PH-controlled aggregation polymorphism of amyloidogenic Aβ (16-22): Insights for obtaining peptide tapes and peptide nanotubes, as function of the N -terminal capping moiety

Article abstract of DOI:10.1016/j.ejmech.2014.07.089

Peptide and protein self-assembly resulting in the formation of amyloidogenic aggregates is generally thought of as a pathological event associated with severe diseases. However, amyloid formation may also provide a basis for advanced bionanomaterials, since amyloid fibrils combine unique material-like properties that make them very useful for design of new types of conducting nanowires, bioactive ligands, and biodegradable coatings as drug-encapsulating materials. The morphology of the supramolecular aggregates determines the properties and application range of these bionanomaterials. An important parameter to control the supramolecular morphology, is the overall charge of the peptide, which is related to the pH of the environment. Herein, we describe the design, synthesis and morphological analysis of a series of N-terminally functionalized Aβ(16-22) peptides (～1/4Lys-Leu-Val-Phe-Phe-Ala-Glu-OH), that underwent a pH-induced polymorphism, ranging from lamellar sheets, helical tapes, peptide nanotubes, and amyloid fibrils as was observed by transmission electron microscopy. Infrared spectroscopy and wide angle X-ray scattering studies showed that peptide self-assembly was driven by β-sheet formation, and that the supramolecular morphology was directed by subtle variations in electrostatic interactions. Finally, a structural model and hierarchy of self-assembly of a peptide nanotube, assembled at pH 1, is proposed.

Full text of DOI:10.1016/j.ejmech.2014.07.089

European Journal of Medicinal Chemistry 88 (2014) 55e65
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
pH-controlled aggregation polymorphism of amyloidogenic
(16e22): Insights for obtaining peptide tapes and peptide
A
b
nanotubes, as function of the N-terminal capping moiety
Ronald C. Elgersma a, Loes M.J. Kroon-Batenburg b, George Posthuma c,
,
, e, **
Johannes D. Meeldijk d, Dirk T.S. Rijkers a *, Rob M.J. Liskamp a
a Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Science,
Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
b Division of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University,
3584 CH Utrecht, The Netherlands
c Department of Cell Biology, Center for Electron Microscopy, University Medical Center, 3508 GA Utrecht, The Netherlands
d Division of Inorganic Chemistry and Catalysis, Department of Chemistry, Faculty of Science, Utrecht University, 3508 TB Utrecht, The Netherlands
e Chemical Biology and Medicinal Chemistry, School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow G12 8QQ,
United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Peptide and protein self-assembly resulting in the formation of amyloidogenic aggregates is generally
thought of as a pathological event associated with severe diseases. However, amyloid formation may also
provide a basis for advanced bionanomaterials, since amyloid fibrils combine unique material-like
properties that make them very useful for design of new types of conducting nanowires, bioactive li-
gands, and biodegradable coatings as drug-encapsulating materials. The morphology of the supramo-
lecular aggregates determines the properties and application range of these bionanomaterials. An
important parameter to control the supramolecular morphology, is the overall charge of the peptide,
which is related to the pH of the environment. Herein, we describe the design, synthesis and morpho-
Received 15 May 2014
Received in revised form
24 July 2014
Accepted 24 July 2014
Available online 25 July 2014
Keywords:
Aggregation
Amyloid
logical analysis of a series of N-terminally functionalized Ab(16e22) peptides (~Lys-Leu-Val-Phe-Phe-
Ala-Glu-OH), that underwent a pH-induced polymorphism, ranging from lamellar sheets, helical tapes,
peptide nanotubes, and amyloid fibrils as was observed by transmission electron microscopy. Infrared
spectroscopy and wide angle X-ray scattering studies showed that peptide self-assembly was driven by
Nanostructures
Peptides and peptidomimetics
Self-assembly
Supramolecular chemistry
b
-sheet formation, and that the supramolecular morphology was directed by subtle variations in elec-
trostatic interactions. Finally, a structural model and hierarchy of self-assembly of a peptide nanotube,
assembled at pH 1, is proposed.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
self-assembly [2], the morphology of these materials is classified in
specific macromolecular structures such as twisted tapes, helical
Peptide self-assembly is now widely recognized as a valuable
tool for the generation of ordered structures through weak, non-
covalent interactions [1]. These self-assembled constructs display
material-like properties that are strongly dependent on the
morphology of the constructs. Depending on the hierarchy of the
ribbons, fibrils, fibers, tubeles and peptide nanotubes [3,4], vesicles,
as well as virus-like capsids. These, so-called peptide-based bio-
nanomaterials [5], find applications in the development of scaffolds
for tissue-engineering and -repair, as biocompatible coatings, as
templates for bone mineralization, and as novel biodegradable drug
delivery systems, like nanocontainers, and stimuli-responsive
hydro/organogels.
The development of these self-assembled bionanomaterials is
often initiated by de novo design of small organic molecules, pep-
tide amphiphiles and peptidomimetics [3a], and the self-assembly
process is driven by the formation of hydrogen bond networks, as is
* Corresponding author.
** Corresponding author. Chemical Biology and Medicinal Chemistry, School of
Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glas-
gow G12 8QQ, United Kingdom.
E-mail addresses: D.T.S.Rijkers@uu.nl (D.T.S. Rijkers), R.M.J.Liskamp@uu.nl,
Robert.Liskamp@glasgow.ac.uk (R.M.J. Liskamp).
present in
b-sheets, via pep- and hydrophobic interactions, by
http://dx.doi.org/10.1016/j.ejmech.2014.07.089
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

56
R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
electrostatic interactions, or by combinations thereof [6]. However,
Nature provides us with fascinating examples of self-assembled
biomaterials like, spider silk, mussel glue, elastin, collagen and
viral capsid proteins, and is highly inspiring for the development of
novel supramolecular peptide-based bionanomaterials with
designed properties [6].
An important parameter in directing self-assembly into poly-
morphous supramolecular assemblies is the overall charge of the
peptide, which is dependent on the pH of the environment [12]. As
nice examples of this principle, among others, studies by Kelly and
co-workers with a designed dibenzofuran template have shown the
control over the morphology of
b-sheet assemblies by varying the
Although, amyloid fibrils and amyloidogenic peptides are
related to severe diseases [7] like Alzheimer- and Parkinson dis-
ease, late-onset diabetes (type II diabetes), and bovine spongiform
encephalopathy (BSE), their use in the design of bionanomaterials
has begun to pique renewed interest among biomaterial scientists,
since these peptides bear an intrinsic tendency for self-assembly,
pH [13]. The development of pH-responsive
design isotropic or nematic organogels and hydrogels has been
reported by Boden and Aggeli [14]. Finally, model peptides char-
acterized by an a-helical coiled-coil motif undergo conformational
transitions and amyloid formation triggered by changes in pH value
as reported by the group of Koksch [15].
b
-sheet peptides to
due to their high
b
-sheet propensity,
p
-stacking interactions and
Herein, we describe the design and synthesis of a series of N-
hydrophobic properties. In the literature, several examples can be
found in which short amyloidogenic peptides have been used as
template for self-assembled bionanomaterials [8]. Prominent ex-
amples are among others, the work of Gazit and co-workers [9], and
terminally functionalized Ab(16e22) peptides (~Lys-Leu-Val-Phe-
Phe-Ala-Glu-OH) that undergo pH-dependent changes as function
of the structural properties of the N-terminal capping moiety.
€
Gorbitz [10], who have studied the Phe-Phe dipeptide core-
2. Results and discussion
recognition motif of the Ab-peptide for the formation of peptide
nanotubes. Furthermore, Lynn and co-workers have demonstrated
that the heptapeptide Ac-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2,
2.1. Rationale for design
derived from the highly amyloidogenic A
b(16e22) sequence, is able
The heptapeptide Ac-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2, repre-
to form helical ribbons that progress into peptide nanotubes [11].
senting the amino acid residues 16e22 of the amyloidogenic A
b
Fig. 1. Structural formulas of the peptides 1e5 that have been synthesized in this study. The right panel represents a schematic representation of the peptides and their charge
distribution at pH 7.4.

R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
57
peptide, has been used by Lynn and co-workers as a building block
for the supramolecular assembly into peptide nanotubes and other
amyloid-like aggregates [11]. Several lines of evidence indicate that
respectively (Fig. 2). To obtain insight into other factors that play a
role in the self-assembly process, a series of N-terminal modified
peptides was synthesized. Peptide 2 (N-
dates the influence of the additional positive charge compared to 1,
while peptide 3 (N- -acetyl-lysyl) emphasizes the role of the aro-
matic moiety compared to 2. The combination of peptides 3 and 4
(N- -acetyl-glutamyl) gives insight into the role of the positively-
a-phenoxyacetyl) eluci-
self-assembly of the A
mation via hydrogen bond interactions of the peptide backbone,
interactions of the internal dipeptide Phe-Phe and, at an
b(16e22) peptide is based on b-sheet for-
a
pep
appropriate pH, electrostatic interactions via the Lys/Glu side
chains. Since it has been demonstrated that electrostatic in-
teractions can direct self-assembly at the molecular level, e.g.
a
or negatively charged N-terminus. Finally, acetylation of the N-
a-
amino functionality will be used as a control (peptide 5).
directing
b-sheet formation either in the antiparallel or parallel
orientation [16], we investigated the role of the N-terminal capping
moiety on the morphology of the supramolecular assemblies as
function of the pH (Fig. 1). It should be noted that the peptide in our
study was a C-terminal acid, while Lynn and co-workers [11] used
an amidated peptide. This modification introduced an additional
negative charge which was assumed to be beneficial for control of
self-assembly into triangular-shaped assemblies involving head-to-
tail arrangement required for these entities (vide infra, Fig. 2).
The initial idea was to modify the N-terminal amino group with
a 2-(2-(3-aminoprop-1-ynyl)phenoxy)acetyl functionality in which
the aminopropyne moiety forms an angle of 60ꢀ with the peptide
backbone (in peptide 1) to arrive at either triangular-shaped as-
semblies [17] or lamellar sheets, which, on their turn, could form
either spherical particles of virus capsid-like dimensions, or fold
into supramolecular sheets, helical ribbons and peptide nanotubes,
2.2. Synthesis
The N-terminal capping moiety 10 was synthesized as depicted
in Scheme 1. The synthesis was started with the O-alkylation of 2-
iodophenol (6) in the presence of tert-butyl bromoacetate and
K2CO3 as base to give aryl iodide 7 in quantitative yield. Then, Boc-
protected propargylamine (8) was coupled to aryl iodide 7 via a Pd-
catalyzed cross-coupling under Sonogashira conditions [18], and
alkyne 9 was obtained in 72% yield. Finally, both the Boc- and tert-
butyl functionalities were simultaneously removed by TFA and the
amine was protected by treatment with Fmoc-ONSu to obtain a
building block suitable for SPPS. Fmoc derivative 10 was obtained in
an overall yield of 65% over two steps.
The amyloidogenic A
Phe-Phe-Ala-Glu, was synthesized on the solid support via
b(16e22) peptide sequence: Lys-Leu-Val-
Fig. 2. Schematic representation of the self-assembly process of peptide 1 at pH 7.4. Note: capsid-like nanostructures were not observed, instead lamellar sheets, helical ribbons,
and nanotubes were the predominantly observed supramolecular assemblies.

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R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
Scheme 1. Synthesis of building block 10.
Fmoc/tBu SPPS protocols [19] to obtain semi-protected peptide
resin 11 with a free N-terminal amine, as shown in Scheme 2. Then,
resin 11 was split into five portions and each portion was subjected
to a particular sequence of reactions to obtain the protected peptide
resins 12aee. Finally, after global deprotection and cleavage from
the resin, peptide derivatives 1e5 were obtained in an overall yield
varying between 36 and 71%. Their purity was analyzed by HPLC
(>95%) and their identity was characterized by mass spectrometry.
concentrated peptide solution (10 mg/mL) in a buffer of appropriate
pH (1, 7.4 and 11) was prepared.
Peptide 1 did not dissolve completely in buffer pH 7.4 and the
turbid solution/suspension remained fluid upon standing. Inspec-
tion by TEM showed that mainly amyloid-like fibrils were formed,
while spherical nanoparticles were not observed (Fig. 3 1B). Fibril
formation was further confirmed by FTIR (Table 1), since the typical
amide I absorption [20] for an antiparallel
n
b-sheet conformation at
1628 cmꢁ1 was found, which was in agreement with the solid-
2.3. Self-assembly and supramolecular morphologies
state NMR data reported by Meredith and co-workers [16] and
with the X-ray diffraction data of Lynn and co-workers [11]. In an
acidic buffer (pH 1), peptide 1 was highly soluble in water and
besides the fact that a translucent gel was formed, the morphology
of the supramolecular assemblies was also different. Under these
The aggregation- and gelation behavior of peptides 1e5 was
investigated by transmission electron microscopy (TEM) and the
results are summarized in Table 1 and Fig. 3. For that purpose, a
Scheme 2. Synthesis of peptide derivatives 1e5.

R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
59
Table 1
Physicochemical properties of peptide 1e5.a
Entry
Peptide
pH
Overall chargeb
Gel formation
Morphology
FTIR/cmꢁ1c
1
2
3
4
5
6
7
8
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
1
þ2
0
Yes
No
Yes
No
No
No
Nanotubes
Fibers
1631 (s), 1673 (m/s)
1628 (s), 1683 (m/s)
7.4
11
1
7.4
11
1
7.4
11
1
7.4
11
1
ꢁ2
þ1
ꢁ1
ꢁ2
þ2
0
ꢁ2
þ1
ꢁ2
ꢁ3
þ1
ꢁ1
ꢁ2
Twisted tapes
Helical ribbons
Fibers
Lamellar sheets
Helical ribbons, nanotubes
Lamellar sheets, nanotubes
lamellar sheets
Helical ribbons
Nanotubes
1635 (m), 1653 (m), 1670 (m)
1628 (s), 1679 (m), 1693 (m)
1629 (s), 1686 (m/s)
1629 (m), 1684 (m)
1628 (s), 1677 (m)
1627 (s), 1688 (s)
1628 (s), 1685 (s)
1628 (s), 1678 (m)
1627 (s), 1687 (m/s)
1628 (s), 1684 (s)
Viscous solution
Yes
Yes
No
9
10
11
12
13
14
15
No
Yes
Yes
Yes
Yes
Lamellar sheets
Nanotubes
Nanotubes
1628 (s), 1672 (w), 1693 (w)
1628 (s), 1679 (m)
1631 (s), 1685 (m)
7.4
11
Nanotubes
a
Peptide concentration: 10 mg/mL.
b
c
pKa values: propargylamine: 7.89, εNH2 Lys: 10.53,
a
COOH Glu: 2.19,
gCOOH Glu: 4.25, according to reference [34].
Typical amide I absorption frequency (cmꢁ1): 1630: aggregated
b
-sheet, 1640: unordered structure, 1675: antiparallel
b
-sheet, according to reference [20], s: strong, m;
medium, w; weak.
conditions, broad flat ribbons, up to 5
mm in length and 90e120 nm
ribbon progressing into a closed tubular structure, was observed
in width, were observed by TEM (Fig. 3 1A). These uniform struc-
tures could also be a result of the sample preparation since a drying
step was included, and to obtain a more realistic view of these
supramolecular assemblies in solution, cryo-TEM images were
generated. Instead of flat ribbons, highly uniform peptide nano-
tubes were observed (Fig. 4A). Moreover, tilting of the sample-grid
to 45ꢀ confirmed the hollow tubular structure (Fig. 4B), and in some
cases, the ‘opening’ of some tubes, with a diameter of ~100 nm,
could be seen (indicated by arrows in Fig. 4B and inset of Fig. 4A).
Another example of polymorphism as function of the pH became
evident when peptide 1 was dissolved in a basic buffer at pH 11. In
this case, tape-like twisted sheets were observed by TEM (Fig. 3 1C).
Although a distinct variation of the supramolecular morphology
could be observed as function of the pH, the driving force of self-
(Fig. 3 4B). Both nanostructures were 80e100 nm in diameter
and up to 3
mm long. At pH 11, this peptide formed lamellar
sheets (~140 nm in width) and some of them were twisted (Fig. 3
4C).
In case of peptide 5, all solutions turned instantaneously into a
turbid gel. Analysis by TEM made clear that in all buffer solutions,
peptide nanotubes were observed as the dominant supramolecular
morphology. However, subtle differences in dimensions and
translucence were visible. At pH 1, striated nanotubes (helical
marking is visible) were observed (Fig. 3 5A), a clear indication that
these constructs were built up from entwined sheets/ribbons. This
observation was in agreement with the data of Lynn and co-
workers, who observed helical ribbons progressing into peptide
nanotubes in case of peptide Ac-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2
in aqueous buffer at pH 2 [11]. The striated tubes had a diameter
that ranged from 100 to 150 nm (80e90 nm was reported in the
assembly is merely based on
backbone then electrostatic interactions, since in all three cases
strong absorptions at
1628e1635 cmꢁ1 were found by FTIR.
b-sheet formation by the peptide
n
literature [11]) and a length up to 5 mm. At pH 7.4, a dense network
The absence of the aminopropyne moiety in peptide 2 signifi-
cantly reduced its water solubility, and more often than not, a fine
precipitate rather than a gel was formed during the period of in-
cubation. At pH 1, aggregation into short helical ribbons, with a
diameter of approx. 2.5 nm, was observed (Fig. 3 2A), while at pH
7.4, well-ordered thin fibers could be seen (Fig. 3 2B). At pH 11 on
the contrary, lamellar sheets (some of them were twisted) were
observed by TEM (Fig. 3 2C).
The water solubility of peptide 3 was significantly improved
compared to 2, and after dissolution in an acidic buffer, the solution
became very viscous but remained translucent. However, the
peptide solutions in neutral and basic buffer systems turned
instantly into turbid gels. Inspection by TEM, clearly revealed
different supramolecular morphologies as function of the pH, but
also compared to peptides 1 and 2. In an acidic buffer, short peptide
nanotubes together with left-handed helical ribbons were
observed. These ribbons and tubes were generally ~70 nm in
of tubular structures were observed (Fig. 3 5B), while at pH 11
translucent nanotubes with a diameter that ranged between 130
and 180 nm (Fig. 3 5C).
FTIR analysis (Table 1) of the peptide solutions/gels revealed the
characteristic amide I absorption at n
1627e1635 cmꢁ1 which was a
strong indication for the presence of
strong absorptions that were found at
b
-sheets [20]. Furthermore,
n
1670e1690 cmꢁ1 could be
assigned to highly ordered antiparallel
strands.
b-sheets and aggregated
2.4. Wide angle X-ray scattering studies (WAXS)
X-ray fiber diffraction studies were performed to pinpoint the
molecular organization of the supramolecular structures formed by
peptide 1 in solution. The solutions/gels of peptide 1 in the three
buffer systems that were used for TEM analyses were also used for
the diffraction studies.
diameter and approx. 1
m
m long (Fig. 3 3A). Increasing the pH
The diffraction pattern of the peptide nanotubes formed at pH 1
(hydrated sample), revealed distinct reflections at 4.4 and 5.1 Å and
a more intense reflection at 10 Å (Fig. 5A). Although the typical
intersheet spacing of 10 Å was clearly visible, the characteristic
resulted in the formation of lamellar sheets (pH 7.4) and tubular
structures (pH 11) as shown in Fig. 3 3B and 3C, respectively.
Peptide 4 slowly dissolved in buffers of pH 1 and 7.4, and upon
standing small aggregates precipitated from the solution while
no gel formation occurred. However, at pH 11, the peptide readily
dissolved and turned the solution into a gel. The morphology of
the supramolecular assemblies formed at pH 1 could be described
as right-handed helical ribbons (Fig. 3 4A), and at pH 7.4, a helical
hydrogen bond spacing reflection of an antiparallel b-sheet (4.7 Å)
was absent. It was not exactly clear how to interpret the reflections
at 4.4 and 5.1 Å, however, Lynn and co-workers described
a hydrogen bond spacing of 5 Å for a similar peptide, Ac-Lys-Leu-
Val-Phe-Phe-Ala-Glu-NH2 in aqueous buffer at pH
2 [11].

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R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
Fig. 3. Polymorphism of the peptides 1e5 at different pH values as visualized by transmission electron microscopy (A: pH 1; B: pH 7.4; C: pH 11). Conditions: the peptides were
dissolved in the appropriate buffer at 10 mg/mL and aged for 3 weeks prior to analysis. The scale bars represent 500 nm.
Additionally, a sample of the peptide gel at pH 1 was air-dried and
the diffraction pattern that was obtained was indicative for a highly
ordered supramolecular structure (Fig. 5B). Equatorial reflections at
4.7 and 25 Å together with the second harmonic at 12.5 Å were
observed. Meridional reflections were present at 4.4, 5.1 and 12.5 Å,
and a strong off-meridional reflection was found at 10 Å. The many
harmonics as present in the diffraction pattern, were strong in-
dications of a high degree of crystalline order. Based on these
reflection data, a molecular model is proposed as shown in Fig. 6. In
this model, approximately 250 peptides are arranged radially as a
monolayer [11d,21] rather than a bilayer [11c,22], which implies a
wall thickness of ~34 Å. In the direction of the tubes, these peptides
way. Within the b-sheet, the peptides are probably oriented in an
antiparallel fashion. Finally, the individual sheets are shifted with
respect to each other over a distance of circa 2.2 Å. Overall, the
peptide nanotube is formed by wrapping-up the individual
sheets helically [23].
b-
At pH 7.4 a fibril-like morphology was observed by TEM. The X-
ray diffraction pattern of this sample showed strong reflections at
4.7 and 10.5 Å, indicating the formation of highly ordered fibrils
(Fig. 5C). Moreover, these reflections were characteristic for a cross
b
-sheet structure. The lamellar sheets as observed at pH 11 by TEM
consisted of antiparallel -sheets, since strong reflections were
found at 4.8 and 10.3 Å (Fig. 5D). These data were in line with those
reported by Tycko and co-workers in case of A (16e22) [24] and
b
are present as lengthy
b-sheets, which are arranged in a parallel
b

R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
61
Fig. 4. Cryo-TEM images of peptide 1 (10 mg/mL in buffer pH 1 and aged for 3 weeks prior to analysis). A: Peptide nanotubes as formed by peptide 1, inset: a top-view of a single
nanotube (scale bar represents 500 nm). B: Peptide nanotubes as formed by peptide 1 shown at 45ꢀ tilt angle, some ‘openings’ of the nanotubes are visible and indicated by arrows
(scale bar represents 200 nm).
A
b
(11e25) [25], which were found to be organized as antiparallel
b
-
arrive at highly ordered bionanomaterials. The aggregation and
self-assembly behavior of these amyloid-based peptides were
studied by TEM and FTIR as function of the pH. It was found that the
N-terminal capping moiety is a major determinant of the supra-
molecular aggregation morphology, ranging from lamellar sheets,
helical tapes, and peptide nanotubes, since this moiety determines
the polarity, amphiphilic character, and overall charge of the
sheets, characteristic for amyloid fibrils.
3. Conclusion
A series of five N-terminally functionalized Ab(16e22) peptides
was designed and synthesized as supramolecular building blocks to
Fig. 5. Two-dimensional WAXS patterns obtained from peptide 1 at three different pH values. A: pH 1 and hydrated sample; B: pH 1 and air-dried sample; C: pH 7.4 and hydrated
sample; D: pH 11 and air-dried sample.

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R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
Fig. 6. Proposed molecular model of peptide 1, assembled at pH 1 and analyzed in the air-dried state, corresponding to the conditions of Fig. 5B.
peptide constructs. Based on wide-angle X-ray scattering studies in
combination with data from the literature, a model of the molecular
organization of a peptide nanotubes is proposed.
electron microscope used for cryo-electron microscopy was a Phi-
lips Tecnai 12 and was equipped with a CCD-camera. Fourier
transform infrared spectra were recorded on a BioRad FTS6000.
4.2. Syntheses
4. Experimental section
4.2.1. tert-Butyl 2-(2-iodophenoxy)acetate (7)
4.1. Chemicals, instruments, and methods
2-Iodophenol 6 (11.3 g, 52 mmol) was dissolved in DMF (20 mL)
and anhydrous K2CO3 (14.6 g, 105 mmol) was added. The suspen-
sion was cooled to 0 ꢀC and tert-butyl bromoacetate (8.4 mL,
52 mmol) was added dropwise. The reaction mixture was stirred
for 16 h at room temperature. Then, the solvent was removed in
vacuo and the residue was redissolved in EtOAc (150 mL) and this
solution was washed with H2O (2 ꢂ 100 mL), 10% NaHCO3
(3 ꢂ 75 mL) and brine (2 ꢂ 100 mL). The EtOAc solution was dried
(Na2SO4) and concentrated in vacuo to give tert-butyl 2-(2-
iodophenoxy)acetate 7 as an orange oil in quantitative yield
(17.1 g). Rf ¼ 0.56 (hexane/EtOAc 9:1); 1H NMR (300 MHz, CDCl3,
Chemicals were obtained from commercial sources and used
without further purification. Peptide grade solvents used for solid
phase peptide synthesis were purchased from Biosolve (The
Netherlands) and were stored on 4Å molecular sieves. Column
chromatography was performed with Silica-P Flash silica gel (Sili-
cycle). Retention factor values (Rf) were determined with thin layer
chromatography (TLC) by using Merck pre-coated silica gel 60F254
plates. Spots were visualized by UV-quenching, ninhydrin, or Cl2/
N,N,N0,N0-tetramethyl-4,40-diaminodiphenylmethane (TDM) [26].
Retention time values (Rt) and analytical HPLC runs were per-
formed on a Shimadzu automated HPLC system. Peptides were
characterized using Electro Spray Mass Spectrometry (EI-MS) and
was performed on a Shimadzu LCMS-QP8000 single quadruple
bench top mass spectrometer operating in a positive ionization
mode. 1H NMR spectra were recorded on a Varian G-300 (300 MHz)
25 ꢀC):
d
¼ 7.78 (d, J ¼ 8.0 Hz, 1H; arom H), 7.27 (t, J ¼ 7.4 Hz, 1H;
arom H), 6.71 (m, 2H; arom H), 4.57 (s, 2H; OCH2), 1.48 (s, 9H;
C(CH3)3); 13C NMR (75.5 MHz, CDCl3, 25 ꢀC):
129.3, 123.2, 112.1, 86.3, 82.5, 66.6, 28.0.
d
¼ 167.2, 156.7, 139.7,
and chemical shift values (
d
) are given in ppm relative to TMS. 13
C
4.2.2. N-tert Butyloxycarbonyl-propargylamine (8)
NMR spectra were recorded on a Varian G-300 (75.5 MHz) spec-
trometer and chemical shift values are given in ppm relative to
CDCl3 (77.0 ppm) or DMSO-d6 (39.5 ppm). The 13C NMR spectra
were recorded using the attached proton test (APT) pulse sequence.
Electron microscopy was performed using a Jeol 1200 EX trans-
mission electron microscope operating at 60 kV. The transmission
Propargylamine hydrochloride (3.6 g, 39 mmol) and triethyl-
amine (11.5 mL, 83 mmol, 2.13 equiv) were dissolved in CH2Cl2
(100 mL) and this solution was cooled on ice. Then, a solution of
Boc2O (8.5 g, 40 mmol), (1.03 equiv) in CH2Cl2 (50 mL) was added
dropwise and the obtained reaction mixture was stirred for 2 h.
After removing the solvent under reduced pressure, the residue

R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
63
was redissolved in EtOAc (150 mL) and this solution was washed
with 1 N KHSO4 (3 ꢂ 75 mL) and brine (150 mL). The EtOAc solution
was dried (Na2SO4) and concentrated in vacuo to give 8 as pink
crystals in 96% yield (5.8 g). Rf ¼ 0.70 (CH2Cl2/MeOH 95:5); 1H NMR
2-chlorotrityl chloride linker [27] by using Fmoc/tBu SPPS protocols
[19]. The first amino acid (Fmoc-Glu(OtBu)-OH, 2.55 g, 3 mmol,
4 equiv) was loaded onto the resin in the presence of DIPEA
(1050 mL, 6 mmol, 8 equiv) in dry CH2Cl2 (25 mL) for 16 h and any
unreacted trityl chloride was capped with MeOH/CH2Cl2/DIPEA
(2:17:1; 20 mL) for 20 min [28]. A synthetic cycle consisted of an N-
a-Fmoc removal by treatment with 20% piperidine in NMP
(3 ꢂ 8 min, 20 mL), a washing step with NMP (3 ꢂ 2 min, 20 mL)
followed by a washing step with CH2Cl2 (3 ꢂ 2 min, 10 mL), a
coupling step (60 min) with the appropriate Fmoc amino acid
(3 mmol, 4 equiv) in the presence of HBTU/HOBt [29] (3 mmol,
(300 MHz, CDCl3, 25 ꢀC):
d
¼ 4.71 (broad s, 1H; urethane NH), 3.93
(m, 2H; CH2), 2.22 (s, 1H; ^CH), 1.46 (s, 9H; C(CH3)3); 13C NMR
(75.5 MHz, CDCl3, 25 ꢀC):
¼ 155.3, 80.0, 69.3, 30.4, 28.3, 27.4.
d
4.2.3. tert-Butyl 2-(2-(3-(tert-butoxycarbonylamino)prop-1-ynyl)
phenoxy)acetate (9)
A solution of 8 (10 g, 30 mmol) and CuI (0.29 g, 1.5 mmol,
0.05 equiv) in CH3CN (120 mL) was degassed by purging with argon
followed by the addition of [Pd(PPh3)4] (1.74 g, 1.5 mmol,
0.05 equiv). In a separate flask, 7 (4.7 g, 30 mmol) and Et3N
(16.6 mL, 120 mmol) were dissolved in CH3CN (60 mL) and this
solution was purged with argon before the solution was transferred
to the Pd-containing reaction mixture. The obtained reaction
mixture was stirred at room temperature for 16 h, followed by
filtration over Hyflo and concentrated in vacuo. The residue was
redissolved in EtOAc (225 mL) and this solution was washed with
1 N KHSO4 (250 mL), 10% NaHCO3 (250 mL) and brine (125 mL). The
organic layer was dried (Na2SO4) and concentrated in vacuo. The
residue was purified by column chromatography (hexane/EtOAc
9:1) to give 9 as a slightly brown solid in 72% yield (7.80 g). Rf ¼ 0.20
4 equiv) and DIPEA (1050 mL, 6 mmol, 8 equiv) in NMP (20 mL),
followed by a washing step with NMP (3 ꢂ 2 min, 20 mL) and
CH2Cl2 (3 ꢂ 2 min, 20 mL). Coupling and deprotection steps were
followed by the Kaiser test [30]. For the N-terminal modification of
peptide resin 11, Fmoc removal was performed with 20% piperi-
dine/NMP (3 ꢂ 2 min, 20 mL) followed by a washing step with NMP
(3 ꢂ 2 min, 20 mL) and CH2Cl2 (3 ꢂ 2 min, 20 mL) before the
appropriate N-terminal capping moiety was introduced.
4.2.6. Peptide resin 12a
Resin 11 (285 mg, 0.10 mmol) was suspended in NMP (10 mL)
and 10 (90 mg, 0.20 mmol, 2 equiv) followed by HATU/HOAt [31]
(hexane/EtOAc 9:1); 1H NMR (300 MHz, CDCl3, 25 ꢀC):
d
¼ 7.39 (d,
(0.2 mmol, 2 equiv) and DIPEA (70 mL, 0.40 mmol, 4 equiv) were
added. After 16 h reaction time, the resin was filtered and subse-
quently washed with NMP (3 ꢂ 2 min, 10 mL) and CH2Cl2
(5 ꢂ 2 min, 10 mL).
J ¼ 6.3 Hz, 1H; arom H), 7.25 (t, J ¼ 5.8 Hz, 1H; arom H), 6.93 (t,
J ¼ 6.6 Hz, 1H; arom H), 6.73 (d, J ¼ 8.5 Hz, 1H; arom H), 4.91 (broad
s, 1H; urethane NH), 4.59 (s, 2H; OCH2), 4.20 (m, 2H; CH2NH), 1.49
t
t
(s, 9H; Bu), 1.46 (s, 9H; Bu); 13C NMR (75.5 MHz, CDCl3, 25 ꢀC):
d
¼ 167.7, 158.6, 155.3, 133.7, 129.5, 121.4, 112.7, 112.2, 89.7, 82.4,
79.8, 79.3, 66.3, 31.6, 28.4, 28.0; elemental analysis calcd (%) for
20H27NO5: C, 66.46, H, 7.53, N, 3.88; found: C 66.28, H 7.54, N 3.79.
4.2.7. Peptide resin 12b
Resin 11 (285 mg, 0.10 mmol) was suspended in NMP (10 mL)
and phenoxyacetic acid (30 mg, 0.20 mmol, 2 equiv) followed by
C
HBTU/HOBt (0.20 mmol, 2 equiv) and DIPEA (70 mL, 0.40 mmol,
4.2.4. 2-(2-(3-(((9H-Fluoren-9-yl)methoxy)carbonylamino)prop-1-
ynyl)phenoxy)acetic acid (10)
4 equiv) were added. After 60 min coupling time, the resin was
filtered and subsequently washed with NMP (3 ꢂ 2 min, 10 mL) and
CH2Cl2 (5 ꢂ 2 min, 10 mL) and dried.
Compound 9 (7.9 g, 21.7 mmol) was dissolved in CH2Cl2 (80 mL)
and TFA (80 mL) was added. The reaction mixture was stirred for
3 h and subsequently concentrated in vacuo and the residue was
coevaporated with toluene (3 ꢂ 30 mL) followed by CH2Cl2
(3 ꢂ 30 mL) to remove any remaining acid. The obtained inter-
mediate was redissolved in H2O/CH3CN (120 mL, 1:1) and Et3N was
added to adjust pH to 9. Then, a solution of Fmoc-ONSu (7.3 g,
21.7 mmol) in CH3CN (50 mL) was added as a single portion. The pH
of the reaction mixture was maintained between 8 and 9 by adding
small portions of Et3N and after 30 min the reaction was complete.
The reaction mixture was concentrated to approx. 50 mL in vacuo
and the aqueous phase was subsequently acidified with 1 N KHSO4
(450 mL) and extracted with EtOAc (250 mL). The EtOAc solution
was washed with 1 N KHSO4 (100 mL) and brine (100 mL), dried
(Na2SO4) and concentrated in vacuo. The residue was purified by
column chromatography (CH2Cl2/MeOH 95:5 / CH2Cl2/MeOH/
AcOH 92:7:1) to give 10 as a yellowish waxy solid in 65% yield
(6.8 g). Rf ¼ 0.80 (CHCl3/MeOH/AcOH 95:20:3), Rf ¼ 0.36 (CH2Cl2/
MeOH/AcOH 95:5:1); 1H NMR (300 MHz, DMSO-d6, 25 ꢀC):
4.2.8. Peptide resin 12c
Resin 11 (285 mg, 0.10 mmol) was suspended in NMP (10 mL)
and Fmoc-Lys(Boc)-OH (94 mg, 0.20 mmol, 2 equiv) followed by
HBTU/HOBt (0.20 mmol, 2 equiv) and DIPEA (70 mL, 0.40 mmol,
4 equiv) were added. After 60 min coupling time, the resin was
filtered and subsequently washed with NMP (3 ꢂ 2 min, 10 mL) and
CH2Cl2 (3 ꢂ 2 min, 10 mL). Then, the Fmoc group was removed with
piperidine (3 ꢂ 8 min, 10 mL) and the resin was washed (NMP
(3 ꢂ 2 min, 10 mL) and CH2Cl2 (3 ꢂ 2 min, 10 mL)). Finally, capping
reagent (a mixture of Ac2O (21.3 mL)/HOBt (1.03 g)/DIPEA (9.82 mL)
in NMP (450 mL); 10 mL) was added and after 30 min reaction time,
the resin was filtered and subsequently washed with NMP
(3 ꢂ 2 min, 10 mL) and CH2Cl2 (5 ꢂ 2 min, 10 mL) and dried.
4.2.9. Peptide resin 12d
d
¼ 7.78e6.78 (m, 12H; arom H (4H)/arom H Fmoc (8H)), 6.93
Resin 11 (285 mg, 0.10 mmol) was suspended in NMP (10 mL)
(broad s, 1H; urethane NH), 4.73 (s, 2H; OCH2), 4.41 (d, 2H; CH2
and Fmoc-Glu(OtBu)-OH (85 mg, 0.20 mmol, 2 equiv) followed by
Fmoc), 4.25 (t, 1H; CH Fmoc), 3.58 (m, 2H; CH2NH); 13C NMR
HBTU/HOBt (0.20 mmol, 2 equiv) and DIPEA (70 mL, 0.40 mmol,
(75.5 MHz, DMSO-d6, 25 ꢀC):
d
¼ 170.0, 158.3, 155.9, 143.8, 140.7,
4 equiv) were added. After 60 min coupling time, the resin was
filtered and subsequently washed with NMP (3 ꢂ 2 min, 10 mL) and
CH2Cl2 (3 ꢂ 2 min, 10 mL). Then, the Fmoc group was removed with
piperidine (3 ꢂ 8 min, 10 mL) and the resin was washed (NMP
(3 ꢂ 2 min, 10 mL) and CH2Cl2 (3 ꢂ 2 min, 10 mL)). Finally, capping
reagent (10 mL) was added and after 30 min reaction time, the resin
was filtered and subsequently washed with NMP (3 ꢂ 2 min, 10 mL)
and CH2Cl2 (5 ꢂ 2 min, 10 mL) and dried.
133.5, 129.7, 127.6, 127.0, 125.2, 120.8, 120.1, 112.1, 111.4, 90.8, 78.3,
65.7, 64.6, 46.6, 30.8; HR-MS: (50 eV) m/z calculated for
C
26H21NO5Na: 450.1317 [MþNa]þ; found: 450.1345 [MþNa]þ.
4.2.5. Peptide resin 11
The fully protected peptide sequence was synthesized
(0.75 mmol scale) on a polystyrene resin functionalized with a

64
R.C. Elgersma et al. / European Journal of Medicinal Chemistry 88 (2014) 55e65
4.2.10. Peptide resin 12e
solution still flowed. If no flow was observed, gelation was said to
have taken place.
Resin 11 (285 mg, 0.10 mmol) was suspended in capping reagent
(10 mL) and after 30 min reaction time, the resin was filtered and
subsequently washed with NMP (3 ꢂ 2 min, 10 mL) and CH2Cl2
(5 ꢂ 2 min, 10 mL) and dried.
4.2.19. Transmission electron microscopy (TEM)
A peptide gel/solution (10 L of a 10 mg/mL solution), which was
m
aged for three weeks in the appropriate buffer, was placed on a
carbon coated copper grid. After 15 min, any excess of peptide was
removed by washing the copper grid on a drop of demi-water (this
was repeated five times). Finally, the samples were stained using
methylcellulose/uranyl acetate (2% aqueous solution) and dried
on air.
4.2.11. Peptide 1
Peptide resin 12a (150 mg, 0.10 mmol) was treated with DBU/
piperidine/DMF (2:2:96; 2 ꢂ 20 min, 10 mL) to remove the Fmoc
group [32] and subsequently washed with NMP (3 ꢂ 2 min, 10 mL)
and CH2Cl2 (5 ꢂ 2 min,10 mL) and dried. Then, the resin was treated
with TFA/TIS/H2O (95:2.5:2.5; 10 mL) for 3 h at room temperature
to detach the peptide from the resin and to remove all protecting
groups. The peptide was precipitated in MTBE/hexane (1:1)
at ꢁ20 ꢀC and finally lyophilized from tert-butanol/H2O (1:1) to give
compound 1 as a fluffy white powder in 51% yield (29 mg).
Rt ¼ 19.12 min; ES-MS (50 eV) m/z (%): 1040.55 [MþH]þ; found:
1040.75 (100) [MþH]þ, 1062.60 (20) [MþNa]þ.
4.2.20. Cryo-TEM
An aliquot of solution or gel (3 mL, 10 mg/mL in the appropriate
buffer) was pipetted onto a glow discharged Quantifoil 2/2 grid in
the environmental chamber of a Vitrobot. The sample was blotted
once during 1 s and rapidly plunged into liquid ethane. The grid was
transferred to a Gatan cryoholder Model 626.
4.2.12. Peptide 2
4.2.21. Fourier transform infrared spectroscopy (FTIR)
Peptide resin 12b (285 mg, 0.10 mmol) was treated with TFA/
TIS/H2O (95:2.5:2.5; 10 mL) for 3 h at room temperature to detach
the peptide from the resin and to remove all protecting groups. The
peptide was precipitated in MTBE/hexane (1:1) at ꢁ20 ꢀC and
finally lyophilized from tert-butanol/H2O (1:1) to give compound 2
as a fluffy white powder in 71% yield (70 mg). Rt ¼ 20.20 min; ES-
MS (50 eV) m/z (%): 987.52 [MþH]þ; found: 987.20 (100) [MþH]þ.
A peptide gel/solution (100
three weeks, was lyophilized and subsequently resuspended in D2O
(150 L) and lyophilized again. This treatment was repeated twice.
A peptide sample was mixed with KBr and pressed into a pellet. The
optical chamber was flushed with dry nitrogen for 1 min before
data collection started. The interferograms from 512 scans with a
resolution of 2 cmꢁ1 were averaged and corrected for H2O and KBr.
mL, 10 mg/mL), which was aged for
m
4.2.13. Peptide 3
4.2.22. Wide angle X-ray spectroscopy (WAXS)
Peptide resin 12c (285 mg, 0.10 mmol) was treated with TFA/TIS/
H2O as described for 2. Compound 3 was obtained as a fluffy white
powder in 60% yield (62 mg). Rt ¼ 17.45 min; ES-MS (50 eV) m/z (%):
1023.56 [MþH]þ; found: 1023.20 (100) [MþH]þ.
A peptide sample (10 mg/mL) was inserted into a 0.5 mm glass
capillary, and the sample was allowed to dry under ambient con-
ditions. Diffraction data were collected on a Mar Image Plate using
CuKa radiation from a BrukerAXS FR591 generator with rotating
anode and Montel 200 mirrors. The capillary was placed in a po-
sition perpendicular to the X-ray beam and at a distance of 250 mm
from the detector. The resolution ranges from approx. 70e2.6 Å.
Scattering from air, capillary, and if applicable, water was sub-
tracted by using an in-house software package (VIEW/EVAL) [33].
4.2.14. Peptide 4
Peptide resin 12d (285 mg, 0.10 mmol) was treated with TFA/
TIS/H2O as described for 2. Compound 4 was obtained as a fluffy
white powder in 36% yield (37 mg). Rt ¼ 18.12 min; ES-MS (50 eV)
m/z (%): 1024.54 [MþH]þ; found: 1024.05 (100) [MþH]þ.
Acknowledgments
4.2.15. Peptide 5
Peptide resin 12e (285 mg, 0.10 mmol) was treated with TFA/TIS/
H2O as described for 2. Compound 5 was obtained as a fluffy white
powder in 59% yield (53 mg). Rt ¼ 18.25 min; ES-MS (50 eV) m/z (%):
895.49 [MþH]þ; found: 895.25 (100) [MþH]þ.
These investigations were supported by the council for Chemical
Sciences of the Netherlands-Organization for Scientific Research
(CW-NWO TOP Grant 700.51.302). Marc van Peski and Rene Scri-
wanek (Center for Electron Microscopy, UMC Utrecht) are
acknowledged for their photographic artwork.
4.2.16. Peptide analysis
The purity (generally >95%) of the peptide sample was evalu-
ated by analytical HPLC on an Adsorbosphere XL C8 column (90Å
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