Microwave-assisted click polymerization for the synthesis of Aβ(16-22) cyclic oligomers and their self-assembly into polymorphous aggregates

Article abstract of DOI:10.1039/b912851d

We report on the design, synthesis, and structural analysis of cyclic oligomers with an amyloidogenic peptide sequence as the repeating unit to obtain novel self-assembling bionanomaterials. The peptide was derived from the Alzheimer Aβ(16-22) sequence si

Full text of DOI:10.1039/b912851d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Microwave-assisted click polymerization for the synthesis of Ab(16–22) cyclic
oligomers and their self-assembly into polymorphous aggregates†
Ronald C. Elgersma,‡§a Maarten van Dijk,‡a,b Annemarie C. Dechesne,a Cornelus F. van Nostrum,b
Wim E. Hennink,b Dirk T. S. Rijkersa and Rob M. J. Liskamp*a
Received 30th June 2009, Accepted 24th July 2009
First published as an Advance Article on the web 27th August 2009
DOI: 10.1039/b912851d
We report on the design, synthesis, and structural analysis of cyclic oligomers with an amyloidogenic
peptide sequence as the repeating unit to obtain novel self-assembling bionanomaterials. The peptide
was derived from the Alzheimer Ab(16–22) sequence since its strong tendency to form antiparallel
b-sheets ensured the formation of intermolecular hydrogen bridges on which the supramolecular
assembly of the individual cyclic oligomers was based. The synthesis of the cyclic oligomers was
performed via a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction of
azido-Lys-Leu-Val-Phe-Phe-Ala-Glu-propargyl amide as the monomer. The formation of cyclic
oligomers, up to pentamers (35 amino acid residues), was verified by MALDI-TOF analysis and the
individual cyclic monomer and dimer could be isolated by HPLC. Gelation behavior and the
self-assembly of the linear monomer and the cyclic monomer and dimer were studied by TEM, FTIR
and CD. Significant differences were observed in the morphology of the supramolecular aggregates of
these three peptides that could be explained by alterations of the hydrogen bond network.
molecular self-recognition motif and, depending on the conditions
(e.g. pH, bivalent metal ions, solvent), peptide nanotubes4 or more
Introduction
The development of peptide-based polymers with unique prop-
erties like self-assembly has received much attention during
recent years. This class of polymers has been used as drug delivery
systems, medical devices, in tissue engineering, and for catalysis.1
Since it has been shown that peptide-based polymers have better
biocompatibility and bioavailability properties than their (semi)-
synthetic congeners, novel applications of this class of biomaterials
are still emerging. Of special interest are polymers that consist of
relatively small building blocks with an intrinsic propensity to
self-assemble into supramolecular constructs.2
fibrillar-like aggregates have been obtained as the supramolecular
assemblies.5 In this context, fragments of the highly amyloidogenic
Ab peptide, such as the dipeptide H-Phe-Phe-OH as described by
Gazit and co-workers6 and Go¨rbitz,4b–d and the heptapeptide Ac-
Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2 (Ab(16–22)) as described by
Lynn,7 have been used to obtain peptide nanotubes. In the current
literature several applications of peptide nanotubes or fibrillar
aggregates as bionanomaterials, ranging from drug containers
and nanoreactors to functional templates and biofilms, have been
described.2,8
A first prominent example of peptide-based supramolecular
polymers has been given by Ghadiri et al.3 They have found
that cyclic peptides with alternating L- and D-amino acids
form peptide nanotubes via hydrogen bond interactions of the
peptide backbone. More recently, designed amphiphilic peptides
and amyloidogenic peptide fragments have been used as the
In previous studies we disclosed an attractive approach for
the synthesis of peptide-based polymers.9a,b Bifunctional peptides,
with an N-terminal azide moiety and a propargyl amide at the
C-terminus, were polymerized via a Cu(I)-mediated click poly-
merization reaction.10,11 Depending on the reaction conditions,
either high molecular weight linear polymers or cyclic medium-
sized oligomers could be obtained from the model dipeptide
azido-Phe-Ala-propargyl amide. In the present study we describe
another application of this click polymerization reaction, this time
to synthesize cyclic oligomeric peptides in which the repeating
motif is derived from the amyloidogenic Ab(16–22) sequence, i.e.
azido-Lys-Leu-Val-Phe-Phe-Ala-Glu-propargyl amide (1, Fig. 1),
to obtain a novel class of building blocks for the supramolecular
assembly of peptide-based bionanomaterials. After running the
click polymerization reaction, the isolated cyclic monomer and
dimer have been individually used in a self-assembly process
to explore their biomimetic organization behavior into peptide
nanotubes and fibrillar aggregates and differences in morphology
could be attributed to alteration of the intra- and intermolecular
hydrogen bond network.
aDivision of 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. E-mail: R.M.J.Liskamp@uu.nl; Fax: +31 30 253 6655;
Tel: +31 30 253 7396/7307
bDivision of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences,
Department of Pharmaceutical Sciences, Faculty of Science, Utrecht
University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
† Electronic supplementary information (ESI) available: Copies of the
1H-, 13C- and COSY NMR spectra, HPLC chromatograms, EI-MS
and MALDI-TOF mass spectra and FTIR spectra and details of the
computational modeling. See DOI: 10.1039/b912851d
‡ These authors contributed equally to this work.
§ Present address: Syntarga B.V., Toernooiveld 1, 6525 ED Nijmegen, The
Netherlands.
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(90% yield) was performed essentially as described by Lundquist
and Pelletier,12 which was based on previous reports by Wong and
co-workers.13
The synthesis of Ab(16–22) peptide 1 was performed on the
solid support. Since the C-terminus of this peptide is modified
into the propargyl amide, building block 3 was connected to
the resin via its g-COOH side chain functionality and the
2-chlorotrityl chloride resin14 (Barlos’ resin) is ideally suited for
this purpose. After loading the resin with Fmoc-derivative 3
(loading was determined15 to be 0.46 mmol g-1), the peptide
sequence was synthesized by Fmoc/tBu chemistry.16 After cleavage
from the resin, peptide 1 was analyzed by HPLC and characterized
by mass spectrometry. The presence of the azide moiety was
confirmed by FTIR, since the characteristic N3-stretch vibration
was found at n 2107 cm-1.
Fig. 1 Structural formula of the Ab(16–22) peptide sequence, decorated
with an N-terminal azido and a C-terminal alkyne moiety.
Results
Synthesis
Peptide Ab(16–22) 1 was used as the monomer in the microwave-
assisted click polymerization reaction (as shown in Scheme 2). The
reaction conditions to obtain cyclic oligomers from bifunctional
monomers, as recently described by van Dijk et al.,9 were used:
a low monomer concentration of 20 mg mL-1 and CuOAc as
the catalytic species under microwave-irradiation for 30 min at
100 ◦C. Attempts to run the click reaction in DMF or DMF–
H2O mixtures failed, mainly due to the low solubility of 1. Since
the Ab(16–22) peptide sequence has a high intrinsic tendency to
The synthesis of Ab(16–22) peptide 1 and the required building
blocks is depicted in Scheme 1. First, glutamic acid building block
3 and lysine derivative 5 were prepared starting from commer-
cially available Fmoc-Glu(OtBu)-OH (2) and H-Lys(Boc)-OH (4),
respectively. Fmoc-Glu(OtBu)-propargyl amide was obtained via
a BOP-mediated coupling of 2 and propargyl amine. The fully
protected intermediate was treated with TFA to remove the Bu-
functionality, and the desired building block 3 was obtained in 70%
overall yield. The diazo-transfer reaction to convert 4 into azide 5
t
Scheme 1 Synthesis approach for the preparation of Ab(16–22) peptide 1. (A) Synthesis of glutamic acid building block 3. (B) Synthesis of lysine
building block 5. (C) Solid phase synthesis of Ab(16–22) peptide 1.
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Scheme 2 Microwave-assisted click polymerization of Ab(16–22) peptide 1 and the structural formulas of the isolated cyclic peptides 8 and 9.
aggregate, DMSO was used as an alternative solvent to disrupt any
premature formation of secondary structure of the monomer, and
the click reaction was run in this solvent with an increased amount
(50 mol%) of CuOAc. The click polymerization reaction product
was analyzed by MALDI-TOF mass spectrometry and revealed
the presence of at least four different compounds (Fig. 2). Based
(isolated yield: 15%) and cyclic dimer 9 (isolated yield: 33%),
since the characteristic N3-stretch vibration was absent. Although
the formation of tri-, tetra- and pentamers was demonstrated by
MALDI-TOF, their isolation by HPLC was unsuccessful.
The higher isolated yield of the cyclic dimer 9 compared to
8 might be explained by a mechanism that was proposed by
Finn and co-workers17 and which was corroborated by others.18
This mechanism assumes that the favored dimerization involves
the formation of a dialkyne–Cu(I) complex which rearranges
via the energetically more favored exo-like conformation to give
preferentially the cyclic dimer.19 Furthermore, the 1,4-substituted
triazole functionality has been described as a good b-turn inducing
moiety,20 thereby promoting the antiparallel organization of the
newly formed dimer which ultimately results in the preferential
formation of the cyclic dimer 9 (vide infra).
on the molecular mass of the monomer ([M + H]+ 916.50),
ave
the observed peaks could be assigned to the cyclic dimer ([M +
H]+ave 1834.01), trimer ([M + H]+ave 2750.47), tetramer ([M + H]+
ave
3666.94) and pentamer ([M + H]+ 4581.58). Purification by
ave
preparative HPLC resulted in the isolation of two compounds,
characterized by a molecular mass of [M + H]+
916.672
monoisotopic
and [M + H]+
1831.542, corresponding to the monomer
monoisotopic
and dimer, respectively. Based on FTIR analysis, it was concluded
that the isolated compounds represented the cyclic monomer 8
Fig. 2 Composition of the crude microwave-assisted click polymerization reaction product of peptide Ab(16–22) 1, as analyzed by MALDI-TOF
spectrometry.
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Supramolecular assembly
The supramolecular assembly of peptides 1, 8 and 9 was studied
by preparing a concentrated aqueous solution (6 mg mL-1 in
H2O–DMSO 9 : 1 v/v, pH 7) of each individual peptide. These
samples were stored for three weeks at 4 ◦C prior to analysis.
Gelation of the solution at sufficiently high concentration is
a first indication of peptide aggregation and the formation of
supramolecular assemblies. Although reference peptide 1 did not
form a gel, analysis by TEM clearly indicated the presence of sheet-
like lamellar assemblies (Fig. 3A). Analysis by FTIR21 revealed,
in addition to the azide and alkyne absorptions, the presence
of a strong absorption at n 1628 cm-1 of the peptide amide
bond, indicative of aggregated antiparallel b-sheets with a lamellar
supramolecular morphology. This result was in agreement with
the X-ray powder diffraction data of Lynn and co-workers, who
found that Ab(16–22) formed supramolecular assemblies, with an
antiparallel orientation of the assembled peptides.7
Likewise, cyclic peptide 8 did not form a gel, although broad
fiber-like assemblies (up to 80 nm in width) were observed by
TEM (Fig. 3B). However, the morphology of these assemblies
was clearly different from that observed with peptide 1. Moreover,
FTIR analysis indicated that the amide I absorption was shifted
from n 1628 to 1672 cm-1. This shift might be explained by
assuming that self-assembly is largely based on hydrophobic
interactions (Val/Leu), p–p interactions (Phe), and electrostatic
interactions (Glu/Lys) rather than on intermolecular hydrogen
bond formation via the peptide backbone. Additional evidence was
obtained by CD spectroscopy which showed a positive maximum
at l 193 nm and a negative minimum at l 205 and 235 nm
of peptide 8 (Fig. 4), which implied an a-helical- rather than a
b-sheet-like conformation.
Fig. 3 TEM pictures of peptide 1 (A), cyclic peptide 8 (B), and cyclic
dimer 9 (C). Scale bars represent 500 nm (conditions: 6 mg peptide/mL
H2O–DMSO 9 : 1 v/v, pH 7; peptide samples were aged for three weeks
prior to analysis).
with the amyloid fibrils formed by full length Ab.22 The formation
of b-sheets was apparent by FTIR, from the presence of the
characteristic absorptions at n 1636 and 1686 cm-1 indicating an
intra- and interstrand hydrogen bond pattern.21 Moreover, CD
spectroscopy showed a positive maximum at l 195 nm and a
negative minimum at l 230 nm (Fig. 4), which is also a strong
indicator of b-sheet formation.
The cyclic dimer 9 indeed formed a gel after three weeks of
aging, and a dense network of fibrils was observed by TEM
(Fig. 3C). Generally, these fibrillar entities were thinner and also
longer than those formed by cyclic monomer 8. In addition, the
supramolecular assemblies as formed by 9 showed a high similarity
Fig. 4 CD spectra of cyclic peptide 8 (filled squares) and cyclic dimer 9 (filled triangles). Conditions: 0.3 mg peptide/mL H2O–HFIP 9 : 1 v/v, pH 7;
peptide samples were freshly prepared prior to analysis.
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Fig. 5 (A) Lowest energy conformation of cyclic monomer 8. (B) Structural formula of cyclic monomer 8 with the proposed intramolecular hydrogen
bond indicated.
peptide 8 in which a single intramolecular hydrogen bond is
Discussion
present (Fig. 5B). The calculated lowest energy conformation is
The bifunctional heptapeptide azido-Lys-Leu-Val-Phe-Phe-Ala-
in agreement with the FTIR data, indicating an intramolecular
hydrogen bond (n 1672 cm-1). In addition, this conformation
Glu-propargyl amide (1) was designed and synthesized as a novel
monomer to be used in the microwave-assisted click polymeriza-
hints at an amphiphilic structure since the triazole moiety, as an
amide bond mimic and hydrogen bond acceptor,28 as well as the
lysine and glutamic acid side chains are located on the same side
of the molecule, while the hydrophobic side chains of leucine,
valine, phenylalanine, and alanine are grouped on the other side
of the molecule. Analogous to the model proposed by Vale´ry
et al.,27 monocyclic peptide 8 may form supramolecular assemblies
as schematically shown in Fig. 6 in which self-assembly is
driven by both electrostatic interactions of the oppositely charged
tion approach toward the synthesis of peptide-triazoles as protein
mimics. The choice of the monomer as the repeating peptide
sequence is of crucial importance to obtain soft material-like
properties,23 such as those displayed by spider silk, elastin and
collagen as the most prominent examples. The sequence of 1
corresponds to residues 16–22 from the highly amyloidogenic
Ab-peptide, which play a prominent role in Alzheimer’s disease.24
The intrinsic propensity of 1 to self-organize into well-defined
nanostructures, as was shown by Lynn and co-workers,7 enticed
us to investigate the relationship between the molecular structure
of 1 (linear, cyclic, or oligomeric) and the supramolecular folding
morphology.
For a rapid access to cyclic oligomers of 1, the microwave-
assisted click polymerization approach as previously reported by
us9 was applied.25,26 Although oligomers up to cyclic pentamers,
i.e. 35 amino acid residues, were identified by MALDI-TOF
analysis, the cyclic monomer 8 and cyclic dimer 9 were isolated
as major components by preparative HPLC in a yield of 15
and 33%, respectively. The head-to-tail cyclodimerization was
found to occur rapidly.17-19 According to Finn and co-workers,19
‘cyclodimerization depends on the ability of the azido–alkyne
peptide to form in-frame hydrogen bonds between chains in order
to place the reacting groups in close proximity and lower the
entropic penalty for dimerization’. The FTIR analysis of cyclic
dimer 9 is in agreement with this and characteristic absorptions at n
1636 and 1686 cm-1 indicated an intra- and interstrand hydrogen
bond pattern.21 Moreover, CD spectroscopy also confirmed the
presence of b-sheets.
Vale´ry et al. described the self-assembly of a cyclic octapeptide
(lanreotide) and proposed a molecular structure of the formed
nanotubes.27 The b-hairpin structure of lanreotide together with
the presence of hydrophobic aromatic side chains as in b-
naphthylalanine, tyrosine and tryptophan, and hydrophilic side
chains as in lysine and threonine, and a C-terminal carbox-
amide, strongly favored segregated p–p interactions and polar
interactions in the self-assembly into peptide nanotubes. Fig. 5A
shows an image of the lowest energy conformation of monocyclic
Fig. 6 Structural representation of the supramolecular assembly in
the case of cyclic monomer peptide 8 (dotted lines: intramolecular
hydrogen bridge, dashed box: electrostatic interactions, brackets: p–p and
hydrophobic interactions).
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Glu/Lys side chains and intermolecular p–p interactions of the
phenylalanine side chains. Since the cyclic dimeric peptide 9 adopts
an antiparallel b-sheet, as judged by FTIR and circular dichroism
(Fig. 4), self-assembly might be due to hydrogen bond formation
via the peptide backbone, as is schematically represented by Fig. 7.
Since it has been shown in this study that the linear monomer,
cyclic monomer and cyclic dimer differ in their self-assembly
behavior, it is expected that controlling the molecular geometry
of the peptide holds promise for directing self-assembly in a
predictable way. This should ultimately lead to the development
of designed nanostructured biological materials with a broad
range of applications such as scaffolds for tissue-engineering and
-repair, biocompatible coatings and films, templates for bone
mineralization, novel biodegradable drug delivery systems, like
nanocontainers, and stimuli-responsive hydro/organogels.1,2,29
Experimental
Chemicals, instruments and general methods
Chemicals were obtained from commercial sources and used
without further purification. Peptide grade solvents used for
˚
solid phase peptide synthesis were stored on 4 A molecular
sieves. Microwave-assisted reactions were performed in a Bio-
tage initiator apparatus with pressure and temperature control.
1H-NMR spectra were recorded either on a Varian G-300
(300 MHz) or on a Varian Inova-500 (500 MHz) spectrometer
and chemical shift values (d) are given in ppm relative to TMS.
13C-NMR spectra were recorded on a Varian G-300 (75.5 MHz)
spectrometer 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. Melting points were measured on a Bu¨chi
Schmelzpunktbestimmungsapparat and are uncorrected. Optical
rotations were measured on a Jasco P1010 polarimeter at ambient
temperature. Column chromatography was performed with Silica-
P Flash silica gel (Silicycle). 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,N¢,N¢-tetramethyl-4,4¢-
diaminodiphenylmethane (TDM).30 Preparative HPLC runs were
carried out on an Applied Biosystems 400 Semi Automated
HPLC System equipped with an Applied Biosystems 757 UV/VIS
Absorbance Detector (l = 214 nm). The crude lyophilized
peptides were dissolved in a minimum amount of TFA and loaded
onto an Alltech Prosphere C4 column (250 ¥ 22 mm, particle size:
Fig. 7 Structural representation of a b-pleated sheet in the case of
cyclic dimer peptide 9 (dotted lines: intramolecular hydrogen bridges,
dashed lines: intermolecular hydrogen bridges, brackets: electrostatic
interactions).
˚
10 mm, pore size: 300 A). The peptides were eluted with a flow
rate of 10 mL min-1 using a linear gradient from 100% buffer
A to 75% buffer B in 120 min (buffer A: 0.1% TFA in H2O–
CH3CN 95 : 5 v/v; buffer B: 0.1% TFA in CH3CN–H2O 95 :
5 v/v). The retention time values (Rt) and purities of the peptides
were evaluated by analytical HPLC on an Adsorbosphere XL C8
In summary, the facile solid phase synthesis of a bifunc-
tional peptide derivative, azido-Lys-Leu-Val-Phe-Phe-Ala-Glu-
propargyl amide, of the amyloidogenic Ab(16–22) fragment
and its subsequent application as monomer in the microwave-
assisted click polymerization to give cyclic oligomers have been
described. Polymerization resulted in the formation of at least
cyclic pentamers as judged by MALDI-TOF analysis. In addition,
the cyclized monomer as well as the cyclic dimer could be
isolated by preparative HPLC. Self-assembly of the cyclized
monomer resulted in broad fiber-like aggregates while a dense
network of amyloid fibrils was observed in the case of the
cyclic dimer. This polymorphism of the supramolecular assemblies
observed by electron microscopy might be attributed to a different
conformation of the individual monomeric and dimeric peptides
based on FTIR, CD and molecular dynamics calculations.
˚
column (250 ¥ 4.6 mm, particle size: 5 mm, pore size: 90 A) at
a flow rate of 1.0 mL min-1 using a linear gradient from 100%
buffer A to 100% buffer B in 20 min (buffer A: 0.1% TFA in
H2O–CH3CN 95 : 5 v/v; buffer B: 0.1% TFA in CH3CN–H2O
95 : 5 v/v). Peptides were characterized either by electrospray
ionization mass spectrometry (EI-MS) on a Finnigan LCQ Deca
XP Max apparatus operating in a positive ionization mode or by
MALDI-TOF analysis which was performed on a Kratos Axima
CFR apparatus. As the external reference, ACTH(18–39) ([M +
H]+monoisotopic: 2465.1989), and as the matrix, a-cyano-4-hydroxy-
cinnamic acid were used, respectively. The mass of each analog
was measured and the observed monoisotopic [M + H]+ value
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was correlated with the calculated [M + H]+ value. Transmission
electron microscopy (TEM) was performed on a Jeol 1200 EX
transmission electron microscope operating at 60 kV. A sample
(10 mL) of a peptide gel/solution (6 mg mL-1 in DMSO–H2O
1 : 9 v/v), which was aged for three weeks at 4 ◦C, was placed
on a carbon-coated copper grid. After 15 min of incubation, the
excess of peptide was removed by washing the copper grid on a
drop of demi-water (this washing step was repeated five times).
Finally, the samples were negatively stained by methylcellulose–
uranyl acetate and dried in air prior to analysis. Fourier transform
infrared spectra (FTIR) were measured on a BioRad FTS 6000
spectrophotometer. A sample (100 mL) of a peptide gel/solution
(6 mg mL-1 in DMSO–H2O 1 : 9 v/v), which was aged for three
in NMP (10 mL) for 2 h at room temperature. All coupling
reactions were monitored by the Kaiser test.32 Resin 7 was treated
with TFA–TIS–H2O (95 : 2.5 : 2.5 v/v/v) for 3 h at room
temperature to deprotect and cleave the peptide from the resin,
and the peptide was precipitated in MTBE–hexane (1 : 1 v/v) at
-20 ◦C. The crude peptide was lyophilized from tert-BuOH–H2O
(1 : 1 v/v). The peptide was purified by HPLC and identified
and characterized by mass spectrometry and NMR spectroscopy,
respectively. Heptapeptide 1 was obtained in 74% yield (310 mg)
after purification. Rt 20.3 min; nmax/cm-1 2108 (N3), 1695 and
1
=
1628 (C O), 1540; H-NMR (300 MHz, DMSO-d6) d = 8.37–
8.30 (m, 2H, amide NH propargyl (1H)/amide NH Val(1H)), 8.18
(d, J = 7.2 Hz, 1H, amide NH Ala), 8.11 (d, J = 8.0 Hz, 1H,
amide NH Phe), 8.00–7.93 (m, 2H, amide NH Phe (1H)/amide
NH Glu (1H)), 7.83 (d, J = 8.8 Hz, 1H, amide NH Leu), 7.24–
7.18 (m, 10H, arom H Phe (2 ¥ 5H)), 4.53 (m, 2H, aCH Phe
(2 ¥ 1H)), 4.38–4.23 (m, 3H, aCH Ala (1H)/aCH Val (1H)/aCH
Glu (1H)), 4.08 (t, J = 7.8 Hz, 1H, aCH Leu), 3.85 (m, 2H,
CH2 propargyl), 3.77 (t, J = 6.9 Hz, 1H, aCH Lys), 3.12 (m,
1H, HC∫), 3.05–2.68 (double m, 4H, bCH2 Phe (2 ¥ 2H)), 2.75
(m, 2H, eCH2 Lys), 2.22 (m, 2H, gCH2 Glu), 1.86–1.73 (m, 3H,
bCH2 Leu (2H)/gCH Leu (1H)), 1.86–1.73 (m, 2H, bCH2 Glu),
1.73–1.30 (m, 6H, bCH2/gCH2/dCH2 Lys (3 ¥ 2H)), 1.53 (m, 1H,
bCH Val), 1.21 (d, J = 7.2 Hz, 3H, bCH3 Ala), 0.84 (dd, 6H,
gCH3/g¢CH3 Val (2 ¥ 3H)), 0.71 (d, J = 4.4 Hz, 6H, dCH3/d¢CH3
Leu); 13C-NMR (75.5 MHz, DMSO-d6) d = 173.9, 171.9, 171.4,
170.8, 170.7, 170.6, 170.4, 169.1, 137.6, 129.1, 128.0, 126.2, 80.9,
73.0, 60.9, 57.5, 53.6, 53.4, 51.7, 51.1, 48.2, 37.5, 30.7, 30.4, 30.2,
28.0, 27.6, 26.6, 24.2, 23.1, 22.2, 21.5, 19.1, 18.0; MALDI-TOF
C46H65N11O9 requires 915.50, found m/z [M + H]+monoisotopic 916.67,
[M + H]+ 918.19, [M + Na]+ 940.21.
◦
weeks at 4 C, was lyophilized and subsequently resuspended in
D2O (150 mL) and lyophilized again. This treatment was repeated
twice. The lyophilized peptide sample was mixed with KBr and
pressed into a pellet. The optical chamber was flushed with
dry nitrogen gas for 1 min before data collection started. The
interferograms from 128 scans with a resolution of 2 cm-1 were
averaged and corrected for H2O and KBr. Circular dichroism (CD)
spectra were measured on an OLIS RSM 1000 CD Spectrometer.
A freshly prepared solution of the peptide (3 mg mL-1) in HFIP
was diluted with HFIP–H2O (1 : 9 v/v) to a final concentration
of 0.3 mg mL-1 and directly placed in a 2 mm cuvette in a
thermostatted optical chamber (20 ◦C), which was continually
flushed with dry nitrogen gas. The CD spectra were measured at
1 nm intervals in the range of 190–250 nm as the average of 10
scans using a spectral bandwidth of 1.0 nm. Calculations of the
lowest energy conformation of the peptides were performed on a
SiliconGraphics O2 workstation with MacroModel 7.0 using the
organic builder and the peptide builder in the grow mode.31
ave
ave
Fmoc-Glu(OH)-propargyl amide (3). Fmoc-Glu(OtBu)-OH 2
(4.25 g, 10 mmol), propargylamine hydrochloride (915 mg,
10 mmol) and BOP (4.42 g, 10 mmol) were dissolved in CH2Cl2
(100 mL) and this solution was cooled on ice before Et3N (4.18 mL,
30 mmol, 3 equiv.) was added. The obtained reaction mixture was
stirred for 1 h at 0 ◦C followed by 16 h at room temperature.
Then, the solvent was removed under reduced pressure and
the residue was redissolved in EtOAc (150 mL). The organic
solution was subsequently washed with 1 M KHSO4 (2 ¥ 75 mL),
sat. aq. NaHCO3 (2 ¥ 75 mL) and brine (1 ¥ 50 mL), dried
(Na2SO4) and concentrated in vacuo. The obtained reaction
product was used without further purification in the next reaction
step. Fmoc-Glu(OtBu)-propargyl amide was dissolved in CH2Cl2–
TFA (100 mL 1 : 1 v/v) and the reaction mixture was stirred
for 2 h at room temperature. After this period of stirring, the
reaction mixture was concentrated in vacuo and the residue was
coevaporated with CH2Cl2 to remove any residual TFA. Then, the
residue was purified by column chromatography (eluent: CH2Cl2–
MeOH 95 : 5 v/v) to give amide 3 as a white solid in 70% overall
yield (2.8 g). Mp 151–154 ◦C; Rf 0.20 (CH2Cl2–MeOH 95 : 5 v/v);
1H-NMR (300 MHz, CDCl3–CD3OD 9 : 1 v/v) d = 7.97 (broad s,
1H, amide NH), 7.78–7.30 (9H, m, urethane NH (1H)/arom H
(8H)), 4.40 (m, 2H, CH2 Fmoc), 4.23 (m, 2H, CH Fmoc (1H)/aCH
Glu (1H)), 4.00 (s, 2H, CH2 propargyl), 2.38 (m, 2H, gCH2 Glu),
2.30 (m, 1H, HC∫), 2.10–1.86 (double m, 2 ¥ 1H, bCH2 Glu);
13C-NMR (75.5 MHz, CDCl3–CD3OD 9 : 1 v/v) d = 175.2, 171.6,
156.5, 143.4, 141.0, 127.5, 126.8, 124.7, 119.6, 78.7, 71.2, 66.7,
Peptide gelation experiments
Each peptide was dissolved in DMSO–H2O (1 : 9 v/v) to obtain
a final concentration of 6 mg mL-1. The aggregation state was
determined by eye at regular time intervals by tilting the test tube
and checking if the solution still flowed. If no flow was observed,
gelation was said to have taken place. The gelation experiments
were performed at room temperature.
Syntheses
Azido-Lys-Leu-Val-Phe-Phe-Ala-Glu-propargyl amide (1).
A
polystyrene resin, functionalized with a 2-chloro trityl chloride
linker (1 g, initial loading: 1.0 mmol g-1, 1.0 mmol), was loaded
with Fmoc-Glu(OH)-propargyl amide 3 (1.22 g, 3.0 mmol,
3 equiv.) in dry CH2Cl2 (10 mL) in the presence of DIPEA
(1.57 mL, 9.0 mmol, 3 equiv.) as base for 16 h at room temperature.
After filtration, the resin was washed with CH2Cl2 (3 ¥ 10 mL
for 5 min each) and any unreacted trityl chloride was capped
by treatment with a mixture of MeOH–CH2Cl2–DIPEA (2 :
17 : 1 v/v/v; 2 ¥ 10 mL for 10 min each). After drying resin
6, the amount of 3 coupled to the resin was calculated by an
Fmoc-determination according to Meienhofer15 and was found
to be 0.46 mmol g-1. The peptide sequence was synthesized
by Fmoc/tBu SPPS protocols on a 0.25 mmol scale (540 mg
resin). The N-terminal N3-Lys(Boc)-OH 5 (136 mg, 0.50 mmol,
2 equiv.) was coupled with HBTU–HOBt–DIPEA (2 : 2 : 4 equiv.)
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Org. Biomol. Chem., 2009, 7, 4517–4525 | 4523
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53.6, 46.8, 29.7, 28.6, 27.5; EI-TOF-LCMS [M + H]+ C23H23N2O5
requires m/z 407.1607, found 407.1626.
Chem. Commun., 2006, 2332–2334; (e) D. Gauthier, P. Baillargeon,
M. Drouin and Y. L. Dory, Angew. Chem., Int. Ed., 2001, 40, 4635–
4638.
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M. Reches, J. Rishpon and E. Gazit, Nano Lett., 2005, 5, 183–186; (c) N.
Kol, L. Adler-Abramovich, D. Barlam, R. Z. Shneck, E. Gazit and I.
Rousso, Nano Lett., 2005, 5, 1343–1346; (d) O. Carney, D. E. Shalev
and E. Gazit, Nano Lett., 2006, 6, 1594–1597; (e) M. Reches and E.
Gazit, Nat. Nanotechnol., 2006, 1, 195–200.
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J. Am. Chem. Soc., 2003, 125, 6391–6393; (b) J. Dong, J. E. Shokes,
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3542; (c) K. Lu, L. Guo, A. K. Mehta, W. S. Childers, S. N. Dublin, S.
Skanthakumar, V. P. Conticello, P. Thiyagarajan, R. P. Apkarian and
D. G. Lynn, Chem. Commun., 2007, 2729–2731; (d) A. K. Mehta, K.
Lu, W. S. Childers, Y. Liang, S. N. Dublin, J. Dong, J. P. Snyder, S. V.
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9829–9835 (correction to reference 7d: J. Am. Chem. Soc., 2009, 131,
8333).
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T. C. B. McLeish, A. N. Semenov and N. Boden, Proc. Natl. Acad.
Sci. U. S. A., 2001, 98, 11857–11862; (b) D. M. Marini, W. Hwang,
D. A. Lauffenburger, S. Zhang and R. D. Kamm, Nano Lett., 2002,
2, 295–299; (c) C. W. G. Fishwick, A. J. Beevers, L. M. Carrick, C. D.
Whitehouse, A. Aggeli and N. Boden, Nano Lett., 2003, 3, 1475–1479.
9 (a) M. van Dijk, K. Mustafa, A. C. Dechesne, C. F. van Nostrum, W. E.
Hennink, D. T. S. Rijkers and R. M. J. Liskamp, Biomacromolecules,
2007, 8, 327–330; (b) M. van Dijk, M. L. Nollet, P. Weijers, A. C.
Dechesne, C. F. van Nostrum, W. E. Hennink, D. T. S. Rijkers and
R. M. J. Liskamp, Biomacromolecules, 2008, 9, 2834–2843.
Azido-Lys(Boc)-OH (5). This compound was synthesized ac-
cording to the procedure of Lundquist and Pelletier,12 starting with
commercially available H-Lys(Boc)-OH (4) on a 12 mmol scale and
compound 5 was obtained as a pale yellow oil in 90% yield. Rf
25
0.57 (CHCl3–MeOH–AcOH 95 : 18 : 3 v/v/v); [a]D -20.9 (c 1.04
CHCl3) (lit.,12 -19.0 (c 1.0 CHCl3)); 1H-NMR (300 MHz, CDCl3)
d = 10.45 (broad s, 1H, COOH), 4.71 (m, 1H, urethane eNH),
3.91 (m, 1H, aCH), 3.13 (m, 2H, eCH2), 1.84 (m, 2H, bCH2), 1.52
(m, 4H, gCH2/dCH2 (2 ¥ 2H), 1.44 (s, 9H, (CH3)3 Boc); 13C-NMR
(75.5 MHz, CDCl3) d = 174.4, 156.3, 79.7, 61.8, 40.2, 30.9, 29.4,
28.4, 22.8.
Microwave-assisted polymerization reaction of N3-Lys-Leu-Val-
Phe-Phe-Ala-Glu-propargyl amide. Peptide monomer 1 (40 mg,
43.7 mmol) was dissolved in N2-purged DMSO (2 mL) and CuOAc
(3 mg, 24.6 mmol, 0.56 equiv.) was added. The reaction mixture
was placed in the microwave reactor and irradiated for 30 min
◦
at 100 C. The clear solution was transformed into a turbid gel.
The gel was dissolved in an additional amount of DMSO (3 mL)
and the solution was concentrated in vacuo (SpeedVac) to obtain
a solid pellet. The greenish solid was redissolved in CH3CN–H2O
(5 mL, 1 : 1 v/v) and lyophilized. After purification by HPLC, two
off-white peptides could be isolated, identified and characterized
by analytical HPLC, FTIR and MALDI-TOF analysis.33
10 (a) C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002,
67, 3057–3064; (b) V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B.
Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–2599.
Cyclic peptide (8). This peptide, the cyclic monomer, was
isolated in 15% yield (6 mg, 6.6 mmol). Rt 19.6 min; nmax/cm-1
1672 (C O), 1548; MALDI-TOF C46H65N11O9 requires 915.497,
found m/z [M + H]+monoisotopic 916.672, [M + Na]+monoisotopic 938.627.
11 (a) For a selection of general reviews, see: H. C. Kolb and K. B.
Sharpless, Drug Discovery Today, 2003, 8, 1128–1137; (b) V. D. Bock,
H. Hiemstra and J. H. van Maarseveen, Eur. J. Org. Chem., 2006, 51–68;
(c) M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108, 2952–3015;
(d) for reviews with an emphasis on polymer and materials science,
see: J.-F. Lutz, Angew. Chem., Int. Ed., 2007, 46, 1018–1025; (e) W. H.
Binder and R. Sachsenhofer, Macromol. Rapid Commun., 2007, 28, 15–
54; (f) W. H. Binder and R. Sachsenhofer, Macromol. Rapid Commun.,
2008, 29, 952–981; (g) J. A. Johnson, M. G. Finn, J. T. Koberstein and
N. J. Turro, Macromol. Rapid Commun., 2008, 29, 1052–1072; (h) B.
Le Droumaguet and K. Velonia, Macromol. Rapid Commun., 2008, 29,
1073–1089; (i) for reviews that describe the general synthetic utility of
click chemistry across the fields: H. C. Kolb, M. G. Finn and K. B.
Sharpless, Angew. Chem., Int. Ed., 2001, 40, 2004–2021; (j) M. V. Gil,
=
Cyclic peptide (9). This peptide, the cyclic dimer, was isolated
in 33% yield (13 mg, 7.1 mmol). Rt 19.1 min; nmax/cm-1 1687
=
and 1635 (C O), 1542; MALDI-TOF C92H130N22O18 requires
1830.994, found m/z [M + H]+
1831.542.
monoisotopic
Acknowledgements
These investigations were supported by the Council for Chemical
Sciences of the Netherlands Organization for Scientific Research
(CW-NWO). Hans Hilbers (Medicinal Chemistry & Chemical
Biology) is gratefully acknowledged for his help with the mod-
eling studies. Dr George Posthuma, Marc van Peski and Rene
Scriwanek (Center for Electron Microscopy, UMC Utrecht) are
acknowledged for their help with the TEM measurements and
photographic artwork.
´
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no reliable conclusions could be drawn from these measurements.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 4517–4525 | 4525
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