Synthesis and Characterization of Hairpin Mimics that Modulate the Early Oligomerization and Fibrillization of Amyloid β-Peptide

Article abstract of DOI:10.1002/ejoc.201700010

Alzheimer's disease is a neurodegenerative disorder that is linked to oligomerization and fibrillization of different amyloid β-peptide isoforms. Among these amyloid peptides, Aβ_1–42 is considered the most aggregative and neurotoxic species. We

Full text of DOI:10.1002/ejoc.201700010

A Journal of
Accepted Article
Title: Synthesis and characterization of hairpin mimics that modulate
amyloid beta-peptide early oligomerization and fibrillization
Authors: Umberto Piarulli, Leila Vahdati, Julia Kaffy, Dimitri Brinet,
Guillaume Bernadat, Isabelle Correia, Silvia Panzeri, Roberto
Fanelli, Olivier Lequin, Miriam Taverna, and Sandrine Ongeri
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700010
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10.1002/ejoc.201700010
European Journal of Organic Chemistry
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Hairpins hold it firmly The small
hairpin mimic containing a cis-
diketopiperazine scaffold as a turn
inducer, is able to decrease the
aggregation of amyloid peptide
A1-42 and prevent the formation of
oligomeric and neurotoxic species.
Amyloid-Oligomerization
Modulators
Leila Vahdati, Julia Kaffy, Dimitri
Brinet, Guillaume Bernadat,
Isabelle Correia, Silvia Panzeri,
Roberto Fanelli, Olivier Lequin,
Myriam Taverna, Sandrine
Ongeri,* and Umberto Piarulli*
.
Page No. – Page No.
Synthesis and characterization
of hairpin mimics that
modulate amyloid -peptide
early oligomerization and
fibrillization
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10.1002/ejoc.201700010
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Synthesis and characterization of hairpin mimics that modulate
amyloid -peptide early oligomerization and fibrillization
Leila Vahdati,[a,b] Julia Kaffy,[b] Dimitri Brinet,[b,c] Guillaume Bernadat,[b] Isabelle Correia,[d] Silvia
Panzeri,[a] Roberto Fanelli,[a] Olivier Lequin,[d] Myriam Taverna,[c] Sandrine Ongeri,*[b] and Umberto
Piarulli*[a]
Dedicated to Professor Cesare Gennari on the occasion of his 65th birthday
amyloidogenesis) can be associated to more than 20 serious
human diseases and can play leading role in
Abstract: Alzheimer’s disease is a neurodegenerative disorder
linked to oligomerization and fibrillization of different amyloid 
peptide isoforms. Among these amyloid peptides, A1-42 is
considered as the most aggregative and neurotoxic species. We
report herein the synthesis of four -sheet mimics composed of a
peptidomimetic arm based on a 5-amino-2-methoxy benzhydrazide
derivative, a 2,5-diketopiperazine scaffold (either cis-DKP or trans-
DKP) and a tetrapeptide sequence (either Gly-Val-Val-Ile, GVVI, or
Lys-Leu-Val-Phe, KLVF). The derivatives containing the cis-DKP
were shown by NMR and computational studies to adopt a stable -
hairpin conformation in solution, whereas the trans-DKP scaffold
promoted the formation of extended structures. The activity of these
compounds in modulating the aggregation of A1-42 peptide was
investigated by Thioflavin T fluorescence assay to measure the
kinetics of aggregation. Capillary electrophoresis (CE) and
transmission electron microscopy (TEM) were then used to monitor
the formation of small soluble Aoligomers and higher molecular
weight and insoluble A aggregates, respectively. As a result, small
hairpin mimics containing the cis-DKP scaffold are able to prevent
the formation of small A1-42 oligomeric and neurotoxic species.
a
neurodegenerative diseases, such as Alzheimer’s disease (AD),
Parkinson’s disease (PD), as well as other systemic disorders
characterized by the accumulation of insoluble protein deposits
(e.g. type II diabetes mellitus).[1] AD, which is the most common
form of late-life dementia,[2] is associated with the accumulation
of intraneuronal neurofibrillary tangles composed of paired
helical filaments of hyperphosphorylated tau protein and
extracellular plaques containing insoluble fibrils made of 40 or
42-residue amyloid peptides (A1-40 or A1-42).[3] A1-42 is the
predominant form in amyloid plaques and is by far more
[4,5]
aggregative and neurotoxic than A1-40
.
The conversion of
monomeric A peptides into fibrils proceeds through a complex
nucleation process and involves the formation of various
aggregated species such as soluble oligomers and protofibrils of
increasing size,[ 6 , 7 , 8 ] which are structured in -sheet-rich
conformations involving the hydrophobic central (K16LVFFA21)
and C-terminal (I31IGLMVGGVVIA42) sequences.[9,10,11,12,13] Low
molecular weight oligomers have been considered for the last
decade by several groups, as primarily responsible for the
neurodegeneration observed in AD, by inducing physical
changes in the cell membrane leading to dysfunction and cell
death.[3,4,14,15,16,17] In addition, fibrils are not inert species as they
have been demonstrated to generate damaging redox activity
Introduction
18
]
and promote the nucleation of toxic oligomers.[
As a
consequence, reducing the prevalence of both fibrils and small
transient oligomers has become an essential criterion for the
evaluation and selection of small therapeutic molecules. Much of
the research effort aimed at targeting the aggregation process of
A1-42 has been focused on retarding the fibrillogenesis, [19,20,21]
and, although many compounds have been reported to inhibit
the aggregation, their mechanism of action and effects on
oligomers formation remain unclear to date, thus hampering
their development as drug candidates.[ 22 , 23 ] Some peptide
derivatives have also shown interesting inhibition activities on
The role of protein-protein interactions in protein aggregation
has been extensively investigated, suggesting that cumulative
protein misfolding in the aggregation process (i.e.
[a]
Dr. L.Vahdati, Dr S. Panzeri, Dr. R. Fanelli, Prof. Dr. U. Piarulli
Università degli Studi dell’Insubria
Dipartimento di Scienza e Alta Tecnologia
Via Valleggio 11, 22100 Como (Italy)
E-mail: umberto.piarulli@uninsubria.it
Url: http://www4.uninsubria.it/on-line/home/rubrica/home-page-
personale.html?P1_m=P000228
24
]
A1-42 aggregation.[
Recently, the assumption that
preorganized strands in -hairpin mimics will promote the
association and sheet formation with A1-42 thus preventing, or at
least delaying its aggregation, has been validated by
macrocyclic -sheet mimic structures.[ 25 , 26 ,] However, to our
knowledge, only rare examples of acyclic -hairpins have been
demonstrated as A1-42 binders and inhibitors of aggregation.
[27,28,29,30]. On the other hand, the exclusive or very high peptidic
character of these compounds would definitely hamper their
development as drugs. We hypothesized that maintaining an
acyclic -hairpin while decreasing the peptidic character could
provide valuable insights to develop -hairpin mimics with higher
drug-like characteristics. For that purpose, we describe the
[b]
[c]
[d]
Dr. J. Kaffy, Dr. D. Brinet, Dr. G. Bernadat, Prof. Dr. S. Ongeri
BioCIS, Univ. Paris-Sud, CNRS, Université Paris-Saclay, 92290,
Châtenay-Malabry, FRANCE
E-mail: Sandrine.ongeri@u-psud.fr
Dr. D. Brinet, Prof. Dr. M. Taverna
Protéins and Nanotechnology in analytical science, Institut Galien
de Paris Sud, Univ. Paris-Sud, CNRS, Université Paris-Saclay,
92290, Châtenay-Malabry, FRANCE
Dr. I. Correia, Prof. Dr. O. Lequin
Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale
Supérieure, PSL Research University, CNRS, Laboratoire des
Biomolécules, 4 place Jussieu, 75252 Paris Cedex 05, France
Supporting information for this article is given via a link at the end of the
document.((Please delete this text if not appropriate))
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10.1002/ejoc.201700010
European Journal of Organic Chemistry
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synthesis of a new class of peptidomimetics (Figure 1), synthesis of the remaining derivatives 2-4: Boc-5-amino-2-
containing a bifunctional diketopiperazine scaffold DKP, as a methoxy benzhydrazide 5 was treated with acetic anhydride to
potential -turn inducer, a peptidomimetic arm and a tetrapeptide afford compound 6a,[ 34 ] or, in alternative, with trifluoroacetic
arm. As peptidomimetic arm, we inserted the 5-amino-2- anhydride in the presence of triethylamine, to introduce a
methoxybenzhydrazide unit, which is a part of the -strand trifluoroacetate group and yield 6b. Cleavage of the Boc moiety
mimic (“Hao” unit) used by Nowick and co-workers.[26] The four and coupling of 6a and 6b to N-Boc-Val-OH afforded the
amino acid residues were selected from hydrophobic A1-42 peptidomimetic strands 7a and 7b, which, after removal of the
sequences playing a crucial role in the -sheet structuration of Boc protecting group were coupled to either diketopiperazine
A1-42 aggregates. The bifunctional diketopiperazines introduced scaffold cis-DKP or trans-DKP. This coupling was realized by
in these constructs, were recently developed in our group as the use of the triazine-based reagent DMTMM(BF4), 35 since
effective inducers of the secondary protein structure. In fact, other activating reagents failed to achieve the coupling or
short peptidic sequences containing the cis-DKP scaffold (Figure afforded the products in low yield and/or with major impurities.
1), were shown by NMR and molecular modeling studies to Conversely, through the use of DMTMM(BF4), compounds 8a-8d
adopt very stable -hairpin structures,[31] whereas the trans-DKP were obtained in satisfactory yields (40-54%). The tetrapeptide
scaffold effectively induced -turn conformations in short cyclic sequences Gly-Val-Val-Ile and Lys-Leu-Val-Phe were then
peptides.[32] The activity of these compounds in modulating A1- constructed in the lower arm in a step by step solution phase
early oligomerization and fibrillization was finally investigated synthesis. The N-Boc protected amino acids were coupled in
42
using a combination of different assays: thioflavin T fluorescence good yields using HATU and HOAt in DMF, and the protected
assay, capillary electrophoresis (CE) and transmission electron modulator precursors 13a-13d were eventually obtained. Final
microscopy (TEM).
deprotection of the main and side chain protecting groups
afforded compounds 1-4. Purification via preparative HPLC and
lyophilization gave the pure TFA salts, for the subsequent
conformational analysis and aggregation inhibition studies.
The conformation of 1 was recently studied by NMR and the
formation of a stable hairpin 1 verified and corroborated by
computational studies, which also assessed the safe
extrapolation of the structural characteristics found in the NMR
studies in methanol to the aqueous environment.[33] The
conformation of the derivative 2 was then investigated in
methanol (see Figure 2 for the structure of compound 2 and the
residue numbering). Attempts to perform these studies in
aqueous solutions were hampered by the low solubility of these
compounds and the broad line-widths of the resulting spectra.
Figure 1. Structure of compounds 1-4 containing
a cis or trans
diketopiperazine scaffold, a peptidomimetic arm and a peptidic arm
Results and Discussion
Synthesis and characterization of peptidomimetics 1-4
To establish a relationship between the structure of these
peptidomimetics with respect to the affinity towards A1-42
,
several structural elements of these compounds were taken into
consideration: 1) the importance of the -hairpin structure: cis
versus trans-DKP scaffold, the presence of two arms linked to
the scaffold versus only one arm, the activity of the isolated
tetrapeptide and peptidomimetic arms; 2) the hydrophobic
peptidic sequence: Gly-Val-Val-Ile versus Lys-Leu-Val-Phe, that
are part of the C-terminal (G38VVI41) and the central (K16LVF19)
A1-42 sequences respectively, both involved in stabilizing A1-42
aggregates;[9-13] 3) the effect of introducing a hydrophobic
trifluoromethyl group versus a methyl group; and 4) the
possibility of forming electrostatic interactions with acidic
residues of A1-42, by cleaving the N-protecting groups in all the
final products. In this way the structures of compounds 1-4 were
devised.
Figure 2. Structure of hairpin mimic 2 highlighting the residue numbering and
the most important interstrand ROEs. Residue abbreviations: Dapa, 2,3-
diaminopropionic acid; Amb, 5-amino-2-methoxy benzhydrazide unit
1
The H and 13C chemical shift deviations (CSD, defined as
the differences between experimental chemical shifts and
corresponding random coil values) of Leu2, Val3, Phe4 and Val7
residues proved that extended conformations of amino acids
predominate (see supporting information, Table S3), as
supported by upfield shifted C carbons (negative CSD values
between –4.6 and –1.9 ppm), and downfield shifted H protons
3
(positive CSD values > 0.1 ppm). The large vicinal JHN-H
Compound 1, containing the cis-DKP linked to the tetrapeptide
GVVI and a peptidomimetic arm was recently prepared[ 33 ]
according to Scheme 1, which was also followed for the
coupling constants (7.8–9.7 Hz) also reflected  dihedral angles
corresponding to  conformations.
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Scheme 1. a) Ac2O, THF, quantitative yield; b) (CF3CO)2O, TEA, THF, 89%; c) 1) TFA, CH2Cl2, quantitative yield; 2) Boc-Val-OH, HBTU, HOBt, DIPEA, DMF,
overnight, 69% (7a), 80% (7b); d) 1) TFA, CH2Cl2, quantitative yield, 2) H2N-DKP-COOCH2CH=CH2 DMTMM(BF4), NMM, DMF, overnight, 0 °C to r.t., 45% (8a),
40% (8b), 54% (8c), 52% (8d); e) TFA, CH2Cl2, quantitative yield, f) HATU, HOAt, DIPEA, Boc-Ile-OH or Boc-Phe-OH, DMF, 0 °C to r.t., overnight, 78% (10a),
87% (10b), 98% (10c), 98% (10d); g) 1) TFA, CH2Cl2, quantitative yield, 2) HATU, HOAt, DIPEA, Boc-Val-OH, DMF, 0 °C to r.t., overnight, 83% (11a), 76% (11b),
80% (11c), 85% (11d); h) 1) TFA, CH2Cl2, quantitative yield, 2) HATU, HOAt, DIPEA, Boc-Val-OH or Boc-Leu-OH, DMF, 0 °C to r.t., overnight, 74% (12a), 75%
(12b), 60% (12c), 90% (12d); i) 1) TFA, CH2Cl2, quantitative yield, 2) HATU, HOAt, DIPEA, Boc-Gly-OH or Boc-Lys(Cbz)-OH, DMF, 0 °C to r.t., overnight, 77%
(13a), 75% (13b), 67% (13c), 65% (13d); j) TFA, CH2Cl2, overnight, quantitative yield, 1, 3, 4; k) Pd/C, H2, MeOH, then TFA, CH2Cl2, overnight, quantitative yield
2.
respectively. HN hydrogen atoms are shown in grey. The figure was prepared
with PyMOL.
Finally, the analysis of H-HN ROE correlations showed strong
sequential and medium intra-residual H-HN ROEs, which are
characteristic of extended backbone conformations. The most
compelling evidence for the formation of a -hairpin came from
the observation of numerous ROEs between the two arms.
Indeed, 14 interstrand ROEs could be detected, involving
backbone and side-chain protons of Val7 with Phe4-Dapa5, and
Amb8 H6 proton with Leu2-Val3 protons (see Figure 2 for residue
numbering and the most important interstrand ROEs and
supporting information, Figure S1, Table S5). The NMR
structures of 2 were then calculated by restrained molecular
dynamics based on distance and dihedral angle restraints
inferred from NMR data (see supporting information, Table S5).
Structures adopt a well-defined -hairpin conformation (Figure 3),
Figure 3. Structure of 2 calculated using NMR conformational parameters
observed in methanol. The 20 conformers were superimposed by best fitting of
backbone heavy atoms. Fluorine, nitrogen, oxygen, backbone and side-chain
carbon atoms are coloured in green, blue, red, cyan and magenta,
with the formation of 3 to 4 inter-strand hydrogen bonds. The
conformers show fraying at the extremities of both arms, the
hydrogen bond between Lys1 and the terminal acetamido group
being not systematically observed. Dapa5 amide proton exhibits
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a small negative temperature coefficient (HN/T value of –4.3
ppb/K, see supporting information, Table S3), in agreement with
its involvement in a hydrogen bond. Other amide protons show
larger variations, which may be ascribed to an increased
flexibility of the -sheet toward the extremities. The structures
are further stabilized by van der Waals interactions between
Leu2, Phe4 and Val7 residues and the DKP-N-benzylic group,
forming a large hydrophobic face. The NMR conformers show
well-defined side-chain conformations, 1 rotamers being
gauche– (∼ –60°) for Leu2 and Phe4, trans (∼180°) for Val7 and
gauche+ (∼ +60°) for Val3.
material formed (Table 1). On the contrary, no fluorescence was
observed, up to the maximum 100 M concentration used in this
assay, in the solutions containing compounds 1-4 alone,
presuming that no extensive self-aggregation occured. The
activity of -hairpin 1 based on the cis-DKP and bearing the
peptidic sequence GVVI and the peptidomimetic 5-acetamido-2-
methoxy benzhydrazide was then investigated.
A modest
inhibitory effect on A1-42 fibrillization was observed, albeit at the
high 1/A1-42 ratio of 10/1 (Table 1). At this ratio, 1 delayed the
rate of aggregation of A1-42 (t1/2 was increased by almost a
factor 2, 186 %) and slightly decreased the fluorescence plateau
(equal to 71 % of F of the control experiment).
We next examined the conformational preferences of the trans-
DKP derivative 4. The Val2,3,7 and Ile4 residues tended to adopt
extended backbone conformations, as indicated by upfield
shifted C carbons (negative CSD values between –3.5 and –
1.7 ppm), and downfield shifted H protons (positive CSD
values, excepted Ile4, see supporting information, Table S4).
These extended conformations were further supported by the
Table 1. Effects of compounds 1-4 on A1-42 fibrillization assessed by ThT-
fluorescence spectroscopy at compound/A ratios of 10/1 and 1/1).
Compound
Compound/
A1-42 / ratio [a]
t1/2
variation
F variation factor (%)
[b]
factor (%) [b]
3
large JHN-H coupling constants (> 8 Hz) and the analysis of 2D
1
2
3
4
10 / 1
1 / 1
186±22 %
ne
71±3 %
ne
ROESY spectrum revealing strong sequential and medium
intraresidual H-HN correlations. However, no long-range ROEs
could be detected between the two arms, proving that 4 did not
adopt an intramolecular -hairpin conformation. The amide
protons also showed a large variation of their chemical shift with
temperature (HN/T between –9 and –6 ppb/K) confirming
that they were not involved in intramolecular hydrogen bonds
(see supporting information, Table S4). Notably, significant
10 / 1
1 / 1
NA
222±36 %
3±2 %
29±2 %
10 / 1
1 / 1
160±12 %
ne
72±18 %
ne
10 / 1
1 / 1
23±4 %
ne
67±4 %
ne
1
chemical shift changes were observed on 1D H spectra upon
concentration variation (10 M – 0.4 mM) suggesting that 4
could be prone to association through intermolecular hydrogen
bonding.
ne = no effect, NA = no aggregation. Variation factors are expressed as mean
± SE, n=3-6. [a] The concentration of A1-42 in this assay is 10 M. [b] See
supporting information for the calculation of the t1/2 and F variation factors
(<100% means a decrease, >100% means an increase).
ECD spectra were also measured for compounds 2 and 4 (0.1
mM in methanol, see Figure S2 in the Supporting Information).
The two curves display similar features, which can hardly be
traced back to any of the canonical secondary structures of
peptides. This can probably be ascribed to the presence of the
the 5-amino-2-methoxybenzhydrazide chromophore contributing
to the observed CD signal in the far UV region.
In summary, peptidomimetics containing the cis-DKP scaffold
can exist in -hairpin conformation and, as such can be
expected to act as -sheet binders and A1-42 aggregation
inhibitors. On the contrary, peptidomimetics containing the trans-
DKP are unable to form -hairpin structures although the two
arms adopt extended conformations which, in principle, could
still actively interact with A1-42 amyloid peptide.
Peptidomimetic 3, containing the trans-DKP scaffold and the
same two arms as 1, was then studied. Also in this case, a
minimal delay in the aggregation process (even lower than 1,
160 vs 186 % of the t1/2 of the control) was observed with almost
no effect on the intensity of the fluorescence plateau. In order to
evaluate the role of the two arms, the tetrapeptide sequence
GVVI-NH2 (primary amide at the C-terminus), and the
deprotected peptidomimetic arm 7a (after TFA cleavage of the
Boc protecting group), were tested. A modest acceleration in the
aggregation rate and a more pronounced increase of the
fluorescence plateau were observed with the tetrapeptide
sequence, whereas the deprotected peptidomimetic arm showed
no activity (see supporting information Table S6). In order to
study the effect of introducing a hydrophobic trifluoromethyl
group versus a methyl group, the acetyl capping group at the 5-
amino-2-methoxybenzhydrazide unit (R1 = CH3, compound 3 in
Figure 1) was replaced by a trifluoroacetyl group (R1 = CF3,
Modulation of amyloid aggregation by peptidomimetics 1-4
Thioflavin T fluorescence assays are a benchmark methodology
to monitor the kinetics of aggregation of amyloid proteins [36] and
their modulation by amyloid -sheet mimics.[26, 27, 28, 29] The ability
of compounds 1-4 and of a few of their synthetic precursors or
fragments to interfere with the in vitro A1-42 fibrillization process,
was thus investigated. The fluorescence curve for A1-42 alone at
a concentration of 10 μM followed the typical sigmoid pattern
with a lag phase (between 4 to 10 h, depending on the
experiment), an elongation phase and a final plateau reached
after 12 to 20 h. Two parameters were derived from the ThT
curves: t1/2, defined as the time at which the half maximal ThT
fluorescence is reached, is a measure of the aggregation
process rate; and F, the fluorescence value of the final plateau
which is assumed to be dependent on the amount of fibrillar
compound
4 in Figure 1). Interestingly, the activity was
significantly affected. Compound 4 (Table 1 and supporting
information Figure S3) promoted the aggregation of Aβ1-42 at a
10/1 ratio, with a significant reduction of t1/2 (equal to only 23%
of t1/2 of the control). In addition, the final fluorescence intensity
was also slightly decreased (equal to only 67% of F of the
control) indicating that the amount and/or morphology of the
fibers might be different from those of the control sample. The
beneficial effect of triflluoromethylation of the 5-amino-2-
methoxybenzhydrazide unit was further corroborated by the
observation that compounds 9b (cis-DKP) and 9d (trans-DKP),
i.e. the synthetic intermediates containing the DKP scaffolds and
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the peptidomimetic arm, displayed a significant activity on the
aggregation process while 9a and 9c (their methylated analogs)
had no effect. In fact, the cis analog 9b slightly accelerated the
aggregation while the trans analog 9d significantly delayed it
even at the low 1/1 9d/A1-42 ratio (see supporting information
Table S6 and Figure S3). Analyzing these first results,
compounds 1, 3 and 4, containing the GVVI tetrapeptide
sequence, behave as modest modulators of the aggregation of
A1-42 at the high 10/1 compound/A1-42 ratio, while they
displayed no activity at the stoichiometric 1/1 ratio.
Summarizing, among the four peptidomimetics prepared, 2 and
4 showed interesting properties in modulating A1-42 aggregation
process, where compound 2 inhibited the aggregation, while
compound 4 accelerated it. For this reason, we decided to
investigate in more detail their mechanism of aggregation
modulation.
It is well known that the Tht- binding assays present limitations
and drawbacks in the evaluation of amyloid aggregation: Indeed,
some small aromatic compounds were observed to displace the
thioflavin from its binding site into the amyloid beta-sheet,
decreasing the fluorescence signal, and thus producing false
positives.[37] A deeper insight in the extent and pathways of A
aggregation can be obtained by a combination of different
methodologies and in particular of capillary electrophoresis (CE)
and transmission electron microscopy (TEM).
Figure 4. Representative curves of ThT fluorescence assays over time
showing Aβ1-42 (10 µM) aggregation in the absence (purple curves) and in the
presence of compounds 2 (black curves) at compound/Aβ1-42 ratios of 10/1 and
1/1.
For this reason, we decided to investigate the KLVF tetrapeptide
sequence, alone or inserted in peptidomimetic derivatives. The
tetrapeptide KLVF-NH2 very slightly delayed the aggregation (t1/2
was increased by 38%, see supporting information) at the ratio
of 10/1 (KLVF/A1-42), although it had no significant effect on the
fluorescence plateau. This result indicates that the sequence
KLVF might be more favorable to decrease the aggregation
process than GVVI which is more likely to accelerate it. Indeed,
hairpin 2 (Figure 1), where the KLVF tetrapeptide was linked to
the cis-DKP scaffold and to the 5-trifluoroacetamido-2-
methoxybenzhydrazide
peptidomimetic
arm
completely
suppressed the ThT fluorescence at a 2/A1-42 ratio of 10/1 (See
Table 1 and Figure 4) indicating or a total inhibition of A1-42
fibrillization or a different pathway of aggregation conducting to
aggregates with different morphology than the one of classical
fibers and that do not bind ThT. Hairpin 2 still displayed a very
good efficiency at a stoichiometric ratio (t1/2 was more than
doubled, 222% and the final fluorescence intensity was only of
29% of the control value, see Table 1 and Figure 4). This result
confirms that the cis configuration of the DKP scaffold combined
with the tetrapeptide KLVF and the trifluoroacetamido-2-
methoxybenzhydrazide-based peptidomimetic are beneficial for
the modulation of A1-42 fibrillization. The Boc-protected
compounds 13a-13d, precursors of the final compounds 1-4
were also evaluated. However, they all tended to self-aggregate
(at 100 μM and even slightly at 10 μM) and to be self-organized
in structures that bind the ThT dye already in the control
experiments (see supporting information Table S6), preventing
their evaluation.
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Figure 5. Electrophoretic profile obtained immediately (0 h), 4 h and 6 h after
sample reconstitution (t0) of A1-42 peptide (100 μM) alone (A), in the presence
of compound 2 at 2/A1-42 ratio of 1/1 (B) and in the presence of compound 4
at 4/A 1-42 ratio of 1/1 (C).
overall kinetics of aggregation and to change the pathway of
fibrillization forming more globular aggregates.
Specifically, while transmission electron microscopy (TEM)
analyses have been used to reveal the formation and
morphology of higher molecular weight and insoluble A
aggregates, we have recently reported a CE method to monitor
the early steps of the oligomerization process over time, and in
particular the formation of small soluble oligomers.[ 38 ] This
technique allows the evaluation of the impact of small molecules
on three key species, (i) the monomer of A1-42 (peak ES), (ii)
different small metastable oligomers grouped under peak ES’
and (iii) transient later species corresponding to oligomers larger
than dodecamers (peak LS).[38, 39] Aggregation kinetics of A1-42
alone showed that overtime, the peak of monomer ES
decreased in favor of the peaks of oligomers ES’ and LS, and
that soluble species were no longer visible after 6 h (Figure 5A).
The electrophoretic profiles obtained in the presence of the cis-
DKP derivative 2 indicated that 2 delayed the aggregation
process and maintained the presence of the monomer ES which
was still present after 6 h (Figure 5B). Small and large
oligomeric species were only slightly visible after 4 and 6 h.
These results suggest that 2 prevents small oligomeric species
to be formed even at the 2/A1-42 ratio of 1/1 small oligomeric
species being considered as mainly responsible for the
Figure 6. Effects of the hairpin 2 on A1-42 fibril formation visualized by TEM.
Negatively stained images were recorded at 42 h of incubation of A1-42 (10
μM in 10 mM Tris.HCl, 100 mM NaCl at pH = 7.4) alone (left row), in the
presence of 100 μM (middle row), and 10 µM (right row) of 2. Scale bars, 500
nm.
Compound 4 was studied at the higher ratio (4/A1-42 ratio of
10/1) since it displayed no significant effect at the stoichiometric
ratio in the ThT experiment. TEM images were recorded at 5 h
(during the lag time of A1-42 alone) and 29 h (once the
fluorescence plateau was reached) of the fibrillization kinetics
(see the ThT curves, Figure S3 in supporting informations).
Major differences were observed in the morphology and in the
quantity of aggregates formed in presence of 4 (Figure 7). After
5 h of aggregation whereas the sample of A1-42 alone had
almost aggregated into short fibrils, the sample incubated with 4
showed a denser network of fibers which are substantially
thicker. At 29 h, once the maximum aggregation was reached, a
dense network of fibers with the classical morphology is
observed for A1-42 alone. A network of comparable density,
composed of significantly thicker fibers, was observed in the A1-
neurotoxicity associated with AD.[3,4,14-15 17] The electrophoretic
,
profiles obtained in the presence of the trans-DKP derivative 4
(Figure 5C) indicated that 4 modulated the oligomerization
process. Indeed, as early as the beginning of the kinetics (t0),
the CE profile of the sample containing 4 exhibited already the
presence of ES together with several oligomeric species as ES´
and LS and with new late migrating species. The most
remarkable one is the new later species observed after 4.2 min,
which did not appear in the control profile. Interestingly, the ES
peak (monomer form) started to decrease only after the period
of 6 h and therefore late compared to the control. The overall
results indicate that compound 4 accelerates the oligomerization
process but probably through a different pathway than the one
observed for A1-42 alone.
sample incubated with 4. This different morphology might
42
explain the different plateau of fluorescence observed in the ThT
assays. We hypothesized that 4 having the propensity to self-
associate, as observed by NMR (see above), it could be
involved in nucleus formation triggering A1-42 oligomerization,
as seen in the CE experiments, and accelerating aggregation.
Although accelerating the aggregation pathways is less intuitive,
[40]
this strategy has recently aroused interest
and needs further
investigations. Indeed, methylene blue (MB), which has reached
clinical trials, promotes fibrillization,[40c] and few compounds that
accelerate Aβ1-42 fibril formation reduce the concentration of
soluble Aβ1-42 oligomers and demonstrate a reduced toxicity for
mammalian cells.[40a-b]
TEM analyses were performed on compounds 2 and 4 under the
same conditions employed for the ThT studies. For compound 2,
images were recorded after 42 h of preincubation with the
peptide, corresponding to the end of the ThT-fluorescence
kinetics (Figure 6). A typical and very dense network of fibers
was observed in the A1-42 control sample (Figure 6, left image).
On the grid containing A1-42 incubated with compound 2 at a
2/A1-42 ratio of 10/1 (Figure 6, middle image), the network was
substantially less dense with the presence of globular
aggregates rather than mature fibrils observed in the control
sample. This globular morphology was also observed in the
sample containing A1-42 incubated with compound 2 at a
stoichiometric ratio (Figure 6, right image), where the network
was also less dense than in the control sample. This is in
accordance with the ThT assays where 2 was shown to totally
inhibit the ThT fluorescence at a 2/A1-42 ratio of 10/1 and still
decrease the final fluorescence plateau at a 2/A1-42 ratio of 1/1.
5h
29h
Figure 7. Effects of the hairpin 4 on Aβ1-42 fibril formation visualized by TEM.
Negatively stained images were recorded at 5 h and 29 h of incubation of Aβ1-
42 (10 μM in 10 mM Tris.HCl, 100 mM NaCl at pH = 7.4) alone (left row) and in
the presence of 100 μM of 4 (right row). Scale bars, 500 nm.
As the CE analysis showed
a decrease of the early
oligomerization process, and as both ThT and TEM experiments
showed a decrease of the fibrillization process, the combination
of the three methods indicate that 2 is able to decrease the
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700010
European Journal of Organic Chemistry
FULL PAPER
Bruker 400 (1H, 400 MHz, 13C, 100 MHz, 19F 376 MHz).
Chemical shift  are in parts per million (ppm) and the following
abbreviations are used: singlet (s), doublet (d), doublet of
doublet (dd), triplet (t), multiplet (m), and broad singlet (bs).
Melting points were determined on Kofler melting point
apparatus and are uncorrected.
Conclusions
We described the design and the synthesis of four new acyclic
peptidomimetics composed of a bifunctional diketopiperazine
scaffold DKP, as a potential -turn inducer, a peptidomimetic
arm and a tetrapeptide arm. The compounds containing the cis-
DKP (namely
1 and 2) adopted stable -hairpin mimic
General procedures
conformations, and the present work validates the capacity of
small -hairpin peptidomimetics to interact with A1-42 peptide in
order to modulate its aggregation. On the other hand,
peptidomimetics containing the trans-DKP are unable to form -
hairpin structures, although the two arms adopt extended
conformations, which could still actively interact with A1-42
amyloid peptide. And in fact, compound 4 accelerated the
aggregation by promoting the early oligomerization process,
albeit probably through a different pathway than the one of A1-42
alone. We confirmed that in both cis and trans series, the whole
molecules were essential for the activity, as fragments or
truncated compounds displayed no or lower efficacy to modulate
A1-42 aggregation. We clearly demonstrated that both
hydrophobic peptide KLVF and GVVI inspired by the central and
C-terminal sequence of A1-42 respectively, and involved in the
-sheet structuration of A1-42 aggregates, were good
recognition elements to bind A1-42, although, KLVF was shown
to be more beneficial for inhibiting the aggregation process. The
capacity of the N-terminus of peptidomimetics and of the lysine
residue to form ionic interactions with acidic residues of A1-42
seems an essential criterion for a good activity. In both cis and
trans series, the introduction of a hydrophobic trifluoromethyl
group in the peptidomimetic arm increased the affinity for A1-42
and thus notably influenced the activity of the molecules. In
conclusion, this work demonstrates that compound 2, a small -
hairpin peptidomimetic is able to modulate and decrease
fibrillization and early oligomerization process of A1-42,.
preventing the formation of small and soluble A1-42 oligomers,
which are described by several groups as primarily responsible
for neurotoxicity. Maintaining an acyclic -hairpin while
decreasing the peptidic character could provide valuable insights
to develop druggable -hairpin mimics as aggregation inhibitors
of amyloid-forming proteins.
General procedure A for deprotection reactions: To a solution of
the N-Boc-protected amino acid or peptide in CH2Cl2 (0.13 M)
was added a half volume of TFA and the reaction was stirred at
r.t. for 1-3 h. The solvent was evaporated, toluene (2×) was
added followed by evaporation, and then ether was added and
evaporated to afford the corresponding TFA salt.
General procedure B for coupling reactions: The N-protected
amino or peptide acid (3 equiv) was dissolved in DMF (0.1 M)
under the nitrogen atmosphere and the solution was cooled in
an ice bath, followed by adding HOAt (3 equiv), HATU (3 equiv)
and DIPEA (5 equiv). The solution was stirred at 0 oC for 1 h and
then was added the solution of TFA salt in DMF. The reaction
was stirred at 0 ºC for 30 minutes to 1 h and at r.t. overnight.
The DMF was evaporated and the mixture was diluted with
EtOAc and consecutively extracted with 1 M KHSO4 (2×) or citric
acid (10% solution), aqueous NaHCO3 (2×) and brine (1×), dried
over Na2SO4 and the solvent evaporated under reduced
pressure to afford the crude product.
General procedure C for coupling reactions: To a solution of the
N-protected amino acid in DMF (0.1 M), under nitrogen
atmosphere and at 0 ºC, was added DMTMM (BF4) (1equiv) and
NMM (3 equiv). After 30 min, a solution of the TFA salt of the
peptide in DMF was added and the reaction mixture was stirred
at 0 ºC for 1 h and at r.t. overnight. The solvent was evaporated
and the residue was diluted with EtOAc and consecutively
extracted with 1 M KHSO4 (2×) or citric acid (10% solution),
aqueous NaHCO3 (2×) and brine (1×), dried over Na2SO4 and
the solvent evaporated under reduced pressure to afford the
crude product.
Synthesis of 2
To a solution of 13b (59 mg, 0.043 mmol) in 3 mL MeOH was
added Pd/C (6 mg, 10% weight). The reaction flask was flushed
three times with hydrogen, and the reaction was stirred, under
hydrogen atmosphere, for 1 h at r.t. The mixture was filtered
through an HPLC filter, then the filtrated solution was
evaporated under reduced pressure to afford product without
Cbz protecting group that undergoes the deprotection reaction
according to general procedure A. The reaction is left overnight
under stirring to obtain product 2 as a light pink solid (60mg,
Experimental Section
Chemistry
Usual solvents were purchased from commercial sources and
dried and distilled by standard procedures. The following
compounds were prepared according to published methods: Cis-
DKP,[31] trans- DKP,[32b] compounds 6a-13a and 1.[33] Pure
products were obtained after flash chromatography using Merck
silica gel 60 (40-63 μm). TLC analyses were performed on silica
gel 60 F250 (0.26 mm thickness) plates. The plates were
visualized with UV light ( = 254 nm) or revealed with a 4 %
solution of phosphomolybdic acid or ninhydrin in EtOH.
Elemental analyses (C, H, N) were performed on a Perkin-Elmer
CHN Analyser 2400 at the Microanalyses Service of the Faculty
of Pharmacy in Châtenay-Malabry (France). HRMS were
obtained using a TOF LCT Premier apparatus (Waters), with an
electrospray ionization source. NMR spectra were recorded on
an ultrafield AVANCE 300 (1H, 300 MHz, 13C, 75 MHz) or a
1
quantitative yield over two steps). H NMR (400 MHz, DMSO) 
11.33 (s, NH, 1H), 10.46 (s, NH, 1H), 10.13 (s, NH, 1H), 8.51 (d,
J= 4 Hz, 1H), 8.38-8.35 (m, 3H), 8.21-8.09 (m, 7H), 7.97 (d, J= 4
Hz, 2H), 7.85-7.79 (m, 4H), 7.34-7.14 (m, 15H), 5.03 (d, J= 16
Hz, 1H), 4.72 (m, 1H), 4.49 (t, J= 8 Hz, 1H), 4.42 (m, 1H), 4.31
(m, 1H), 4.18 (t, J= 8 Hz, 1H), 4.09 (d, J= 16 Hz, 1H), 3.90 (s,
3H), 3.83-3.79 (m, 2H), 3.66-3.56 (m, 2H), 2.93-2.73 (m, 7H),
2.02 (m, 1H), 1.91 (m, 1H), 1.70-1.67 (m, 2H), 1.59-1.52 (m, 4H),
1.44-1.34 (m, 5H), 1.28-1.23 (m, 4H), 0.98-0.93 (m, 6H), 0.87-
0.74 (m, 12H); 13C NMR (100 MHz, DMSO)  171.6, 171.4,
171.0, 170.9, 169.3, 169.0, 168.4, 168.3, 166.2, 165.3, 162.9,
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700010
European Journal of Organic Chemistry
FULL PAPER
158.8, 158.5, 154.7, 154.6, 137.5, 136.7, 129.4, 129.2, 129.0,
128.6, 128.3, 128.1, 127.9, 127.4, 126.3, 125.7, 125.4, 123.6,
121.5, 112.8, 62.2, 62.1, 58.3, 57.8, 56.3, 53.6, 53.5, 51.9, 51.7,
51.3, 48.0, 46.8, 41.9, 40.6, 40.4, 39.2, 38.5, 38.4, 37.9, 32.5,
31.0, 30.7, 30.6, 29.4, 26.5, 24.1, 23.1, 21.5, 21.0, 19.2, 19.1,
spectra. 1H and 13C chemical shifts were calibrated using the
solvent residual peak (CHD2OH,  1H 3.31 ppm,  13C 49.5 ppm).
The chemical shift deviations were calculated as the differences
between observed chemical shifts and random coil values
reported in water.[41] The temperature gradients of the amide
18.5, 18.1, 16.7, 12.4; 19F NMR (188 MHz, d6-DMSO) 
−73.92 (s, 6F, CF3COOH); -74.26 (s, 3F, CF3CO) ; HRMS Calc
for C55H75F3N12O11 [M+H]+ (1137.5630): found 1137.5709; IR
νmax: 3280, 2975, 1688, 1647, 1497, 1202, 1163.
=
proton chemical shifts were derived from 1D H WATERGATE
1
spectra recorded over a 25°C interval. 3JHN-Hcoupling constants
were measured on 1D 1H WATERGATE or 1D 1H selective
TOCSY spectra.
NMR structure calculation
Synthesis of 3
Inter-proton distance restraints were derived from ROESY cross-
peak volumes integrated using Sparky. Upper bounds for proton
pairs were calculated using the isolated spin pair approximation
with an additional 20% tolerance. Upper bounds involving
equivalent methyl or aromatic protons were set to 3.5, 4.0 or
5.0 Å for strong, medium and weak ROE cross-peak intensities,
respectively. The lower bounds were set to the sum of van der
Waals radii of protons. Phi angle restraints were derived from
Compound 13c (0.014 mmol,
1 equiv) was deprotected
according to general procedure A affording compound 3 (14 mg,
92%) as a light yellow solid. mp: 186-190 oC; 1H NMR (400 MHz,
DMSO)  9.94 (d, J= 8 Hz, NH, 1H), 8.38 (d, J= 8 Hz, NH, 1H),
8.24 (t, J= 4 Hz, NH, 1H), 8.12-8.06 (m, NH, 3H), 7.96 (d, J= 4
Hz, 1H), 7.77 (dd, J= 8, 20 Hz, 2H), 7.34-7.26 (m, 5H), 7.11 (d,
J= 8 Hz, 1H), 5.28 (d, J= 16 Hz, 1H), 4.39-4.33 (m, 3H), 4.23-
4.15 (m, 2H), 4.00 (d, J= 16 Hz, 1H), 3.86 (s, 3H), 3.68-3.61 (m,
3H), 3.52 (m, 1H), 2.87-2.78 (m, 2H), 2.02 (s, 3H), 1.98-1.91 (m,
2H), 1.68 (m, 1H), 1.38 (m, 1H), 0.96-0.91 (m, 6H), 0.87-0.76 (m,
18H); 13C NMR (101 MHz, DMSO)  171.8, 170.7, 170.4, 169.3,
169.2, 168.1, 166.6, 165.9, 165.7, 163.4, 152.7, 132.6, 128.4,
127.9, 127.3, 123.4, 123.1, 121.4, 112.5, 66.0, 58.6, 58.1, 57.4,
56.6, 56.2, 56.0, 50.4, 46.4, 40.1, 38.8, 36.5, 34.1, 30.9, 30.1,
24.1, 23.8, 19.3, 19.2, 19.1, 18.5, 18.2, 17.9, 15.2, 10.7; HRMS
3JHN-H coupling constants using
a
Karplus relationship.
Structures were calculated using Amber 14 program.[42] Amino
acid residues were built using ff99SB force field. The 5-
trifluoroacetamido-2-methoxybenzhydrazide
unit
was
parameterized using gaff force field atom types and partial
charges were computed via the AM1-BCC method implemented
within Antechamber. Improper angles were added to maintain
the planarity around the hydrazide N–N bond and the phenyl-
acetamido moiety, (n order to facilitate this parametrization task,
conformational preference and rotation barriers of a minimal
structure containing the Hao unit were studied by performing a
DFT potential energy surface scan, see supporting information).
A set of 50 structures was calculated by simulated annealing at
1000 K in vacuo using 43 NMR experimental restraints (40
distances and 4  angles, listed in table S5), and 7 dihedral
angle restraints to fix the trans or cis configuration of amide
bonds. Structures were then refined in implicit aqueous solvent
using GBSA (Generalized Born Surface Area) model. The
refinement protocol consisted of 60 ps restrained molecular
dynamics at 300 K followed by energy minimization. The best 20
structures exhibiting the lowest potential energy and no restraint
violation (< 0.1 Å and < 5° for distances and dihedral angles
restraints, respectively) were selected to represent the final
NMR family.
+
+
Calcd for C47H70N11O11 [M (NH3 )] (964.5251): found 964.5274;
IR νmax: 3279, 2966, 2359, 1634, 1541, 1497, 1470, 1152.
Synthesis of 4
Compound 13d (0.019 mmol,
1 equiv) was deprotected
according to general procedure A affording compound 4 (20 mg,
92%) as a yellow solid. mp: 216-220 oC; 1H NMR (400MHz,
DMSO)  11.32 (s, NH, 1H), 10.31 (s, NH, 1H), 9.97 (s, NH, 1H),
8.40 (d, J= 8 Hz, NH, 1H), 8.25 (t, J= 4 Hz, 1H), 8.11 (d, J= 8 Hz,
1H), 8.07 (m, 5H), 7.81-7.79 (m, 2H), 7.33-7.27 (m, 5H), 7.22 (d,
J= 8 Hz, 1H), 5.28 (d, J= 16 Hz, 1H), 4.41-4.34 (m, 3H), 4.23-
4.15 (m, 2H), 4.00 (d, J= 16 Hz, 1H), 3.89 (s, 3H), 3.54 (m, 2H),
2.89-2.73 (m, 2H), 2.06-1.95 (m, 3H), 1.68 (m, 1H), 1.41 (m, 1H),
1.28-1.23 (m, 2H), 1.09 (m, 1H), 0.97-0.91 (m, 6H), 0.87-0.76 (m,
18H); 13C NMR (101 MHz, DMSO)  171.9, 170.7, 170.5, 169.4,
169.3, 166.6, 165.9, 165.7, 163.1, 158.4, 154.5, 136.5, 129.3,
128.5, 127.9, 127.3, 125.5, 123.4, 121.7, 114.4, 112.7, 58.6,
58.1, 57.5, 56.7, 56.3, 56.1, 53.6, 50.4, 46.4, 40.1, 38.9, 36.5,
36.4, 31.0, 30.9, 30.1, 24.1, 19.3, 19.2, 19.1, 18.5, 18.2, 17.9,
15.2, 10.7; 19F NMR (376.3 MHz, CD3OD)  −74.51 (s, 3F), -
Thioflavin T assay
Thioflavin T was obtained from Sigma. A1-42 was purchased
from American Peptide. The peptide was dissolved in an
aqueous 1% ammonia solution to a concentration of 1 mM and
then, just prior to use, was diluted to 0.2 mM with 10 mM Tris-
HCl, 100 mM NaCl buffer (pH 7.4). Stock solutions of tested
compounds were dissolved in DMSO with the final concentration
kept constant at 0.5% (v/v) (1µL of DMSO in 200 µL). Thioflavin
T fluorescence was measure to evaluate the development of
Aβ1-42 fibrils over time using a fluorescence plate reader
(Fluostar Optima, BMG labtech) with standard 96-wells black
microtiter plates. Experiments were started by adding the
peptide (final Aβ1-42 concentration equal to 10 µM) into a mixture
containing 40 µM Thioflavin T in 10 mM Tris-HCl, 100 mM NaCl
buffer (pH 7.4) with and without the tested compounds at
different concentrations (100 and 10 µM) at room temperature.
The ThT fluorescence intensity of each sample (performed in
+
+
74.56 (s, 3F) ;HRMS Calc for C47H67F3N11O11 [M (NH3 )]
(1018.4968): found 1018.5098; IR νmax: 3292, 2967, 2360, 2341,
1647, 1541, 776, 652.
NMR Spectroscopy
Lyophilized compounds 2 and 4 were dissolved at 0.5 mM
concentration in 550 μL of CD3OH (Eurisotop, Saint-Aubin,
France). NMR experiments were recorded on a Bruker Avance
III 500 MHz spectrometer equipped with a TCI 1H/13C/15N
cryoprobe. NMR spectra were processed with TopSpin 2.0
software (Bruker) and analysed with Sparky program
(http://www.cgl.ucsf.edu/home/sparky/). 1H and 13C resonances
were assigned using 1D 1H WATERGATE, 2D 1H-1H TOCSY
(MLEV17 isotropic scheme of 66 ms duration), 2D 1H-1H ROESY
1
1
(300 ms mixing time), 2D H-13C HSQC and 2D H-13C HMBC
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201700010
European Journal of Organic Chemistry
FULL PAPER
duplicate or triplicate) was recorded with 440/485 nm
excitation/emission filters set for 42 hours performing a double
orbital shaking of 10 s. before the first cycle. The fluorescence
assays were performed between 2 and 3 times over different
days. The ability of hairpin compounds to modulate Aβ1-42
aggregation was assessed considering both the time of the half-
life of aggregation (t1/2) and the intensity of the experimental
fluorescence plateau (F). See supporting information for the
calculation of the t1/2 and F variation factors.
thanked for financial support for DB. The Laboratory BioCIS is a
member of the Laboratory of Excellence LERMIT supported by a
Grant from ANR (ANR-10-LABX-33).
Keywords: Amyloid -peptide • Alzheimer’s disease •
Foldamers • Hairpins • Peptidomimetics
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Transmission electron microscopy
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Capillary electrophoresis
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Sample preparation: The commercial A1-42 was dissolved upon
reception in 0.16% NH4OH (at 2 mg/mL) for 10 minutes at 20°C,
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pretreatment. CE experiments were carried out with a PA800
ProteomeLab instrument (Beckman Coulter Inc., Brea, CA,
USA) equipped with a diode array detector. UV detection was
performed at 190 nm. The prepared sample (as previously
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containing DMSO (control or Stock solutions of glycopeptides
dissolved in DMSO) to kept constant the DMSO/ phosphate
buffer ratio at 2.5% (v/v) and the final peptide concentration at
100 μM regardless the peptide/compound ratio. For the CE
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to the detector) 50 mm I.D. were used. The background
electrolyte was a 80 mM phosphate buffer, pH 7.4. The
separation was carried out under -30 kV at 20°C. The sample
was injected from the outlet by hydrodynamic injection at 3.44
KPa for 10 s. After each run, the capillary was rinsed for 5 min
with water, 1 min with SDS 50 mM, 5 min with NaOH 1 M and
equilibrated with running buffer for 5 min.
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