Studies of Amyloid-like fibrillogenesis through β-sheet-mediated self-assembly of short synthetic peptides

Article abstract of DOI:10.1002/cbdv.200900050

Three structurally different types of small peptides, namely, i) Boc-Ile-Aib-Ile-OMe (Aib = α-aminoisobutyric acid), ii) Boc-Xx-m-aminobenzoic acid (Xx = β-Ala and γ-aminobutyric acid), and iii) Boc-Xx-m-nitroaniline, were found to exhibit β-sheet-mediated fibrillogenesis in the solid state, revealed by FT-IR, single-crystal X-ray diffraction, and FE-SEM studies. Interestingly, the fibrils grown from peptides 2 and 3 were found to bind with the physiological dye Congo red, a characteristic feature of amyloid fibrils.

Full text of DOI:10.1002/cbdv.200900050

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Studies of Amyloid-Like Fibrillogenesis through b-Sheet-Mediated
Self-Assembly of Short Synthetic Peptides
by Anita Dutta), Elinor C. Spencerb), Judith A. K. Howardb), and Animesh Pramanik*a)
a) Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata-700009, India
(phone: þ91-33-24841647; fax: þ91-33-23519755; e-mail: animesh_in2001@yahoo.co.in)
b) Department of Chemistry, University of Durham, South Road, Durham, UK-DH1 3LE
Three structurally different types of small peptides, namely, i) Boc-Ile-Aib-Ile-OMe (Aib¼a-
aminoisobutyric acid), ii) Boc-Xx-m-aminobenzoic acid (Xx¼b-Ala and g-aminobutyric acid), and iii)
Boc-Xx-m-nitroaniline, were found to exhibit b-sheet-mediated fibrillogenesis in the solid state, revealed
by FT-IR, single-crystal X-ray diffraction, and FE-SEM studies. Interestingly, the fibrils grown from
peptides 2 and 3 were found to bind with the physiological dye Congo red, a characteristic feature of
amyloid fibrils.
Introduction. – Currently, considerable research has been directed towards
designing and constructing suitable peptide subunits, which can self-assemble into
supramolecular helices [1–3] and b-sheets [4–10]. Peptide-based supramolecular b-
sheets are important for their numerous potential applications in material [11][12] and
biological sciences [13][14]. They are also utilized in fabricating nano-biomaterials
[15–21]. Sometimes, self-aggregating, b-sheet-forming peptides create fibrous net-
works that can trap solvent molecules to form gels [22]. Under suitable conditions, they
can also provide molecular scaffolds for growing neurons and cartilage [23][24]. It has
been established that b-sheet-driven aggregation of misfolded proteins is responsible
for various neurodegenerative diseases such as Alzheimerꢀs disease [25–28], Parkin-
sonꢀs disease [29][30], and prion-related diseases [31][32].
The design and construction of supramolecular b-sheets is important for a better
understanding of the peptide self-aggregation mechanism. Therefore, in this study, we
have chosen three different types of peptides to explore the self-assembly mechanism in
the solid state (Fig. 1). The tripeptide Boc-Ile-Aib-Ile-OMe (1), which incorporates the
helicogenic Aib (a-aminoisobutyric acid), was expected to adopt a bent structural self-
assembling subunit. In peptides 2, Boc-b-Ala-m-ABA (m-ABA¼m-aminobenzoic
acid), and 3, Boc-g-Abu-m-ABA (g-Abu¼g-aminobutyric acid), the w-amino acids b-
Ala and g-Abu, known for adopting extended conformations [4][33], were attached to
m-ABA, a template molecule to create a b-sheet-like structure through intermolecular
H-bonding. In peptides 4, Boc-b-Ala-m-NA (m-NA¼m-nitroaniline), and 5, Boc-g-
Abu-m-NA, m-ABA of peptides 2 and 3 was replaced by m-NA, to explore the
influence of the NO2 group on the self-assembling process. All the peptides were
synthesized by conventional solution-phase methodology, and their structures were
determined by single-crystal X-ray diffraction studies. The morphological studies of the
ꢁ 2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

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dried fibrous materials generated from 1–5 were carried out with field-emission
scanning electron microscopy (FE-SEM).
Fig. 1. Schematic representation of peptides 1–5
Results and Discussion. – Solid-State FT-IR Studies. Solid state FT-IR is a useful tool
for obtaining preliminary information about the peptide conformation. In the solid
state (KBr matrix), the peptides 1–5 showed intense bands at 3326–3387 cmꢀ1
,
indicating the presence of strongly H-bonded NH groups (Fig. 2). Characteristic IR
data for all the peptides are listed in Table 1. The absence of a band attributable to free
NH groups (over 3430 cmꢀ1) indicated that all the NH groups in 1–5 were involved in
intermolecular H-bonding. The CO stretching band at ca. 1667–1696 cmꢀ1 (amide I),
and the NH bending peak near 1535 cmꢀ1 (amide II) suggested the presence of
intermolecular H-bonded supramolecular b-sheet-like conformations for all the
peptides in the solid state (Fig. 2) [34–36]. Therefore, from the solid-state FT-IR
data, it can be concluded that all the peptides formed intermolecular H-bonded b-
sheet-like structures. To obtain detailed information about the intermolecular H-
bonding networks and self-assembly mechanisms, single-crystal X-ray diffraction
studies were carried out.
Table 1. Characteristic Frequences [cmꢀ1] of IR Absorption Spectra Obtained from Peptides 1–5 in the
Solid State (KBr pellets; s, strong signal; m, medium signal)
Peptide
CO stretch
NH bend
NH stretch
Boc-Ile-Aib-Ile-OMe (1)
Boc-b-Ala-m-ABA (2)
Boc-g-Abu-m-ABA (3)
Boc-b-Ala-m-NA (4)
Boc-g-Abu-m-NA (5)
1667 (s)
1689 (s)
1687 (s)
1696 (s)
1680 (s)
1523 (s)
1535 (m)
1537 (m)
1546 (m)
1532 (s)
3387 (m), 3320 (s)
3343 (s)
3326 (s)
3326 (s)
3357 (s)
Single-Crystal X-Ray Diffraction Studies. The colorless, orthorhombic crystals of
peptide 1 were obtained from an acetone/H2O mixture by slow evaporation. The
molecule crystallized in space group P212121 with one molecule in the asymmetric unit.
A diagram of 1 is presented in Fig. 3. The incorporation of helicogenic Aib created a

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Fig. 2. FT-IR Spectra of peptides 1–5 in the regions 3000–4000 and 1000–2000 cmꢀ1
bent structure, where the backbone torsion angles (f, y) were: within Ile(1) f1,
ꢀ123.7(2)8 and y1, 165.7(2)8; within Aib(2) f2, 57.2(3)8 and y2, 38.4(2)8; and within
Ile(3) f3, ꢀ124.2(2)8 and y3, 163.4(2)8 (Table 2). The structure corresponded to a
distorted type II b-turn conformation with Ile(1) and Aib(2) occupying the iþ1 and iþ
2 positions, respectively. Due to large deviations of the backbone torsion angles (f, y)
from the ideal values for a type II b-turn conformation (f1, ꢀ608 and y1, 1208; f2, 808

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Fig. 3. ORTEP Diagram of peptide 1 with atom-numbering scheme. Thermal ellipsoids are drawn at the
25% probability level.
Table 2. Selected Backbone Torsion Angles [8]
Residues
f1
y1
q1
f2
y2
f3
y3
Peptide 1
Peptide 2
2A
2B
2C
ꢀ123.7(2)
165.7(2)
57.2(3)
38.4(2)
ꢀ124.2(2)
163.4(2)
86.1(6)
ꢀ112.5(5)
ꢀ80.5(6)
ꢀ136.4(2)
81.4(5)
156.9(4)
ꢀ86.2(5)
ꢀ101.3(5)
140.1(2)
ꢀ164.8(4)
ꢀ157.4(4)
173.4(2)
Peptide 4
and y2, 08), the turn conformation was unable to permit the formation of any
intramolecular H-bonds.
The bent structure of peptide 1 molecules were regularly inter-linked via
intermolecular H-bonds between the Ile(1) NH moiety of one molecule and the
Ile(3) C¼O group of another molecule (N1ꢀH1· · ·O5, 2.18 ꢃ) to create a semi-
cylindrical structure parallel to the crystallographic a-axis (Fig. 4 and Table 3). It was
also observed that the Aib(2) NH group of each turn was further H-bonded to an
Aib(2) C¼O group of a neighboring turn (N2ꢀH2· · ·O4, 2.11 ꢃ) along the crystallo-
graphic b-axis. As a result, the parallel semi-cylindrical structures were connected to
produce a corrugated b-sheet structure along the crystallographic b-axis (Figs. 4 and 5).
The colorless, triclinic crystals of peptide 2 were grown from an acetone/toluene
mixture. The space group is P1 with three molecules in the asymmetric unit. A diagram
of one of the isomers of 2 is shown in Fig. 6. All three molecules adopted extended
conformations with similar backbone torsion angles at b-Ala, e.g., in 2A: f1, 86.1(6)8;
q1, 156.9(4)8; y1, 81.4(5)8 (Table 2). The molecules were self-assembled through
intermolecular H-bonds, to create parallel layers of supramolecular b-sheets (Fig. 7).
In each layer, the molecules were arranged in an antiparallel fashion, so that the m-

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Fig. 4. Packing diagrams of peptide 1 showing a) the formation of semi-cylindrical structures parallel to
the crystallographic a-axis and b) the self-association of turns parallel to the crystallographic b-axis,
forming a layer of b-sheet. Side chains of amino acids and H-atoms are omitted for clarity.
Table 3. H-Bonding Parameters for Peptides 1, 2, and 4
DꢀH···A
H···A [ꢃ]
D···A [ꢃ]
DꢀH···A [8]
Peptide 1
N1ꢀH1···O5i
N2ꢀH2···O4ii
Peptide 2
2.18
2.11
3.032(2)
2.949(2)
164.31
159.40
N1ꢀH1···O1iii
N2ꢀH2···O13iv
O2ꢀH2···O4iii
N5ꢀH5···O6v
N6ꢀH6···O3vi
O11ꢀH11···O9vii
N3ꢀH3···O12viii
N4ꢀH4···O8ix
O7ꢀH7···O14viii
Peptide 4
2.03
1.98
1.83
2.03
1.98
1.83
2.13
2.00
1.81
2.908(6)
2.854(5)
2.620(5)
2.898(6)
2.854(5)
2.626(5)
2.941(5)
2.874(5)
2.609(5)
173.71
175.02
155.73
168.24
169.60
157.50
152.60
172.50
159.30
N2ꢀH2···O3i
2.11
2.11
2.964(2)
2.956(2)
162.30
160.40
N3ꢀH3···O4i
Symmetry codes: (i) xꢀ1, y, z; (ii) 1ꢀx, yꢀ1/2, 1/2ꢀz; (iii) 2ꢀx, ꢀy, 2ꢀz; (iv) 1þx, y, z; (v) xꢀ1, 1 þ
y, z; (vi) xꢀ1, y, z; (vii) xꢀ1, yþ1, z; (viii) 1þx, yꢀ1, z; (ix) 2ꢀx, 1ꢀy, 1ꢀz.
ABA units of two neighboring molecules could recognize each other through
intermolecular H-bonds (Fig. 7 and Table 3). The arrangement was further stabilized
by two additional intermolecular H-bonds between the carboxylic OH and the Boc
C¼O groups, to create a molecular duplex. These duplexes were further inter-linked

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Fig. 5. Packing diagram of peptide 1 showing the formation of corrugated b-sheets parallel to the
crystallographic b-axis. Side chains of amino acids are omitted for clarity. The dotted lines indicate H-
bonding.
through two intermolecular H-bonds between the b-Ala C¼O and the b-Ala NH
groups to create a layer of b-sheets (Fig. 7). Several such parallel layers were stacked
one on top of the other and linked through Van der Waals interactions (Fig. 8). We
were unable to grow single crystals of Boc-g-Abu-m-ABA (3). However, since both 2
and 3 are structurally similar, the latter is expected to produce a b-sheet-like structure
as observed in the solid state structure of 2. This hypothesis is supported by the FT-IR
data (Table 1 and Fig. 2).
Fig. 6. ORTEP Diagram of peptide 2 with atom-numbering scheme. Thermal ellipsoids are drawn at the
25% probability level.
The colorless, monoclinic crystals of peptide 4 were obtained from a MeOH/H2O
mixture by slow evaporation. The space group was P21 with one molecule in the
asymmetric unit. The crystal structure of 4 (Fig. 9), where the m-ABA of 2 has been
replaced by m-NA, showed an infinite ribbon-like b-sheet structure through
intermolecular H-bonding (Fig. 10). The backbone torsion angles, which characterize
the extended conformation, were f1, ꢀ136.4(2)8; q1, 173.4(2)8; and y1, 140.1(2)8 at b-
Ala (Table 2). The incorporation of b-Ala helped to attain a fully extended
conformation, which was necessary for b-sheet formation. There were two intermo-
lecular H-bonds between N2 and O3 (xꢀ1, y, z) and between N3 and O4 (xꢀ1, y, z)
with donor–accepter distances of 2.964 and 2.956 ꢃ, respectively (Table 3). These H-
bonds resulted in the formation of a 14-membered ring that connects neighboring

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Fig. 7. Packing diagram of peptide 2 showing the formation of a b-sheet layer through the antiparallel self-
assembly of peptides. The dotted lines indicate H-bonding.
Fig. 8. Packing diagram of peptide 2 showing the stacking of parallel layers of b-sheets through Van der
Waals interactions
Fig. 9. ORTEP Diagram of peptide 4 with atom-numbering scheme. Thermal ellipsoids are drawn at the
25% probability level.
molecules. A parallel b-pleated sheet parallel to the crystallographic a-axis was thus
formed with the strands running in the same direction (Fig. 10). It is interesting that the
space group was P21 with just one molecule in the unit cell, which facilitated this
packing formation. The molecular arrangement was further stabilized by p–p
interactions between the phenyl rings. It is a well-documented fact that p–p
interactions have a significant role to play in amyloid aggregation [37]. The peptide
5, Boc-g-Abu-m-NA, where the b-Ala of 4 was replaced by g-Abu, was expected to

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Fig. 10. Packing diagram of peptide 4 showing the formation of a ribbon-like b-sheet through H-bonding
and p–p interactions
adopt a similar ribbon-like b-sheet structure in the solid state like that of 4. Although it
was not possible to grow single crystals of 5, the FT-IR data indicated a b-sheet-like
structure in the solid state (Table 1). These studies clearly demonstrate that, although
the self-assembling patterns for peptides 1, 2, and 4 in the solid state are significantly
different, all of them form layers of b-sheets through different kinds of two-dimensional
H-bonding networks.
Scanning Electron-Microscopic Study. Field-emission scanning electron-micro-
scopic (FE-SEM) images of the dried fibrous materials of peptides 1–5, grown slowly
from acetone, clearly demonstrated that the aggregates in the solid state were bunches
of fibrillar structures (Fig. 11). Peptides 2 and 3, with free COOH groups, showed
excellent fibril-forming properties. Peptides 4 and 5, having NO2 in place of COOH
groups, could also produce fibrillar structures. Interestingly, the peptide fibrils
generated from 2 and 3 exhibited an amyloid-like behavior, as they bind to the
physiological dye Congo red (Fig. 12). These results demonstrate that supramolecular
b-sheet forming small peptides with diverse functionalities could promote fibrillar
structures in the solid state.
Conclusions. – Solid-state FT-IR data for all peptides revealed that they self-
assemble to form intermolecular H-bonded supramolecular b-sheet structures. Single
crystal X-ray diffraction studies showed that although peptides 1, 2, and 4 are
structurally different, all of them formed b-sheet-like structures through molecular self-
assembly. While the bent structure of 1 self-assembled into a b-sheet-like structure, the
extended structure of 2 formed a b-sheet layer through the self-association of
molecular duplexes. The preferred structure of 4 was an infinite ribbon-like b-sheet

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Fig. 11. FE-SEM Images of the fibrous materials of peptides 1–5 showing the formation of the fibrillar
structures
Fig. 12. Light microscope images (ꢁ1000 magnification) of Congo red stained fibrils of a) peptide 2 and
b) peptide 3, a characteristic feature of amyloid fibrils
structure through molecular self-assembly. SEM studies revealed that the peptides 1–5
could form fibrillar structures in the solid state. The fibrils grown from 2 and 3 were
found to bind with the physiological dye Congo red, a characteristic feature of amyloid
fibrils. The insertion of w-amino acids, such as b-Ala and g-Abu, into the peptide
backbone not only helped to form H-bonded supramolecular b-sheet structures, but
also provided proteolytic resistance by replacing regular peptide bonds with CꢀC
bonds. The investigation of the pathway(s) of amyloid-fibril formation has a major role
in therapeutics of the amyloid diseases. In this context, developing easily modifiable
molecular systems that will self-assemble to amyloid-like fibrils is very important. The
amyloid-like fibrils generated from the small peptides 1–5 may serve as screening tools
in the search for anti-neurodegenerative drugs acting as inhibitors of misfolded protein
aggregation.

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The financial assistance of the UGC, New Delhi is acknowledged (Major Research Project, No. 32-
190/2006 (SR)). A. D. thanks the UGC, New Delhi, India, for providing her a senior research fellowship,
and E. C. S. thanks the EPSRC for funding.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2; 100–200 mesh; Spectrochem, India). IR
Spectra: Perkin Elmer 782 FT-IR spectrometer; ˜n in cmꢀ1. 1H- and 13C-NMR Spectra: Bruker Avance 300
NMR spectrometer, at 300 and 75 MHz, resp.; d in ppm, J in Hz; peptide concentrations 1–10 and 30–
40 mm for 1H- and 13C-NMR, resp.
Peptide Synthesis. Peptides 1–5 were synthesized by conventional soln.-phase procedures [38]. Boc
and Me ester groups were used to protect the NH2 and COOH groups, resp., and dicyclohexylcarbo-
diimide (DCC) and 1-hydroxybenzotriazole (HOBT) were employed as coupling agents. Methyl ester
hydrochlorides of Aib, Ile, and m-ABAwere prepared by the SOCl2-MeOH procedure. The purities of all
intermediates obtained were checked by TLC on silica gel (SiO2) and used without further purification.
The final peptide products were purified by CC (SiO2; AcOEt/petroleum ether (PE)). Peptides 1–5
were fully characterized by IR, 1H- and 13C-NMR. Moreover, the structures of peptides 1, 2, and 4 were
confirmed by X-ray diffraction analysis.
Boc-Ile-Aib-Ile-OMe (1). Boc-Ile-Aib-OH [6] (1.0 g, 3.16 mmol) was dissolved in DMF (3 ml). Ile-
OMe (0.46 g, 3.16 mmol), obtained from the corresponding HCl salt, was added, followed by the addition
of DCC (0.65 g, 3.16 mmol). The mixture was stirred at r.t. for 5 d. The precipitated dicyclohexylurea
(DCU) was filtered and diluted with AcOEt. The org. layer was washed with an excess of H2O, 1m HCl
(3ꢁ30 ml), 1m Na2CO3 soln. (3ꢁ30 ml), and then again with H2O. The filtrate was then dried (Na2SO4)
and evaporated in vacuo to give a light yellow solid. Purification was performed by CC (SiO; AcOEt/
PE). Single crystals of 1 were grown from acetone/H2O 9 :1 by slow evaporation and were stable at r.t.
1
(1.29 g, 92%). M.p. 77–788. IR (KBr): 1523, 1667, 1712, 3320, 3387. H-NMR (CDCl3): 0.90–0.97 (m,
12 H, Me(g), Me(d) of Ile); 1.14–1.22 (m, 4 H, CH2(g) of Ile); 1.46 (s, 3 Me of Boc); 1.59 (s, 2 Me(b) of
Aib); 1.86–1.92 (m, 2 H, HꢀC(b) of Ile); 3.75 (s, MeO); 3.87–3.90 (m, HꢀC(a) of Ile(1)); 4.53–4.62
(m, HꢀC(a) of Ile(3)); 5.02 (d, J¼6.9, NH of Ile(1)); 6.55 (s, NH of Aib); 7.10 (d, J¼8.4, NH of Ile(3)).
13C-NMR (CDCl3): 11.3; 11.5; 15.4; 15.5; 24.7; 24.8; 25.1; 25.5; 28.2; 36.9; 37.7; 51.9; 56.7; 57.5; 59.8; 80.2;
155.8; 171.5; 172.2; 173.8. Anal. calc. for C22H41N3O6 (443.57): C 59.57, H 9.32, N 9.48; found: C 59.52, H
9.28, N 9.52.
Boc-b-Ala-m-ABA (2). Boc-b-Ala-m-ABA-OMe [4] (1.0 g, 3.10 mmol) was dissolved in MeOH
(15 ml), and 2m NaOH (10 ml) was then added. The mixture was stirred at r.t. for 2 d. The progress of the
reaction was monitored by TLC. After completion of the reaction, MeOH was evaporated. The residue
obtained was diluted with H2O and washed with Et2O. The aq. layer was neutralized with 2m HCl and
then extracted with AcOEt. The solvent was evaporated in vacuo to give a light yellow solid. Purification
was performed by CC (SiO; AcOEt/PE). Single crystals of 2 were grown by slow evaporation from
acetone/toluene and were stable at r.t. (0.76 g, 80%). M.p. 182–1848. IR (KBr): 1535, 1689, 3200, 3343.
1H-NMR ((D6)DMSO): 1.33 (s, 3 Me of Boc); 2.44 (t, J ¼ 6.9, CH2(b) of b-Ala); 3.19 (q, J ¼ 6.6, CH2(a)
of b-Ala); 6.84–6.91 (m, NH of b-Ala); 7.37 (t, J¼7.8, HꢀC(5) of m-ABA); 7.57 (d, J¼7.5, HꢀC(4) of
m-ABA); 7.76 (d, J¼7.8, HꢀC(6) of m-ABA); 8.20 (s, HꢀC(2) of m-ABA); 10.01 (s, NH of m-ABA).
13C-NMR ((D6)DMSO): 28.7; 36.9; 37.3; 78.1; 123.7; 124.4; 129.3; 131.7; 139.8; 156.0; 167.7; 170.1. Anal.
calc. for C15H20N2O5 (308.32): C 58.42, H 6.53, N 9.08; found: C 58.30, H 6.39, N 8.88.
Boc-g-Abu-m-ABA (3). From Boc-g-Abu-m-ABA-OMe, 3 was synthesized following a similar
1
procedure to that of 2. Yield: 0.94 g (82%). M.p. 172–1748. H-NMR ((D6)DMSO): 1.23 (s, 3 Me of
Boc); 1.51–1.60 (m, CH2(b) of g-Abu); 2.17 (t, J ¼ 7.2, CH2(a) of g-Abu); 2.82 (q, J ¼ 6.6, CH2(g) of g-
Abu); 6.70–6.80 (m, NH of g-Abu); 7.27 (t, J¼7.8, HꢀC(5) of m-ABA); 7.47 (d, J¼7.5, HꢀC(4) of m-
ABA); 7.67 (d, J¼7.7, H ꢀC(6) of m-ABA); 8.09 (s, HꢀC(2) of m-ABA); 9.95 (s, NH of m-ABA).
13C-NMR ((D6)DMSO): 25.9; 28.7; 34.3; 39.9; 77.9; 120.3; 123.6; 124.3; 129.4; 131.7; 139.9; 156.1; 167.7;
171.7. Anal. calc. for C16H22N2O5 (322.35): C 59.61, H 6.87, N 8.69; found: C 59.75, H 6.99, N 8.85.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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Boc-b-Ala-m-NA (4). Boc-b-Ala-OH (0.95 g, 5 mmol) was dissolved in DMF (3 ml), and m-
nitroaniline (m-NA; 0.68 g, 5 mmol) was added, followed by DCC (1.0 g, 5 mmol). The mixture was
stirred at r.t. for 3 d. The precipitated DCU was filtered and diluted with AcOEt (40 ml). The org. layer
was washed with an excess of H2O, 1m HCl (3ꢁ30 ml), 1m Na2CO3 soln. (3ꢁ30 ml), and then again with
H2O. The solvent was then dried (anh. Na2SO4) and evaporated in vacuo, giving 4 as a brown solid (1.32 g,
85%). Single crystals were grown by slow evaporation from MeOH/H2O and were stable at r.t. M.p. 155–
1568. IR (KBr): 1546, 1696, 3326. 1H-NMR (CDCl3): 1.44 (s, 3 Me of Boc); 2.69–2.75 (m, CH2(b) of b-
Ala); 3.54–3.60 (m, CH2(a) of b-Ala); 5.37 (br. s, NH of b-Ala); 7.47 (t, J¼8.1, HꢀC(5) of m-NA); 7.91–
7.97 (m, HꢀC(4), HꢀC(6) of m-NA); 8.51 (s, HꢀC(2) of m-NA); 9.12 (br. s, NH of m-NA); 13C-NMR
(CDCl3): 28.3; 36.4; 37.8; 80.1; 114.5; 118.6; 125.4; 129.7; 139.3; 148.4; 156.7; 170.3. Anal. calc. for
C14H19N3O5 (309.32): C 54.36, H 6.19, N 13.58; found: C 54.18, H 6.04, N 13.43.
Boc-g-Abu-m-NA (5). Peptide 5 was synthesized following a similar procedure to that of peptide 4.
1
Yield: 1.41 g (87%). IR (KBr): 1532, 1680, 3303, 3357. H-NMR (CDCl3): 1.47 (s, 3 Me of Boc); 2.03–
2.09 (m, CH2(b) of g-Abu); 2.43–2.55 (m, CH2(a) of g-Abu); 3.26–3.35 (m, CH2(g) of g-Abu); 4.91–
5.50 (m, NH of g-Abu); 7.45 (t, J¼7.5, HꢀC(5) of m-NA); 7.91 (d, J¼7.2, HꢀC(4) of m-NA); 8.01 (d, J¼
6.9, HꢀC(6) of m-NA); 8.55 (s, HꢀC(2) of m-NA); 9.77 (br. s, NH of m-NA). 13C-NMR (CDCl3): 27.6;
28.4; 34.6; 39.1; 80.3; 114.5; 118.3; 125.4; 129.6; 139.8; 148.6; 157.8; 171.8. Anal. calc. for C15H21N3O5
(323.34): C 55.71, H 6.54, N 12.99; found: C 55.56, H 6.38, N 12.87.
Field-Emission Scanning-Electron Microscopy. The morphologies of the peptides 1–5 were
investigated using field-emission scanning electron microscopy. For the study, fibrous materials were
dried and gold coated. The micrographs were taken with a JEOL JSM 6700F apparatus.
Congo Red-Binding Study. The fibrils generated from peptides 2 and 3 were stained with alkaline
Congo red soln. (MeOH/glass dist. H2O 8 :2, containing 10 ml of 1% NaOH) for 2 min and then the
excess stain was removed by rinsing the stained fibrils with MeOH/glass dist. H2O 8 :2 several times. The
stained fibrils were dried in vacuo at r.t. for 24 h and then visualized under a light microscope at ꢁ1000
magnification [39–42].
Single-Crystal X-Ray Diffraction Study. Single crystals of 1, 2, and 4 were obtained by slow
evaporation from acetone/H2O 9 :1, acetone/toluene 9 :1, and MeOH/H2O 9 :1, resp. Data for 1, 2, and 4
Table 4. Crystallographic Refinement Details for Peptides 1, 2, and 4
1
2
4
Formula
C22H41N3O6
443.58
Orthorhombic
P212121
4
C15H20N2O5
308.33
C14H19N3O5
309.32
Monoclinic
P21
Formula weight [g molꢀ1
]
Crystal System
Space group
Z
Triclinic
¯
P1
6
2
a [ꢃ]
b [ꢃ]
c [ꢃ]
a [8]
9.2700(2)
12.0462(3)
23.2439(5)
90
90
90
10.798(2)
13.490(2)
17.694(3)
82.535(3)
82.003(3)
75.588(3)
2460.0(7)
0.094
5.0378(3)
26.274(1)
5.5682(3)
90
96.056(2)
90
b [8]
g [8]
V [ꢃ3]
2595.6(1)
0.082
732.92(7)
0.108
m (MoKa) [mmꢀ1
]
Collected reflections
Unique reflections
No. Parameters
Rint
18634
5666
290
0.0343
1.052
17410
7719
604
0.0809
1.077
4692
2845
202
0.0231
1.060
GoF
R1 [I>2s(I)]
wR2 [I>2s(I)]
0.0531
0.1336
0.0908
0.1509
0.0363
0.0946

374
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
were collected at 120(2) K with graphite monochromated X-radiation on Bruker SMART diffractom-
eters (SMART 6K for 1 and 4, and SMART 1K for 2). Data processing was performed with standard
Bruker software [43]. Structure solution was by direct methods and refinement was on F2 using full-
matrix least-squares techniques. All H-atoms were placed at calculated positions and had been refined
with a riding model. All non-H-atoms were refined anisotropically (see Table 4 for further crystallo-
graphic details). The crystallographic data of 1, 2, and 4 have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication numbers CCDC-673918–CCDC-673920 and
can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif.
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Received March 2, 2009