Synthesis of chemically-tethered amyloid-β segment trimer possessing amyloidogenic properties

Article abstract of DOI:10.1016/j.bmcl.2015.05.029

As amyloid-β (Aβ) undergoes dynamic aggregation, it is impossible to isolate ('hook') the transient Aβ oligomer in an assembly state-pure form (e.g., sole Aβ dimer, trimer, tetramer, etc.). Obtaining such a pure Aβ oligomer would allow us to establish an in vitro system to perform a more detailed investigation of the pathogenic properties of the oligomer. A chemically-tethered Aβ oligomer, constructed only by covalent bonds, could satisfy this demand. Here we designed a chemically-tethered trimer of a pathogenic Aβ fragment (Aβ_25-35) (1) and successfully generated it in situ from its precursor (4), a water-soluble and non-aggregative O-acyl isopeptide of 1, in neutral aqueous media. Chemically-tethered 1 possessed stronger amyloidogenic properties, that is, potential for β-sheet structure, fibril formation, and cytotoxicity, than the corresponding monomer Aβ_25-35 (6). Trimerization of Aβ_25-35 sequence might affect both the aggregative properties and cytotoxicity, based on the present results. This work opens the door for chemical synthesis of oligomers bigger than trimers in an assembly state-pure form, allowing for identification of the most toxic Aβ oligomer.

Full text of DOI:10.1016/j.bmcl.2015.05.029

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2976–2979
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of chemically-tethered amyloid-b segment trimer
possessing amyloidogenic properties
a
a,b,
⇑
, Motomu Kanai a,b,
⇑
Kiyomichi Shinoda , Youhei Sohma
a
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
ERATO (Japan) Science and Technology Agency (JST), Kanai Life Science Catalysis Project, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
As amyloid-b (Ab) undergoes dynamic aggregation, it is impossible to isolate (‘hook’) the transient Ab oli-
gomer in an assembly state-pure form (e.g., sole Ab dimer, trimer, tetramer, etc.). Obtaining such a pure
Ab oligomer would allow us to establish an in vitro system to perform a more detailed investigation of the
pathogenic properties of the oligomer. A chemically-tethered Ab oligomer, constructed only by covalent
bonds, could satisfy this demand. Here we designed a chemically-tethered trimer of a pathogenic Ab frag-
ment (Ab25–35) (1) and successfully generated it in situ from its precursor (4), a water-soluble and non-
aggregative O-acyl isopeptide of 1, in neutral aqueous media. Chemically-tethered 1 possessed stronger
amyloidogenic properties, that is, potential for b-sheet structure, fibril formation, and cytotoxicity, than
the corresponding monomer Ab25–35 (6). Trimerization of Ab25–35 sequence might affect both the aggrega-
tive properties and cytotoxicity, based on the present results. This work opens the door for chemical syn-
thesis of oligomers bigger than trimers in an assembly state-pure form, allowing for identification of the
most toxic Ab oligomer.
Received 20 April 2015
Revised 9 May 2015
Accepted 12 May 2015
Available online 19 May 2015
Keywords:
Amyloid
Oligomer
Alzheimer
Aggregation
Peptide synthesis
Ó 2015 Published by Elsevier Ltd.
Amyloid-b peptide (Ab) is widely considered the etiological
experimental system without other untargeted assembly states
of Ab. Such a system would facilitate detailed investigations of
specific oligomers and allow for the discovery of aggregation inhi-
bitors targeting the oligomer.
cause of Alzheimer disease (AD), an incurable neurodegenerative
1
disorder. AD accounts for 60–70% of dementia, and there are cur-
2,3
rently more than 35 million AD patients world-wide. Once gen-
erated by proteolytic processing of the amyloid precursor protein,
Ab monomers aggregate to form oligomers and then fibrils, which
eventually construct senile plaques, a pathological hallmark of AD
in the brain. Among the aggregates formed by Ab monomers, oligo-
mers are the most neurotoxic,4 and therefore the most important
potential target for the development of drugs to treat AD. The abil-
ity to further study the most toxic Ab oligomer species is currently
a highly attractive aim.
One possible strategy toward achieving this aim is to
synthesize chemically-tethered Ab oligomers (Fig. 1b). The
chemically-tethered Ab oligomers are constructed only by covalent
bonds and not from naturally oligomer-constructing non-covalent
bonds (i.e., hydrogen bonds and hydrophobic interactions).
Therefore, in principle, using the chemically-tethering method,
we can isolate defined and unique Ab oligomers in an assembly
state-pure form using affordable analytical instruments. There
are several previous reports of chemically-tethered Ab oligomers,
5
Despite a number of studies of mixtures of Ab oligomers, none,
7
–11
to our knowledge, have successfully isolated an assembly state-
but they are limited to dimers
and there has been no report
6
pure oligomer (e.g., sole Ab dimer, trimer, tetramer, etc.) from a
on the synthesis of oligomers larger than dimers, as the synthesis
of trimers is considered more difficult than that of dimers. Some
reports, however, advocate an important role for Ab trimers in
mixture of oligomers (Fig. 1a). This is largely due to the extensive
aggregation of Ab oligomers, which results in high heterogeneity of
the oligomer species. Therefore, it is technically impossible to
1
2–15
Ab pathogenicity.
Based on this background, here we report
‘
hook’ transient oligomers composed of non-covalent bonds in an
the synthesis of Ab trimers.
assembly state-pure form. The ability to obtain assembly state-
pure Ab oligomers would allow us to set up an in vitro
We planned to tether Ab chains to the cyclic framework at their
N-termini via flexible linkers and designed the chemically-tethered
trimer (1; Fig. 2). As the Ab chain, we used the pathogenic segment
1
6–18
19–21
Ab25–35, which has been used in both in vitro
and in vivo
⇑
Corresponding authors. Tel.: +81 3 5841 4830; fax: +81 3 5684 5206.
E-mail addresses: ysohma@mol.f.u-tokyo.ac.jp (Y. Sohma), kanai@mol.f.u-tokyo.
experiments, and which retains the same toxic effect in rat hip-
2
2
pocampal compared to Ab1–42
.
ac.jp (M. Kanai).
http://dx.doi.org/10.1016/j.bmcl.2015.05.029
0
960-894X/Ó 2015 Published by Elsevier Ltd.

K. Shinoda et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2976–2979
2977
included an essential purification step by reverse-phase (RP)
HPLC, was difficult, and resulted in a low isolation yield (13%).
More problematic, however, was the fact that isolated 1 was not
very soluble in various solvents (e.g., 10% aqueous TFA, 0.02% aque-
3
ous NH , DMSO, hexafluoroisopropanol), and therefore we could
not prepare the concentrated stock solution that was necessary
for conducting the subsequent biological experiments with 1.
These observations reflected the strong aggregative properties of 1.
Therefore, we planned to apply the O-acyl isopeptide method24
to 1. The structure of the O-acyl form of 1 (4) is shown in Figure 3a.
The O-acyl structure was introduced to Ab chains in 1 to the bond
between Gly25 and Ser26. The O-acyl form of Ab1–42 has good sol-
ubility and no aggregative properties, whereas native Ab1–42 has
poor solubility and is strongly aggregative. Moreover, in neutral
aqueous solution, O-acyl isoAb1–42 is quickly converted to native
Ab1–42 via an O-to-N acyl rearrangement, thus acting as a precursor
of native Ab1–42. We expected that 4 could act as a dissolvable pre-
cursor of 1. Indeed, when we subjected 2 and 5 (O-acyl form of 3)
to the Huisgen cycloaddition, no gel was generated and 4 was
obtained without difficulty via RP-HPLC purification with an
improved isolated yield (31%, Fig. S3).
Figure 1. (a) It is impossible to ‘hook’ native Ab trimers in an assembly state-pure
form from a mixture of oligomers. (b) Chemically-tethered Ab trimers would be
obtained in an assembly state-pure form.
Next, we prepared a stock solution of 4 to conduct physico-
chemical and biochemical assays investigating the aggregative
properties of 1. First, 4 was easily dissolved in 0.1% aqueous TFA
affording a highly concentrated solution (150–200 lM). To prevent
any possible aggregation, this solution was subjected to ultracen-
trifugation (4 °C, 100,000 rpm, 1 h). The quantity of 4 was not
changed by centrifugal treatment, suggesting that 4 formed no
pre-aggregate species. We used this solution for several assays
after diluting it with neutral (pH 7.4) aqueous media, by which
condition 4 was quantitatively converted to 1 (Fig. 3b), allowing
us to investigate the properties of 1 in the physiological environ-
ment. In size exclusion chromatography (SEC), 1 gave a single peak
at the elution volume corresponding to Ab25–35 trimers just after its
in situ generation, suggesting high assembly state-purity of 1
(Fig. S4).
Figure 2. Chemically-tethered Ab25–35 trimer (1).
To synthesize 1, we prepared the following two peptides by
Fmoc chemistry-based solid phase peptide synthesis: cyclic pep-
tide 2 and azide functionalized Ab25–35 3 (for their structures, see
Fig. S1). When 2 and 3 were subjected to Cu(I)-catalyzed Huisgen
The cross b-sheet structure is characteristic of Ab aggregates,
2
5
especially large oligomers and fibrils. To compare the amount
of cross b-sheet structures between 1 and the corresponding
monomers Ab25–35 (6), we performed a thioflavin-T (ThT) fluores-
cence assay; cross b-sheet structures are recognized by ThT,
2
3
cycloaddition (Fig. S2), the reaction mixture turned to gel as
the reaction proceeded. Accordingly, the handling of 1, which
Figure 3. (a) In situ generation of 1 from 4 by an intramolecular O-to-N acyl rearrangement. (b) HPLC charts of the conversion from 4 to 1. The HPLC analysis was performed
using a C18 reverse phase column (4.6 Â 150 mm; YMC-Triart C18 column) with a linear gradient of MeCN (0–100% over 40 min) in 0.1% aqueous TFA at a flow rate of 1.0 mL/
min at 40 °C, and peaks were detected at 214 nm. Stock solution of 4 was diluted with 0.1% aqueous TFA and 0.1 M phosphate buffer (pH 7.4) (final concentration: 10 lM),
and the solutions were immediately subjected to analytical HPLC to obtain the charts designated as ‘before rearrangement’ and ‘after rearrangement’, respectively. The
retention time of in situ generated 1 was identical to that of the authentic sample.

2
978
K. Shinoda et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2976–2979
b-sheet structures. As a control, chemically-tethered Ab1–11 trimer
7; the sequence of Ab1–11 is H-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-
(
Gly-Tyr-Glu-OH; for its synthesis, see Supporting information) was
synthesized. The FI in 7 was not increased (Fig. S5), suggesting that
the enhanced propensity of 1 to form cross b-sheet structures was
not produced by the tethering. Additionally, as another control, lin-
early-tethered Ab25–35 trimer (Ac-Lys(X)-Gly-Lys(X)-Gly-Lys(X)-
2
Gly-NH where X = triazole linker-Ab25–35; 8) was subjected to
the ThT assay (for the synthesis of its O-acyl form (9), see
Supporting information). 8 showed the identical FI to 1 (Fig. S6),
suggesting that the contribution of the linker unit to form cross
b-sheet was minor. Circular dichroism (CD) spectra also supported
the predominance of b-sheet structures formed in 1; 1 (10
changed its secondary structure from a random-coil to b-sheet-
containing structure, whereas 6 (30 M) retained its random-coil
lM)
l
structure (Fig. S7). These findings suggest that trimerization of
Ab25–35 remarkably increases the propensity of cross b-sheet struc-
ture formation.
Next, to investigate whether 1 formed amyloid fibrils, we con-
ducted atomic force microscopy (AFM). 1 (10
were incubated for 20 min at 37 °C, and subjected to AFM analysis
Fig. 4b; images at t = 10 min and 1 h are shown in Fig. S8). Under
lM) and 6 (30 lM)
(
these conditions, 1 formed a larger quantity of fibrils than 6. The
morphology of the fibrils derived from 1 was robust and they were
up to ca. 2
lm long. These findings indicate that 1 had strong amy-
loid fibril-forming properties.
2
7
We further conducted a sedimentation assay and SEC to
investigate the aggregative properties of 1. In the sedimentation
assay, the quantity of the non-aggregated form of 1 (10
decreased (t = 3 h) when incubated at pH 7.4 and 37 °C, whereas
that of 6 (30 M) was not decreased at all (Fig. S9). In SEC, both
and 6 gave a single peak at the corresponding elution volume
lM) was
l
1
at t = 0 h (Fig. S10), but at t = 3 h, the peak height of 6 was almost
retained, whereas the peak of 1 disappeared. The disappearance
was considered to be due to the progression of aggregation to
higher-molecular-weight assemblies. Such aggregates might be fil-
tered out by the pre-column filter. These findings were consistent
with 1 having higher aggregative properties than 6.
We considered that the stronger aggregative properties of 1
2
8
could be explained by the seeding hypothesis; conversion of
monomers to oligomers is slow, but once oligomers are formed,
they act to seed aggregation and the conversion of oligomers to fib-
rils is quite fast. The present findings are consistent with this
hypothesis, as well as chemically synthesized Ab dimers with
stronger aggregative properties than those of the corresponding
7
,9–11
monomers.
Additionally, the activities of known aggregation inhibitors of
Ab were investigated to identify compounds with the potential to
Table 1
Figure 4. (a) ThT assay (kex = 440 ± 5 nm, kem = 480 ± 10 nm). 1 (derived from 4) or
was incubated at 37 °C in 0.1 M phosphate buffer (pH 7.4) for the indicated times
Activities of aggregation inhibitors on 1 or Ab1–42
6
ThT fluorescence intensitya (%)
*
**
Compound
with agitation by stirring. Mean ± SD, n = 3, p <0.05 versus 6, p <0.01 versus 6
Welch’s t-test). (b) AFM images (amplitude mode). 1 (derived from 4) or 6 was
(
1
Ab1–42
incubated at 37 °C for 20 min in 0.1 M HEPES–NaCl buffer (pH 7.4). (c) Cell viability
29,30
17 ± 8.5b
Curcumin
EGCG
NQTrp
5.9 ± 3.4
64 ± 3.2
17 ± 5.4
77 ± 9.6
20 ± 0.62
96 ± 3.1
assay. Well-seeded PC12 cells were treated with 1 (derived from 4), 6 or 10 at 37 °C
18,31
c
9.5 ± 3.3
2.9 ± 14
**
under 5% CO
2
for 4 d. Mean ± SEM, n = 3, * p <0.001 in the indicated pair (Tukey’s
32,33
test).
35
106 ± 14d
Tetracycline
Thionin34
0.44 ± 9.5
119 ± 20
e
-H-KLVFF-OH36
D
leading to an increase in its fluorescence intensity (FI) (Fig. 4a).26 FI
in 1 (10 M) and in 6 (30 M) was identical just after incubation
started (t = 0 min). With further incubation for up to 3 h at 37 °C,
however, FI in 1 increased in a time-dependent manner and almost
reached a plateau at 20 min, whereas FI in 6 hardly increased at all.
This finding indicated that 1 had a higher propensity to form cross
Mean ± SD, n = 3–6.
a
l
l
Relative FI to that of 1 alone (10
lM) or Ab1–42 alone (10
lM), incubated in the
presence of each inhibitor (50 lM) at 37 °C for 1 h with agitation by stirring.
b
p <0.05 versus Ab1–42 (Welch’s t-test).
p <0.001 versus Ab1–42 (Student’s t-test).
p <0.05 versus Ab1–42 (Student’s t-test).
p <0.01 versus Ab1–42 (Welch’s t-test).
c
d
e

K. Shinoda et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2976–2979
2979
inhibit aggregation of 1. Curcumin,2
9,30
EGCG,18,31 NQTrp (1,4-
Supplementary data
naphthoquinon-2-yl-
L
-tryptophan),3
2,33
and thionin had strong
34
3
5
inhibitory effects on aggregation of 1, whereas tetracycline and
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.05.
3
6
D
-H-KLVFF-OH had no inhibitory activity (Table 1). As cur-
3
0
and EGCG18 is known to inhibit the aggregation of
0
29.
cumin
Ab25–35, these inhibitions on 1 imply that the aggregation pattern
of the original Ab25–35 is preserved in 1. In addition, as EGCG and
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