the DICHROWEB
server.48 Table 1 shows the percentage of each secondary struc-
tural motif at 0 and 72 h for each sample. Initially, Ab40 had pri-
marily (82–87%) unordered secondary structure with negligible
(0–3%) b-structure (b-sheet and b-turn) and a small (11–15%)
a-helical component. These results are consistent with previous
studies, which showed that LMW Ab lacks significant ordered
structure, and that transient
a-helix-containing intermediates
occur ‘on-pathway’ to the formation of b-structure rich fibrils.34,44
After 72 h, the Ab40 control sample transitioned to predominantly
(70%) b-structure with only small unordered (12%) and
a-helical
(18%) components, consistent with prior studies.34,44 Trehalose
had little effect on the b-structure content (65–69%), and trehalose
glycocluster 10 at the two lowest concentrations was only margin-
ally more active (62% b-structure). However, the sample containing
trehalose glycocluster 10 at a ratio of 5:1 (10:Ab40) displayed sub-
stantially less b-structure content (42%) and more unordered (36%)
and a-helical (22%) secondary structure compared to Ab40 control.
This confirms the results of the ThT assay, that trehalose glycoclus-
ter 10 delays but does not prevent the eventual formation of Ab40
fibrils that are rich in b-structure.
The morphology of the Ab40 fibrils at equilibrium was studied
using TEM. Trehalose glycocluster 10 was incubated with Ab40 at
molar ratios of 1:5, 1:1 and 5:1 under quiescent conditions at pH
7.4 and 37 °C for 7 days. Fibrils averaging ꢀ12 nm in width and
Fig. 2. ThT assay lag times and amplitudes obtained by fitting an empirical
sigmoidal model to the fluorescence intensity versus time data using non-linear
regression. The dashed horizontal lines show the control Ab40 (35 lM) value.
>2 lm long were observed for Ab40 alone and when Ab40 was
Values are the mean + SE of four independent experiments with ⁄p <0.05, ⁄⁄p <0.01,
⁄⁄⁄p <0.001 and ⁄⁄⁄⁄p <0.0001 c.f. control. Trehalose was tested at concentrations six
times greater than trehalose glycocluster 10 to ensure an equivalent concentration
of trehalose units.
incubated with trehalose (Fig. 3). Trehalose glycocluster 10 had a
subtle effect on the fibril morphology, with slightly smaller average
fibril width (ꢀ9 nm) and a significant number of shorter (<2
lm)
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H. Rajaram et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1
Conformation of Ab40 determined by deconvolution of CD spectra using the CONTIN algorithm
Sample
Conc (
l
M)
Time (h)
a
-Helix (%)
b-Sheet (%)
b-Turn (%)
Unordered (%)
Ab40
30
0
72
13
18
2
40
0
30
85
12
Ab40 (30
Ab40 (30
l
l
M) + glycocluster 10
6
0
72
0
72
0
72
15
21
13
20
11
22
1
35
0
32
2
16
0
27
0
30
0
26
84
17
87
18
87
36
30
150
M) + trehalosea
36
0
72
0
72
0
72
15
19
14
26
13
18
3
36
3
36
2
36
0
30
0
33
0
29
82
15
83
5
85
17
180
900
a
Trehalose was tested at concentrations six times greater than trehalose glycocluster 10 to ensure an equivalent concentration of trehalose units.
Figure 3. Morphology of Ab40 fibrils after 7 days of incubation. TEM images of: (a) Control Ab40 (60
fibril width 11.8 1.2 nm; and (c) Ab40 (60 M) + trehalose glycocluster 10 (300 M), fibril width 8.7 1.1 nm. Values are the mean SD and the scale bar represents 200 nm.
In all of the samples the majority of the fibrils were >2 m long however the sample containing trehalose glycocluster 10 (300 M) exhibited a significant number of shorter
(<2 m) fibrils. Trehalose was tested at concentrations six times greater than trehalose glycocluster 10 to ensure an equivalent concentration of trehalose units.
lM), fibril width 12.3 1.2 nm; (b) Ab40 (60 lM) + trehalose (1800 lM),
l
l
l
l
l
fibrils observed, and this effect was most pronounced in the sam-
ple containing the highest concentration of 10 (Fig. 3). Taken
together, the results of the ThT, CD and TEM studies reveal that
the principal effect of trehalose glycocluster 10 is inhibition of
the rate of primary nucleation, with no significant effect on the
thermodynamic equilibrium of Ab40 aggregation.
unordered (36%) and a -helical (22%) secondary structure com-
pared to Ab40 control (Table 1). Hence, preferential exclusion of
trehalose glycocluster 10 due to the osmophobic effect may stabi-
lize a compact helical form of Ab40 and retard the unfavorable
exposure of the peptide backbone that is required for the confor-
mational transition to b-structure.58 This may also inhibit the rate
of primary nucleation, resulting in an extended lag phase (Figs. 1
and 2). These explanations are consistent with molecular dynamics
simulations which showed that preferential exclusion of trehalose
is the origin of its inhibition of Ab40 aggregation, partly due to sta-
The preferential exclusion of osmolyte (preferential hydration
of protein) theory explains the mechanism by which osmolytes
protect folded proteins from denaturation.49,50 The theory states
that osmolytes raise the Gibbs energy of the unfolded denatured
state more than they do the folded state,49,50 because of unfavor-
able interactions between the osmolyte and the peptide backbone,
which is much more exposed in the unfolded state than in the
folded state.51 This solvophobic thermodynamic force, called the
osmophobic effect, means that protein denaturation in the pres-
ence of osmolyte is more unfavorable than it is in the absence of
osmolyte and hence osmolytes promote the folded form of the pro-
tein.52 High-resolution NMR spectroscopy showed that in aqueous
solution, synthetic Ab40 adopts a compact, partially folded struc-
ture in which residues 13–23 form a helix and the N- and C-ter-
mini, which contain many bends and turns, are collapsed against
the central helix.53 CD spectroscopy indicates that synthetic Ab ini-
tially has predominantly unordered secondary structure, and the
bilization of monomers with
a-helical structure so that b-sheet
structure is prevented.59 Compounds that retard Ab aggregation
by stabilizing helical structure may be of therapeutic benefit in
AD because nascent Ab generated by c-secretase cleavage of trans-
membrane protein C99 (APP672-770, also called b-CTF) is likely to
comprise substantial helical content, since residues 17–23 of Ab
arise from the C99 ‘N-helix’ (APP688–694) and residues 29–40/42
of Ab (APP700–711/713) originate from within the C99 helical trans-
membrane domain (APP700–723).60
The effect of trehalose glycocluster 10 on Ab40-induced neuro-
toxicity was studied using a cell-based trypan blue dye exclusion
assay.61 Preliminary studies confirmed that the test compounds
alone were devoid of any inherent toxicity at the concentrations
used in this study. Trehalose glycocluster 10 was incubated with
Ab40 for 6 h at 37 °C at molar ratios of 1:1 and 2.5:1 (10:Ab40)
prior to addition to 7–9 day old cultured mouse primary cortical
neurons. After 48 h the cells were fixed with trypan blue dye and
the number of live and dead cells were counted (Fig. 4). The normal
control cell death was very low at 10% and oligomeric
a
-helical content increases with time reaching maximal levels of
19–32% before waning as aggregation proceeds.34,44 It is well
established that eventually, Ab transitions into aggregates that
are rich in b-structure.34,44,54–57 In this study, trehalose glycoclus-
ter 10 incubated with Ab40 at a ratio of 5:1 (10:Ab40) for 72 h dis-
played substantially less b-structure content (42%) and more
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H. Rajaram et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
for assistance with TEM. The authors acknowledge the facilities,
and the scientific and technical assistance, of the Australian
Microscopy & Microanalysis Research Facility at the Centre for
Microscopy and Microanalysis, UQ.
Supplementary data
Supplementary data (including novel and literature
derived18,26–30,35,40,46,48,61,64–66 experimental content) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2014.07.077.
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was incubated with or without test compound for 6 h at 37 °C then added to the
neurons and after 48 h cell death was determined by the trypan blue exclusion
assay. The values are mean + SE with ⁄p <0.05, ⁄⁄p <0.01, and ⁄⁄⁄p <0.001 c.f. Ab40.
Trehalose was tested at concentrations six times greater than trehalose glycocluster
10 to ensure an equivalent concentration of trehalose units.
Ab40-induced cell death was 56%. Both trehalose glycocluster 10
and trehalose15 were neuroprotective with the greatest and most
significant activity observed with 100
10. The percentages of neuroprotection observed with trehalose
glycocluster 10 at 40 M and 100 M were 45% and 59%, respec-
tively, and with trehalose at 240 M and 600 M the percentages
lM of trehalose glycocluster
l
l
l
l
of neuroprotection were 39% and 55%, respectively. The mecha-
nism of neuroprotection may be attributed to the ability of treha-
lose glycocluster 10 to inhibit primary nucleation resulting in the
formation of fewer toxic Ab aggregates during the 6 h incubation
period used in the study. However, trehalose was also neuropro-
tective despite trehalose not exhibiting significant activity in the
ThT fluorescence assay and the CD spectroscopy study. Trehalose
is an activator of autophagy62 and increased autophagy has been
found to reduce Ab levels and improve cognitive function in a
mouse model of AD,63 therefore further work is required to eluci-
date the precise mechanism of the neuroprotection observed in
this study. Retardation of Ab fibril formation in combination with
enhanced clearance of Ab due to activation of autophagy may be
beneficial in the treatment of AD.62
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