Synthesis and structural investigation of 2-aminomethyl-3-(4-methoxy-phenyl)-propionic acid containing a peptide analogue of the amyloidogenic AS(6-7) sequence: Inhibition of fibril formation

Article abstract of DOI:10.1039/c7ob00797c

The incorporation of a single β-amino acid moiety in a highly amyloidogenic peptide sequence resulted in the complete inhibition of amyloid fibril formation. The Boc-l-Phe-l-Leu-OMe sequence 1, which has sequence identity with the N-terminal AS(6-7) of th

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Synthesis and structural investigation of 2-aminomethyl-3-(4-methoxy-
phenyl)-propionic acid containing peptide analogue of amyloidogenic
AS(6-7) sequence: Inhibition of fibril formation†
Arpita Paikar, Mintu Debnath, Debasish Podder, Supriya Sasmal and Debasish Haldar*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
The incorporation of a single βꢀamino acid moiety in a highly amyloidogenic peptide sequence resulted in
a complete loss of amyloid fibril formation. The BocꢀLꢀPheꢀLꢀLeuꢀOMe 1 having sequence identity with
Nꢀterminal AS (6ꢀ7) of the nonꢀimmunoglobulin amyloid fibril protein AS responsible for Rheumatoid
10 Arthritis, selfꢀassociate to produce fibrils. The DꢀPhe analogue peptide 2 shows elongated ribbon like
morphology. But, 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic acid containing analogue peptide 3
exhibits polydisperse microspheres morphology. Moreover, fibrils from peptides 1 and 2 exhibit typical
greenꢀgold birefringence on Congo red (CR) binding assay and showing amyloidꢀlike morphological
resemblance. But, the 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic acid modified peptide 3 does not
15 response to this Congo red assay. From Xꢀray crystallography, the peptide 1 having LꢀPhe residue adopts
extended structure whereas the DꢀPhe analogue 2 adopts kinkꢀlike structure. Both peptides 1 and 2 show
twisted anti parallel sheetꢀlike structure at higher order assembly. However, the peptide 3 adopts nine
member hydrogen bonded δꢀturn like structure in solid state and selfꢀassociates to form loop like
supramolecular structure through multiple intermolecular hydrogen bonds. The structural analysis
20 presented here may foster new studies for de novo design and therapeutics.
45 protein AS forms amyloid fibril that may be responsible for
Rheumatoid Arthrities.7 Hence, the analysis of the physicoꢀ
chemical principles behind the amyloid formation is highly
important to prevent or cure amyloidꢀrelated disorders.8 But, the
insolubility and nonꢀcrystallinity of the amyloid plaques is the
50 main stumbling block for understanding amyloidogenesis at
atomic level. One of the processes to figure out the mechanism
of amyloid fibril formation is the study of small peptides
fragments as model systems.8h Small peptides adopted from
amyloid sequences were shown to form typical amyloid fibrils
55 that have similar ultrastructural, biophysical, and cytotoxic
properties like a full length amyloid polypeptides. Even, selfꢀ
assembly of only phenylalanine can form toxic fibrillar
aggregates.9a So, the design of model systems having sequence
identity with the amyloid protein and their analogues that provide
60 valuable knowledge about the factors responsible for structural
transition and integration of this knowledge is crucial.9b,c
Introduction
Engineering peptides with backbone modified synthetic amino
25 acids have particular importance for extending our understanding
of protein structure and stabilization into the realm of folded,
nonbiological polymers.1,2 Moreover, the selfꢀassembly of
peptides and proteins may be pathogenic by formation of amyloid
fibrils.3 The symptoms of amyloidosis are highly variable
30 depending on local amyloidosis or systemic amyloidosis.4 These
amyloid fibrils are highly stable and deposition of amyloid plaque
disrupt the tissue architecture, damage the regular tissue function
and causing disease.5 About 30 different proteins are known to
form amyloid fibrils in humans.6 Even a nonꢀimmunoglobulin
35
a Department of Chemical Sciences, Indian Institute of Science Education
and Research Kolkata, Mohanpur, West Bengal 741246, India,Fax:
+913325873020; Tel: +913325873119; E-mail: deba_h76@yahoo.com;
deba_h76@iiserkol.ac.in
Design and synthesis of βꢀamino acids is highly important
because of their presence in various natural products.10 The βꢀ
amino acid containing synthetic peptides display important
65 applications such as inhibition of viral infection11 and mimicry of
somatostatin signalling.12 Moreover, the peptides derived from
40 † Electronic Supplementary Information (ESI) available: Synthesis and
characterization of peptides, H NMR, 13C NMR, Figures ESI S1ꢀS4,
1
Figure S5ꢀS18. CCDC 1533809, 1533669, 1533670 and 1533671 or
other electronic format see DOI: 10.1039/b000000x/
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nonproteinogenic βꢀamino acids show a strong tendency to adopt
conventional solutionꢀphase methodology and purified and
well defined folded conformations.13 Conformationally 55 characterized by 1HꢀNMR, 13CꢀNMR, FTꢀIR and mass
constrained cyclic βꢀamino acid residues lead to extremely stable
βꢀpeptides.14 Particularly peptides containing βꢀamino acids are
very much important because of their immunity towards
enzymatic degradation.15
spectrometry (MS) analysis.
The morphology of the aggregates obtained from peptides
solutions in methanolꢀwater was investigated by atomic force
microscopy (AFM). A solution containing corresponding peptide
5
We are developing synthetic amino acids and analogues and 60 in methanolꢀwater (2:1) was incubated at 30°C over 5 days. A
corresponding peptides with desired functionalities.16 Recently
we have reported the synthesis of 2ꢀacetyl aminoꢀ3ꢀ[4ꢀ(2ꢀaminoꢀ
drop of that solution was drop casted on a clean microscopic
10 5ꢀsulfoꢀphenylazo)ꢀphenyl]ꢀpropionic acid as an HEWL (hen egg
white lysozyme) amyloid inhibitor starting from phenylalanine.17
Here, we wanted to investigate whether βꢀamino acid analogue of
phenylalanine can be designed with chemical modifications and
to study their intrinsic folding nature in peptide backbone. Herein
15 we report the synthesis of 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀ
propionic acid by microwave assisted Knoevenagel reaction.18
Here, we describe the results based on solidꢀstate FTꢀIR
absorption, Congo red (CR) binding assay and morphological
techniques on three terminallyꢀprotected dipeptides BocꢀDꢀPheꢀ
20 LꢀLeuꢀOMe 2 and BocꢀβꢀXaa ꢀLꢀLeuꢀOMe 3 (βꢀXaa = 2ꢀ
65
70
Scheme 2. The synthesis of 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic
acid. Reagents and condition: (a) NH4HCO2, Microwave (800 W), 30
seconds; (b) NaBH4, dry MeOH, Boc2O, NiCl4.6H2O (cat. amt.).
aminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic
acid),
the
prototype of which is BocꢀLꢀPheꢀLꢀLeuꢀOMe 1 (Scheme 1). The
single crystal Xꢀray diffraction analysis shed some light on the
atomic level structures and aggregation of the reported peptides.
cover slip and allowed to dry under vacuum at 30°C for 48 h. The
75 AFM shows that the LꢀPheꢀLꢀLeu 1 exhibits fibrillar morphology
(Fig. 1a).19 The length of the fibers is in several micrometer
ranges. The atomic force microscopic images further revealed
that the Dꢀphenylalanine modified peptide 2 shows fibrillar
morphology from the methanol−water solution (Fig. 1b). But, the
80 AFM images revealed that the 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀ
phenyl)ꢀpropionic acid modified peptide 3 exhibits polydisperse
microsphere morphology from the methanol−water solution (Fig.
1c). The 3D view (Fig. 1d) of peptide 3 microspheres shows the
surfaces of the spheres are comparatively smooth. Hence, the
85 incorporation of βꢀamino acid disrupted the fibril formation.
25
30
35 Scheme 1. The schematic presentation of peptides 1, 2 and 3.
90
Results and discussion
The LꢀPheꢀLꢀLeu motif 1 has been adopted from the Nꢀterminal
AS(6ꢀ7) of the nonꢀimmunoglobulin amyloid fibril protein AS in
Rheumatoid Arthritis.7 For peptide 2, we have replaced the Lꢀ
40 phenylalanine residue by Dꢀphenylalanine to explore the effect of
stereo isomers on the peptide folding and amyloid like fibril
95
formation. For peptide 3, we have incorporated
a
conformationally flexible 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀ
propionic acid residue to study the effect of peptide backbone
45 modification on folding as well as selfꢀassembly propensities.
The 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic acid was
synthesized by microwave assisted Knoevenagel reaction
between pꢀmethoxy benzaldehyde and ethylcyanoacetate in
presence of ammonium formate followed by reduction with
50 NaBH4 (Scheme 2). X–ray crystallography confirmed the
synthesis of the compound 5 as a racemate (ESI Figure 1 and 2).
The reported peptides BocꢀLꢀPheꢀLꢀLeuꢀOMe 1, BocꢀDꢀPheꢀLꢀ
LeuꢀOMe 2 and BocꢀβꢀXaaꢀLꢀLeuꢀOMe 3 were synthesized by
100
Fig 1. (a) AFM image of peptide 1 fibers. (b) AFM image of peptide 2
aggregates. (c) AFM image of polydisperse microspheres from peptide 3.
(d) 3D view of peptide 3 microspheres.
105 The morphologies of the peptides 1, 2 and 3 were also studied by
field emission scanning electron microscopy (FEꢀSEM). The
solutions of the reported peptides in methanol–water (0.5 mg mLꢀ
1) were dropꢀcasted on a microscopic glass cover slip, dried under
vacuum at 30°C for two days and investigated by FEꢀSEM. From
2
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FEꢀSEM, the LꢀPheꢀLꢀLeu motif 1 exhibits fiber like morphology
(Fig. 2a).20 The fibers are about 500 nm wide and several
micrometers in length. Fig. 2b shows the elongated ribbon like
greenꢀgold birefringence on Congo red staining (Fig 3b).
However, the 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic
acid modified peptides 3 does not response to this Congo red
morphology obtained from Dꢀphenylalanine modified peptide 2. 60 binding assay (Fig. 3c). Gazit and Reches have shown the role of
5
The ribbons are about 50 nm wide and several micrometers in
length like a polymer. From FEꢀSEM, the peptide 3 exhibits
polydisperse microspheres morphology (Fig. 2d). The
microsphere has diameter ca 0.5 ꢀm. The inset of Figure 2d
aromatic interactions in the selfꢀassembly and amyloid fibril
formation of BocꢀPheꢀPheꢀCOOH, ZꢀPheꢀPheꢀCOOH and Fmocꢀ
PheꢀPheꢀCOOH.23 Current study supports our hypothesis that
amyloid fibril like nanostructure formation can be modulated by
shows the smooth surface of a microsphere. In comparisons, both 65 simple chemical modification.
10 LꢀPhe and DꢀPhe containing peptides promote fibril formation
however, the replacement by a βꢀamino acid inhibit fibril
formation under same condition.
Short peptide fragments adopted from amyloid sequences
exhibits similar structural, biophysical and cytotoxic properties.9
To investigate and compare the conformational preference of
70 peptides 1, 2 and 3, solid state FTꢀIR spectroscopy was
performed. The frequency region 3500ꢀ3200 cmꢀ1 is responsible
for the N–H stretching vibrations and 1800ꢀ1500 cmꢀ1 is
corresponding to the stretching band of amide I and bending peak
of amide II.23 From FTꢀIR spectra, the bands at 3342 cmꢀ1 for
75 peptide 1 and peptide 2 indicate that NH groups are hydrogen
15
20
25
23
bonded (Fig. 4). There is no peak around 3400 cmꢀ1 for non
hydrogen bonded NH protons. The amide I and amide II bands
appeared at 1653 and 1520 cmꢀ1 for peptide 1 and 1645 cmꢀ1 and
1515 cmꢀ1 for peptide 2 indicate the kinkꢀlike structure for both
80 peptides (Fig. 4).23 This also suggests that the peptides have
extensively hydrogenꢀbonded network structures. The peak
around 1692 cmꢀ1 indicates the antiparallel arrangement. The
ester carbonyl appears at 1750 cmꢀ1. For peptide 3, the band at
3405 cmꢀ1 indicate that all NH groups are not hydrogen bonded
85 (Fig. 4). 23 The amide I and amide II bands appeared at 1700 cmꢀ1
and 1523 cmꢀ1 for peptide 3 indicate the turnꢀlike structure for the
peptide 3.
30 Fig 2. (a) FEꢀSEM image of peptide 1 showing fiber like morphology. (b) and
(c) FEꢀSEM image of peptide 2 showing entangled fiber like morphology.
(d) FEꢀSEM image of polydisperse microspheres of peptide 3. Inset shows
the image of a sphere at high magnification.
90
Moreover, the amyloidꢀlike morphological resemblance of the
35 reported peptides fibrils were studied by a Congo red (CR)
binding assay.21 The aggregated mass obtained from the
corresponding peptide in methanolꢀwater (2:1) solution were
stained by Congo red and were observed through cross polarizers.
Fig. 3a shows the typical greenꢀgold birefringence of Congo redꢀ
40 bound fibrils of LꢀPheꢀLꢀLeu 1 under cross polarizer. These
results are consistent with Congo red binding to an amyloid βꢀ
95
100
45
Fig. 4. The solid state FTꢀIR spectra of aggregates obtained from peptide 1
(black), peptide 2 (red) and peptide 3 (blue).
Fig. 3 (a) Congo red assay of peptide 1 fibrillar aggregates showing greenꢀ
50 gold birefringence. (b) The peptide 2 fibril on staining with Congo red
exhibiting greenꢀgold birefringence under cross polarizer. (c) Peptide 3
aggregates on staining with Congo red do not show greenꢀgold birefringence
under cross polarizer.
105
The solid state conformations and packing of the reported
peptides 1, 2 and 3 at atomic level were studied by Xꢀray
crystallography. Doi and coꢀworkers have reported the crystal
structure of BocꢀLꢀPheꢀDꢀLeuꢀOMe having open conformation
with parallel arrangement.24 Horvat and coꢀworkers have
sheet fibrillar framework with hydrogen bridges and hydrophobic
55 exterior.22 The optical microscopic images further revealed that
the Dꢀphenylalanine modified peptides 2 fiber exhibit typical
110 analyzed the crystal structure of BocꢀLꢀPheꢀLꢀLeuꢀOBzl showing
extended folding.25 The Xꢀray crystallography shows that the
asymmetric unit contains three molecules of peptide 1 (Fig 5a).26
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The molecule A and C of peptide 1 adopts extended structure.
The colourless monoclinic crystals of compound 3 were
But molecule B has kink like structure. The selected backbone 60 obtained from methanolꢀwater solution by slow evaporation. The
torsion angles are listed in Table 1. There is no intramolecular
hydrogen bond, rather intermolecular hydrogen bonds between
Boc C=O and LꢀPhe NH, and LꢀLeu NH and LꢀPhe C=O
(N3−H3———O12, N6−H6A———O8, N1−H1———O7, N4−H4———O3) are
asymmetric unit contains two molecules of peptide 3 as a mixture
of diastereomers (Fig. 7a).26 The solid state conformation of
compound 3 reveals that it adopts nine member week hydrogen
bonded δꢀturn27 like structure through intramolecular N–H⋯O
5
10
15
present. In higher order packing, the individual subunits of 65 hydrogen bonding interactions between 2ꢀaminomethylꢀ3ꢀ(4ꢀ
peptide 1 are themselves regularly interlinked through multiple
intermolecular hydrogenꢀbonding interactions, and thereby form
methoxyꢀphenyl)ꢀpropionic acid NH and ester CꢀO (Fig. 7a).28
The Nꢀterminal 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic
acid adopts anti conformation in both molecules A and B.29 The
important backbone torsion angles are listed in Table 1. There is
a
supramolecular twisted sheet–like structure along the
crystallographic b direction (Fig 5b). The Phe rings are separated
by hydrophobic alkyl chains and there is no π–π interaction (Fig. 70 no hydrogen bond between molecules A and B. Moreover, the
5c). Hydrogen bonding parameters for peptide 1 are also listed in
Table 2.
Colourless monoclinic crystals of compound 2 were obtained
from methanol–water solution by slow evaporation. The Xꢀray
crystallography shows that the asymmetric unit contains two
molecules of peptide 2.26 There is no solvent in the crystal. The
ORTEP diagram (Fig 6a) of peptide 2 shows that the peptide
individual turnꢀlike molecules A and B of compound 3 are
themselves regularly interꢀlinked with molecules A and B
respectively through cooperative multiple intermolecular
75
80
85
90
95
20 backbone adopts
a kinkꢀlike conformation. The important
backbone torsion angles are listed in Table 1. The two molecules
in asymmetric unit are in anti parallel arrangement. There is no
intramolecular hydrogen bond, rather two intermolecular
hydrogen bonds between DꢀPhe NH, LꢀLeu NH and DꢀPhe
25 C=O(N1−H1———O8, N2−H2———O8) that help to form a rigid dimer.
30
35
40
45
Fig. 6. (a) The ORTEP diagram of peptide 2. Ellipsoids are drawn at the 50%
probability level. (b) Side view of supramolecular twisted sheet formed by peptide 2.
The hydrogen bonds are showing as dotted line. (c) The top view of supramolecular
100 twisted sheet formed by peptide 2 showing arrangements of phenyl rings.
hydrogen bonding interactions (N1G–H1G….O19 and N1E–
H1E….O2), thereby forming a supramolecular loop like structure
along the crystallographic a direction (Fig. 7b,c). Figure 7d
shows the schematic representation of the supramolecular loop.
105 Hydrogen bonding parameters for peptides are listed in Table 2.
Fig. 5. (a) The ORTEP diagram of peptide 1. Ellipsoids are drawn at the 50%
probability level. (b) Side view of supramolecular twisted sheet formed by peptide 1.
50 The hydrogen bonds are showing as dotted line. (c) The top view of supramolecular
twisted sheet formed by peptide 1 showing arrangements of phenyl rings.
Table 1. Selected backbone torsion angles (deg) for peptide 1, peptide 2
and peptide 3
θ1
ꢀ
ꢀ
ꢀ
ꢀ
φ1/°
ψ1/°
φ2/°
ψ2/°
In higher order packing, the individual dimeric blocks of peptide
Peptide 1, A ꢀ122.72 137.10
Peptide 1, B ꢀ71.31 153.17
Peptide 1, C ꢀ141.43 141.55
Peptide 2, A 82.69 57.09
Peptide 2, B 74.17 15.86
ꢀ52.31 140.78
ꢀ56.68 41.02
ꢀ58.89 135.91
ꢀ74.74 165.76
ꢀ85.69 173.47
2
are themselves regularly interlinked through multiple
intermolecular hydrogenꢀbonding interactions, N3−H3———O2,
55 N4−H4———O3 and thereby form a supramolecular twisted sheetꢀ
like structure (Fig. 6b) along the crystallographic b direction
where the Phe rings are separated by alkyl chains and there is no
π–π stacking interaction (Fig. 6c).
ꢀ
Peptide 3, LL 91.26 ꢀ132.91 55.40 ꢀ71.42 ꢀ10.85
Peptide 3, DL ꢀ84.88 126.96 ꢀ49.30 54.05
36.42
4
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peptide 3 is a mixture of diastereomers, the CD experiment has
45 not performed. Moreover, a comparison of the spectra obtained
by Xꢀray diffraction data of the fibers (ESI Figure S4a) and the
powder pattern from Xꢀray crystallography of a single crystal of
peptide 2 (ESI Figure S4b) clearly exhibits the existence of the
same structure in both fibril and crystal. We have also performed
50 the Xꢀray diffraction experiment of the spheres of peptide 3
obtained from methanolꢀwater solution. The comparison of the
Xꢀray diffraction spectra of the peptide 3 spheres (ESI Figure
S4c) and the powder pattern obtained from single crystal Xꢀray of
peptide 3 (ESI Figure S4d) shows the same structure in both
55 form.
5
10
15
20
Conclusions
In conclusion, we have designed and synthesized a new βꢀamino
acid namely 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀpropionic
acid by microwave assisted Knoevenagel condensation reaction.
60 We have also incorporated the 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀ
phenyl)ꢀpropionic acid in peptide backbone and compared the
folding and selfꢀassembly propensities of peptide with its
phenylalanine analogues. FEꢀSEM images show that the
phenylalanine analogue peptides 1 and 2 exhibit fibers like
Fig. 7. (a) The ORTEP diagram of peptide 3. Ellipsoids are drawn at the 50%
probability level. (b) Side view of supramolecular loopꢀlike structure formed by
peptide 3. The hydrogen bonds are showing as dotted line. (c) The top view of
supramolecular loopꢀlike structure. (d) The schematic representation of the
65 morphology.
But, 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀ
propionic acid containing analogue 3 exhibits polydisperse
microspheres morphology. The fibrils from peptides 1 and 2
exhibit typical greenꢀgold birefringence on Congo red (CR)
binding assay, but the peptide 3 does not response. The peptide 1
70 and 2 having LꢀPhe and DꢀPhe residues respectively form
supramolecular twisted sheetꢀlike structures in solid state. The Xꢀ
ray crystallography reveals that the peptide 3 exhibits nine
member hydrogen bonded δꢀturn like unit that selfꢀassociates to
form supramolecular loopꢀlike structure. The results presented
75 here shows that there are still many challenges to develop
amyloid inhibitor and therapeutics.
25 supramolecular loop.
Table 2. Hydrogen bonding parameters of peptides 1, 2 and 3a
DꢀH….A
D..H(Å)
H..A(Å)
D..A(Å)
DꢀH..A (°)
1
N1ꢀH1….O7
N4ꢀH4….O3
N6ꢀH6A….O8
N3ꢀH3…O12
N2ꢀH2….O13
N5ꢀH5…O2
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
2.08
2.02
2.00
2.38
2.12
2.00
2.14
2.13
2.11
2.13
2.16
2.27
2.13
2.34
3.10
3.14
2.855(5)
2.862(5)
2.852(6)
2.993(5)
2.913(5)
2.852(6)
2.831(4)
2.981(4)
2.968(4)
2.893(4)
2.961(3)
3.072(8)
2.989(3)
3.168(3)
3.538(3)
3.518(3)
149
165
170
128
153
128
170
168
2
3
N1ꢀH1…O8
N2ꢀH2….O8
N3ꢀH3…O2
173 a
148a
153b
155c
171d
160e
114
N4ꢀH4…O3
Experimental
N1EꢀH1E….O2
N1FꢀH1F….O1
N1GꢀH1G...O19
N6ꢀH6…O
General
All Lꢀamino acids and DꢀPhe were purchased from Sigma
80 chemicals.
HOBt
(1ꢀhydroxybenzotriazole)
and
DCC
N1eꢀH1e…O4
N6ꢀH6…O18
109e
(dicyclohexylcarbodiimide) were purchased from SRL.
Peptide Synthesis
aSymmetry equivalent a = 1ꢀx, ½+y, 1ꢀz, b = ½+x, 1/2+y, z, c =
½+x, ꢀ1/2+y, z, d = ꢀ1ꢀx, y, ꢀ1ꢀz, e = ꢀ1ꢀx, y, ꢀ2ꢀz.
The Boc protected 2ꢀaminomethylꢀ3ꢀ(4ꢀmethoxyꢀphenyl)ꢀ
propionic acid methyl ester (compound 5) was synthesized by
85 microwave assisted Knoevenagel reaction between pꢀmethoxy
benzaldehyde and ethylcyanoacetate in presence of ammonium
formate followed by reduction with NaBH4. The peptides were
synthesized by conventional solutionꢀphase methods using
racemization free fragment condensation strategy. The Boc group
90 was used for Nꢀterminal protection, and the Cꢀterminus was
30
The comparison of the crystal structures of peptides 1, 2, and 3
clearly demonstrates the driving forces responsible for different
structures of the three sequences. The DꢀPhe introduced positive
35 dihedral (φ1 and ψ1) in peptide 2 and the peptide fold
accordingly. The introduction of βꢀamino acids in peptide 3
molecule may induce deviation from the usual folding and the
backbone adopts an overall molecular planarity, which is a
necessary condition for δꢀturn formation.
40 The specific structural properties of the fibrils of peptides 1 and 2
were examined by circular dichroism spectroscopy. ESI Figure
S3 shows the positive Cotton effect observed in peptide 1 fibril,
completely inverse by incorporation of DꢀPhe in peptide 2. As
protected as
a
methyl ester. Coupling was mediated by
benzotriazole
dicyclohexylcarbodiimide/1ꢀhydroxyl
(DCC/HOBt). The products were purified by column
chromatography using silica (100−200 mesh size) gel as a
95 stationary phase and an nꢀhexane-ethyl acetate mixture as an
eluent. The intermediates and final compounds were fully
1
characterized by 500 MHz and 400 MHz H NMR spectroscopy,
125 MHz 13C NMR spectroscopy, FTꢀIR spectroscopy and mass
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spectrometry. The compound 5 is a racemate and the peptide 3 is
a mixture of diastereomers. The peptides 1, 2 and 3 and
compound 5 were characterized by Xꢀray crystallography.
NMR Experiments
All NMR studies were carried out on a Brüker AVANCE 500
MHz and Jeol 400 MHz spectrometer at 278 K. Compound
concentrations were in the range 1–10 mM in CDCl3 and
(CD3)2SO.
CSIR, India for research fellowship. S. Sasmal acknowledges
IISERꢀKolkata for fellowship.
60 Notes and references
1 (a) Foldamers: Structure, Properties, and Applications, Editors S. Hecht
and I. Huc, WileyꢀVCH Verlag GmbH, 2007; (b) Highlights in
Bioorganic Chemistry: Methods and Applications Editors C.
Schmuck and H. Wennemers, WileyꢀVCH Verlag GmbH, 2004.
65 2 (a) S. H. Gellman, Acc. Chem. Res. 1998, 31, 173ꢀ180; (b) D. J. Hill, M.
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FT-IR Spectroscopy
10 All reported solidꢀstate FTꢀIR spectra were obtained with a
Perkin Elmer Spectrum RX1 spectrophotometer with the KBr
disk technique.
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Biomol. Chem. 2013, 11, 219ꢀ232; (f) D. Mandal, A. N. Shirazi and
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Mass Spectrometry
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Mass spectra were recorded on a QꢀTof Micro YA263 highꢀ
15 resolution (Waters Corporation) mass spectrometer by positiveꢀ
mode electrospray ionization.
75
UV/Vis spectroscopy
Absorption spectra were recorded on
spectrophotometer.
a
Perkin Elmer
80
20 Atomic force microscopy
The morphology of the reported compound was investigated by
atomic force microscopy (AFM). A drop of the sample solution
in DCM were placed on a clean microscope cover glass and then
dried by slow evaporation. The material was then allowed to dry
25 under vacuum at 30°C for two days. Images were taken with an
NTMDT instrument, model no. APꢀ0100 by semicontactꢀmode.
Field Emission Scanning Electron Microscopy
Morphologies of the reported peptides were investigated using
field emissionꢀscanning electron microscopy (FEꢀSEM). A small
30 amount of solution of the peptide was placed on a clean glass
cover slip and then dried by slow evaporation. The material was
then allowed to dry under vacuum at 30°C for two days. The
materials were goldꢀcoated, and the micrographs were taken in an
FEꢀSEM apparatus (Jeol Scanning MicroscopeꢀJSMꢀ6700F).
35 Congo red assay
An alkaline saturated Congo red solution was prepared. The dried
peptide aggregates from methanol–water were stained by alkaline
Congo red solution (80% methanol/20% glass distilled water
containing 10 ml of 1% NaOH) for 2 minutes and then the excess
40 stain (Congo red) was removed by rinsing the stained aggregates
with 80% methanol/20% glass distilled water solution for several
times. The stained aggregates were dried under vacuum at room
temperature for 24 h, then visualized at 40× magnification and
birefringence was observed between crossed polarizers (Olympus
45 optical microscope equipped with polarizer and CCD camera).
Single crystal X-ray diffraction study
Intensity data of peptides 1, 2 and 3 were collected with MoKα
radiation using Bruker APEXꢀ2 CCD diffractometer. Data were
processed using the Bruker SAINT package and the structure
50 solution and refinement procedures were performed using
SHELX97. CCDC 1533809, 1533669, 1533670 and 1533671
contain the supplementary crystallographic data for 1, 2, 3 and 5
respectively.
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Acknowledgements
55 We acknowledge the CSIR, India, for financial assistance
(Project No. 02(0206)/14/EMRꢀII). A. Paikar acknowledges the
UGC, India for fellowship. M. Debnath and D. Podder thank
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26 Crystallographic data of Peptide 1: C21H32N2O5, Mw = 392.49,
orthorhombic, space group P 65, a = 25.1584(8), b = 25.1584(8), c =
24.8801(7) Å, α = β = 90°, γ = 120°, V = 13637.9(9) Å3, Z = 18, dm
= 0.860 Mgmꢀ3, T = 100 K, R1 0.0586 and wR2 0.1571 for 15969
data with I> 2σ(I). Peptide 2: C21H32N2O5, Mw = 392.49,
monoclinic, space group P 1 21 1, a = 10.8333(4), b = 18.5688(6), c
= 11.5795(4) Å, α = 90°, β = 104.444 (4), γ = 90°, V = 2255.72(14)
Å3, Z = 4, dm = 1.156 Mgmꢀ3, T = 100 K, R1 0.0410 and wR2
0.1115 for 6142 data with I> 2σ(I). Peptide 3: C23H36N2O6, Mw =
436.55, monoclinic, space group C 1 2 1, a = 18.8735(10), b =
9.5367(4), c = 27.4383(17) Å, α = 90°, β = 104.585 (6), γ = 90°, V =
4779.5(5) Å3, Z = 8, dm = 1.213 Mgmꢀ3, T = 100 K, R1 0.0461 and
wR2 0.1598 for 6507 data with I> 2σ(I). Compound 5: C17H25NO5,
Mw = 323.38, monoclinic, space group P 1 21/c 1, a = 19.4462(7), b
= 10.2917(4), c = 9.0894(2) Å, α = 90°, β = 99.477 (3), γ = 90°, V =
1794.28(10) Å3, Z = 4, dm = 1.197 Mgmꢀ3, T = 293 K, R1 0.0522
and wR2 0.1232 for 4059 data with I> 2σ(I). Intensity data were
collected with MoKα radiation using Bruker APEXꢀ2 CCD
diffractometer. Data were processed using the Bruker SAINT
package and the structure solution and refinement procedures were
performed using SHELX97.30 The data for compounds 1, 2, 3 and 5
have been deposited at the Cambridge Crystallographic Data Centre
with reference number CCDC 1533809, 1533669, 1533670 and
1533671 respectively.
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TOC Graphic
Synthesis and structural investigation of 2-aminomethyl-3-(4-methoxy-
phenyl)-propionic acid containing peptide analogue of amyloidogenic AS(6-7)
sequence: Inhibition of fibril formation
Arpita Paikar, Mintu Debnath, Debasish Podder, Supriya Sasmal and Debasish Haldar
The incorporation of a β-amino acid namely 2-aminomethyl-3-(4-methoxy-phenyl)-propionic
acid inhibits amyloid-like fibril formation.