Peptide nanotube aligning side chains onto one side

Article abstract of DOI:10.1002/psc.2881

A novel pseudo cyclic penta-β-peptide composed of a β-naphthylalanine, two β-alanines, and a sequence of ethylenediamine-succinic acid (CP5ES) is synthesized and investigated on peptide nanotube (PNT) formation. When the PNT is formed with the maximum number of intermolecular hydrogen bonds between the cyclic peptides, the sequence enables the alignment of the side chains, naphthyl groups, on one side of the PNT. Microscopic and spectroscopic observations of CP5ES crystals reveal that CP5ES forms rod- or needle-shaped molecular assemblies showing exciton coupling of the Cotton effect and predominant monomer emission, which are different from a reference cyclic tri-β-peptide composed of a β-naphthylalanine and two β-alanines. Insertion of a sequence of ethylenediamine-succinic acid into β-amino acids in the cyclic skeleton is therefore suggested to be effective to make the side chains aligning on one side of the PNT. Copyright

Full text of DOI:10.1002/psc.2881

Research Article
Received: 4 February 2016
Revised: 8 March 2016
Accepted: 9 March 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2881
Peptide nanotube aligning side chains
onto one side
Yuki Tabata, Shota Mitani and Shunsaku Kimura*
A novel pseudo cyclic penta-β-peptide composed of a β-naphthylalanine, two β-alanines, and a sequence of ethylenediamine-
succinic acid (CP5ES) is synthesized and investigated on peptide nanotube (PNT) formation. When the PNT is formed with the max-
imum number of intermolecular hydrogen bonds between the cyclic peptides, the sequence enables the alignment of the side
chains, naphthyl groups, on one side of the PNT. Microscopic and spectroscopic observations of CP5ES crystals reveal that CP5ES
forms rod- or needle-shaped molecular assemblies showing exciton coupling of the Cotton effect and predominant monomer
emission, which are different from a reference cyclic tri-β-peptide composed of a β-naphthylalanine and two β-alanines. Insertion
of a sequence of ethylenediamine-succinic acid into β-amino acids in the cyclic skeleton is therefore suggested to be effective to
make the side chains aligning on one side of the PNT. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: β-amino acid; cyclic peptide; self-assembly; peptide nanotube
The molecular structure of the cyclic peptide containing the
ethylenediamine-succinic acid sequence is shown in Figure 1.
Introduction
Cyclic β-peptides have been shown to self-assemble into peptide
nanotubes (PNTs) with the cyclic skeletons stacking with each other
through intermolecular hydrogen bonds [1–3]. In these PNTs, all of
the amide bonds are oriented along the nanotube axis and
pointing to the same direction, which endow the PNT with a
macrodipole moment. In general, the side chains of the stacking
cyclic β-peptides, however, are randomly orienting outward from
the nanotube surface. When a regular arrangement of the side
chains is attainable, the PNT can increase its potential to be applied
for functional nanomaterials with using the PNT as a scaffold for
one-dimensional functional molecular array of the side chain.
There have been several reports on making such PNTs regulating
the side-chain orientation. One is D,L-alternating cyclic octa-α-pep-
tide stacking in a well-controlled manner with the help of multiple
electrostatic (salt-bridge) interactions between Lys and Glu residues
of the neighboring cyclic peptides in the PNT. Lys and Glu residues
were designed to flank the residue having pyrene at the side chain
to make an one-dimensional array of pyrenes [4]. This kind of salt-
bridge interactions was also included in the molecular design of
Because the ethylenediamine-succinic acid sequence is regarded
as an analog of β-alanine dimer, the cyclic peptide (CP5ES) is con-
sidered as a pseudo cyclic penta-β-peptide (Figure S1). With this
sequence, successive and maximum intermolecular hydrogen
bonds along the PNT are available only when the cyclic peptides
stack one another with the ethylenediamine-succinic acid units
aligning at the same nanotube side (Figure 1(a)). With
counterbalancing of the reduction of the macrodipole moment of
the PNT from that composed of genuine cyclic β-peptides of the
same ring size, the arrangement of the side chains localizing at
the same PNT side can be attainable. In order to evaluate the
one-dimensional array of the side chains, β-naphthylalanine was in-
corporated in CP5ES. As a reference compound, cyclic tri-β-peptide
containing β-naphthylalanine (CP3, Figure S1), was also prepared.
Cyclic tripeptide was chosen because the small cyclic peptide is
considered to easily take a planar structure, which promotes molec-
ular stacking to form the PNT.
α,γ-cyclic octapeptide to make the fullerene moieties orienting Materials and Methods
toward the same nanotube side [5]. Instead of the salt-bridge inter-
actions, a self-assembling property of long alkyl chains was used for
Spectroscopic Measurements
the oriented stacking of cyclic tetra-β-peptides. A long alkyl chain
with a diacetylene unit in the middle was introduced to one of
the side chains of cyclic tetra-β-peptide. The long alkyl chain associ-
ated together to allow polymerization of neighboring diacetylene
units to afford the conjugate of the PNT and polydiacetylene
connected through covalent bonds [6]. Apart from these side-chain
modifications, we report here to achieve the oriented stacking of
cyclic peptides with a new design of the cyclic skeleton containing
a sequence of ethylenediamine and succinic acid. The sequence
differs from β-alanine dimer about the direction of the connecting
amide bond.
NMR spectra were recorded with a Bruker DPX-400 spectrometer.
TM
ESI and FAB mass spectra were obtained on Thermo Scientific
TM
Exactive Bench-Top LC-MS Spectrometer and a JEOL JMS HX-
110A spectrometer. FT-IR was performed with a Nicolet 6700 FT-IR
*
Correspondence to: Shunsaku Kimura, Department of Material Chemistry,
Graduate School of Engineering, Kyoto University, Kyoto-Daigaku-Katsura,
Nishikyo-ku, Kyoto 615-8510, Japan. E-mail: shun@scl.kyoto-u.ac.jp
Department of Material Chemistry, Graduate School of Engineering, Kyoto
University, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan
J. Pept. Sci. 2016; 22: 391–396
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis of Cyclic Peptide
Syntheses of the cyclic peptides were carried out according to the
Schemes 1 and 2 in the Supporting Information.
Boc-ED-SA-OH (3)
Succinic anhydride (1.00 g, 9.99 mmol) was dissolved in dry CH Cl2
2
(5 ml), Boc-ethylenediamine (1) (1.90 ml, 12.0 mmol) added and
stirred at r.t. for 8 h under Ar atomosphere. After evaporation, the
unreacted Boc-ethylenediamine was removed under reduced pres-
sure to afford the product 3 (2.15 g, 8.26 mmoμ, 83%).
1
H-NMR (400 MHz, CDCl , δ): 1.45 (s, 9H, Boc), 2.52–2.55 (m, 2H,
3
NHCOCH CH CO), 2.67–2.71 (m, 2H, NHCOCH CH CO), 3.24–3.39
2
2
2
2
(
m, 4H, NHCH CH NH), 4.93 (s, 1H, urethane), 6.75 (s, 1H, amide).
2
2
2
Boc-β-Ala -OMe (6)
Boc-β-Ala-OH (1.00 g, 5.29 mmol) and HCl H-β-Ala-OMe (885 mg,
.34mmol) were dissolved in dry dimethylformamide (DMF) under
Ar atmosphere at 0 °C. Then dicyclohexyl carbodiimide (DCC)
1.64 g, 7.93 mmol) and hydroxybenzotriazole (HOBt) (1.07g,
.93mmol) were added to the solution. Triethylamine (TEA)
1.62 ml, 11.6mmol) was added to the mixture and stirred at room
temperature for 20 h. After evaporation, ethyl acetate was added to
the residue and filtered. The filtrate was exchanged with CHCl and
washed with 4% KHSO aq. and saturated NaHCO aq. for three
times each. The organic phase was washed with brine, dried over
MgSO for 30 min and filtered. The residue was dissolved in CHCl
and purified by a column chromatography (silica gel, eluent:
CHCl /CH OH= 15/1v/v) to afford the product 6 (1.32 g, 5.39 mmol,
1%).
6
(
7
(
3
4
3
4
3
3
3
9
1
H-NMR (400 MHz, CDCl
3
, δ): 1.42 (s, 9H, Boc), 2.37–2.53 (m, 4H,
CH CO), 3.70 (s, 3H, OMe),
NHCH CH CO), 3.39–3.52 (m, 4H, NHCH
2
2
2
2
Figure 1. Schematic illustrations of two kinds of CP5ES dimers with (a) five
5.17 (s, 1H, urethane), 6.19 (s, 1H, amide).
or (b) three hydrogen bonds.
HCl H-β-Ala(nap)-OMe (8)
Boc-β-Ala(nap)-OH (7) (200mg, 607 μmol) was dissolved in 4 N
HCl/Dioxane and stirred at r.t. for 1.5h. After evaporation and dried
under reduced pressure for 30 min, methanol (10 ml) and SOCl2
spectrometer. Molecular assembly formation was confirmed by dy-
namic light scattering (DLS) with a Photal DLS-8000. Circular dichro-
ism (CD) spectra were measured at room temperature on a JASCO
J-600 CD spectropolarimeter. UV spectra were measured by a
Shimadzu UV-2450PC spectrometer. The fluorescent spectra were
measured by a JASCO FP-6600 spectrofluorometer. CD, UV and
(
66 μl, 910μmol) was added at 0 °C for 10min and stirred at r.t.
overnight. The solvent was evaporated to afford the product 8
150 mg, 536μmol, 88%).
(
1
H-NMR (400 MHz, CD OD δ): 2.61–2.79 (m, 2H, NHCHCH CO),
3
2
fluorescent spectra of peptide solutions or crystals were using an
3
.08–3.24 (m, 2H, naphtylCH ), 3.67 (s, 3H, OMe), 3.89–4.00 (m, 1H,
2
À5
optical cell of 1-cm path length with concentration of 5 × 10
or a dispersion of crystals on quartz substrate.
M
NHCHCH CO), 7.39–7.91 (m, 7H, naphthyl).
2
2
Boc-β-Ala -β-Ala(nap)-OMe (9)
Boc-β-Ala
2
-OH (212 mg, 814 μmol), HCl H-β-Ala(nap)-OMe (8)
Optical Microscopy
(228 mg,
814μmol), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyl uronium hexafluorophosphate (HATU) (371 mg,
Optical microscopy was performed with an Olympus IX70. A disper-
sion of crystals was placed on a slide glass and observed under
cross-Nicol configuration. The sensitive tint plate was set between
the polarizers.
9
4
77 μmol) and 1-hydroxy-7-azabenzotriazole (HOAt) (66.5mg,
89 μmol) were dissolved in dry DMF and cooled to 0 °C. Then
diisopropylethylamine (DIEA) (439 μl, 2.52 mmol) was added to
the mixture and stirred at 0 °C for 10 min and at r.t. overnight under
Ar atmosphere. After concentration, the residue was dissolved in
ethyl acetate and washed with 4% KHSO
NaHCO aq. (four times) and brine and dried over MgSO
residue was dissolved in CHCl and purified by column chromatog-
raphy (silica gel, eluent: CHCl /CH OH = 10/1 v/v), then
4
aq (five times), saturated
TEM and Electron Diffraction
3
4
. The
The images and diffraction were taken with JEOL JEM-2000EXII
3
(JEOL, Japan) at an accelerating voltage of 100 kV. A drop of the
3
3
sample was dried on a carbon-coated Cu grid. The electron diffrac-
tion diagrams were obtained in the microdiffraction mode. The dis-
tance in the diffractions was calibrated with the (111) diffraction
ring of evaporated Au particles.
concentrated under reduced pressure to afford the product 9
(348 mg, 717μmol, 88%).
1
H-NMR (400 MHz, CDCl
3
, δ): 1.43 (s, 9H, Boc), 2.13–2.27 (m, 4H,
CO), 2.97–3.13 (m, 2H,
NHCH
2
CH CO), 2.61–2.79 (m, 2H, NHCHCH
2
2
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Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2016; 22: 391–396

Peptide Nanotube Aligning Side Chains onto One Side
naphtylCH ), 3.32–3.54 (m, 4H, NHCH CH CO), 3.69 (s, 3H, OMe),
The residue and Boc-β-Ala(nap)-OH (593 mg, 1.80 mmol) were dis-
2
2
2
4
.59–4.68 (m, 1H, NHCHCH CO), 5.13 (s, 1H, urethane), 6.30 and
solved in dry DMF and cooled to 0 °C. HATU (822 mg, 2.16 mmol)
2
6.58 (s, 2H, amide), 7.33-7.83 (m, 7H, naphthyl).
was added to the mixture and stirred at 0 °C for 10 min under N at-
2
mosphere. DIEA (0.98 ml, 5.63 mmol) was added to the mixture and
2
Boc-ED-SA-β-Ala -β-Ala(nap)-OMe (10)
stirred at 0 °C for 3 h and at room temperature for 92 h under N at-
2
mosphere. HATU (200 mg, 0.526 mmol) and HOAt (0.147mg,
The Boc group of Boc-β-Ala
2
-β-Ala(nap)-OMe (9) (326 mg,
1.08 mmol) were added after 4.5h. After concentration, the residue
6
71 μmol) was removed by 4N HCl/dioxane; the solvent was re-
moved under reduced pressure. Boc-ED-SA-OH (3) (174 mg,
71 μmol), HATU (306 mg, 806 μmol) and HOAt (109 mg, 806 μmol)
was dissolved in CHCl and washed with 4% KHSO aq (three times),
3
4
saturatedNaHCO (three times) and brine and dried over MgSO .
6
3
4
After concentration, the residue was washed with diisopropyl ether
were dissolved in dry DMF and cooled to 0 °C under Ar atmosphere.
Then DIEA (240μl, 1.38 mmol, 2.05 eq.) was added to the mixture
and stirred at 0 °C for 15 min, and HCl H-β-Ala(nap)-OMe (0.25 eq.)
and DIEA (240μl, 1.38 mmol, 2.05 eq.) was added at 15-min inter-
vals for three times. Then the mixture was stirred at r.t. overnight.
After concentration, the residue was washed with ethyl acetate.
twice to afford the product 13 (681 mg, 1.40 mmol, 65.1%).
1
H-NMR (400MHz, CDCl δ): 1.38 (s, 9H, Boc), 2.5–2.22 (m, 6H,
3
CH CO), 3.10–2.94 (2H, naphthylCH ), 3.55–3.48 (4H, NHCH CH CO),
2
2
2
2
3
.64 (s, 3H, OMe), 4.22 (s, 1H, NHCHCH CO), 5.49 (s, 1H, urethane
2
NH), 6.41(s, 2H, amide), 7.80–7.34 (m, 7H, naphthyl).
The residue was dissolved in CHCl and purified by a column chro-
3
Boc-β-Ala(nap)-β-Ala -OH (14)
2
matography (silica gel, eluent: CHCl /Methanol = 10/1v/v), then
3
concentrated under reduced pressure to afford the product 10
Boc-β-Ala(nap)-β-Ala -OMe (13) (681 mg, 1.40 mmol), methanol
2
(
392 mg, 624 μmol, 93%).
(5.6ml) and 1,4-dioxane (5.6ml) were stirred at 0 °C for 10 min.
1
H-NMR (400 MHz, CD OD δ): 1.42 (s, 9H, Boc), 2.23–2.28 (m, 4H,
3
Aqueous NaOH (1N, 2.8ml) was added to the solution and stirred
at 0 °C for 30 min and at room temperature for 4 h. After neutraliza-
tion with aqueous 1N HCl, the solvent was removed under reduced
pressure. The residue was dissolved in ethyl acetate and washed
NHCH CH CO), 2.44 (s, 4H, COCH CH CO), 2.48–2.60 (m, 2H,
2
2
2
2
NHCHCH CO), 2.96–3.02 (m, 2H, naphtylCH ), 3.12–3.21 (m, 4H,
2
2
NHCH CH CO), 3.34 (m, 4H, NHCH CH NH), 3.63 (s, 3H, OMe),
2
2
2
2
4
.56–4.58 (m, 1H, NHCHCH CO), 7.37–7.81 (m, 7H, naphthyl).
2
with 4% KHSO aq (three times) and brine. The organic layer was
4
+
ESI-MS (m/z): [M+ H] calcd for C H N O , 628.3341; found,
32
45
5
8
dried over MgSO and concentrated in vacuo to afford the product
4
+
628.3334. [M + Na] calcd, 650.3160; found, 650.3152.
(
517 mg, 1.10 mmol, 78.6%).
1
H-NMR (400 MHz, CD OD δ): 1.31 (s, 9H, Boc), 2.41–2.34 (m, 4H,
3
TFA H-ED-SA-β-Ala -β-Ala(nap)-OH (11)
2
CH CH CO), 2.51 (m, 2H, CHCH CO), 2.97 (m, 2H, naphthylCH )
2
2
2
2
Boc-ED-SA-β-Ala
2
-β-Ala(nap)-OMe (10) (160 mg, 255 μmol) was dis-
3.42 (m, 4H, CH
naphthyl).
2
CH
2
CO), 4.21 (br, 1H, CHCH
2
CO), 7.82–7.37 (m, 7H,
solved in methanol (4ml) and stirred at 0 °C. 1N NaOH aq. (1ml) was
added to the solution and stirred at room temperature overnight.
After neutralization with 1N HCl aq., the solvent was removed un-
der reduced pressure. The residue was purified by a Sephadex
LH20 column with methanol. Then, the Boc group was removed
by trifluoroacetic acid (TFA) and anisole, and the solvent was re-
moved under reduced pressure. The residue was washed with
diisopropyl ether for three times and dried to afford the product
CP3 (15)
The Boc group of compound (14) (90 mg, 0.019mmol) was re-
moved by TFA and anisole; the solvent was removed under re-
duced pressure. The residue was washed with diisopropyl ether
for 3 times and dried up. The product, HATU (704.9mg, 1.85 mmol)
and HOAt (378.5 mg, 2.78mmol) were dissolved in DMF (50 ml),
and stirred at 0 °C for 15 min under N2 atmosphere. DIEA
1
1 (157 mg, 250 μmol, 98%).
H-NMR (400 MHz, CD OD δ): 2.23–2.31 (m, 4H, NHCH CH CO),
1
3
2
2
(431.3 mg, 3.34 mmol) in DMF (30 ml) was added dropwise over
2
2
3
4
.45–2.49 (m, 4H, COCH CH CO), 2.52–2.57 (m, 2H, NHCHCH CO),
.92–2.98 (m, 2H, naphtylCH ), 3.02–3.05 (m, 2H, NH CH CH NH),
.29–3.31 (m, 4H, NHCH CH CO), 3.42–3.46 (m, 2H, NH CH CH NH),
.53–4.57 (m, 1H, NHCHCH CO), 7.37–7.77 (m, 7H, naphthyl).
2 2 2
1
h. The solution was stirred at 0 °C for 1 h and at room temperature
2
2
2
2
for 72h. The solvent was evaporated, and the residue was washed
with ethyl acetate. The product was purified by a Sephadex LH-20
column using DMF as eluent to give 15 (15 mg, 42.4μmol, 23%).
2
2
2
2
2
2
1
H NMR (400 MHz, DMF-d δ): 2.30 (m, 2H, NHCHCH ), 2.51, 2.15
7
2
CP5ES (12)
(
m, 4H, NHCH CH ), 3.09, 2.96 (m, 2H, benzyl), 3.50, 3.36, 3.11 (m,
2 2
2
TFA H-ED-SA-β-Ala -β-Ala(nap)-OH (11) (157 mg, 250μmol), HATU
4H, NHCH ), 4.42 (br, 1H, NHCH), 7.91–7.41 (m, 10H, aromatic and
2
(
951 mg, 2.50 mmol) and HOAt (510mg, 3.75 mmol) were dissolved
NH).
+
in dry DMF (160 ml) and stirred at 0 °C. DIEA (784 μl, 4.50 mmol) in
dry DMF (50 ml) was added dropwise over 1.5h. Then, the solution
was stirred at r.t. for 72 h. After evaporation, the residue was washed
with acetonitrile (three times), ethyl acetate (three times) and meth-
anol to afford the product 12 (15 mg, 30.3 μmol, 12%).
FAB-MS (m/z): [M+ Na] calcd, for C H N O Na, 376.1637;
found, 376.1623.
20 23 3 3
Results and Discussion
1
H-NMR (400MHz, CD OD δ): 2.24–2.56 (m, 10H, CH CO), 2.91–
3
2
IR Analysis
3
.03 (m, 2H, naphtylCH ), 3.25–3.465 (m, 8H, NH CH ), 4.42 (m, 1H,
2 2 2
NHCHCH CO), 7.34–7.78 (m, 7H, naphthyl).
2
A saturated solution of CP5ES in methanol was dried slowly on a
+
ESI-MS (m/z): [M+ H] calcd for C H N O , 496.2554; found,
2
6
33
5
5
CaF plate in air. An FTIR spectrum of the white solid left on the
2
+
4
96.2549. [M + Na] calcd, 518.2374; found, 518.2373.
plate shows the amide I and II bands around 1650 and
À1
1
540 cm , respectively, suggesting parallel β-sheet like hydrogen
Boc-β-Ala(nap)-β-Ala -OMe (13)
2
bond formation (Figure 2a) [7,8]. A sharp N–H stretching peak (am-
À1
Boc group of (6) (589 mg, 2.15mmol) was removed with 4N
HCl/dioxane and the solvent was removed under reduced pressure.
ide A band) appears at 3309 cm , supporting that all the amide
protons are involved in hydrogen bond formation and their
J. Pept. Sci. 2016; 22: 391–396
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Figure 3. Optical microscopic observations at a cross-Nicol configuration
of (a) CP5ES and (b) CP3 crystals. The double-headed allows show the
orientation of z′-axis of a sensitive tint plate. Scale bar = (a) 20 μm (b) 50 μm.
3
peptides, cyclo(aminocyclohexyl carboxylic acid) [9] and cyclo
(
aminoglucosyl carboxylic acid) [10], suggesting the amide bonds
3
aligning along the long axis of the rod crystals, which contribute
to the anisotropic refractive indices of the PNT.
CP3 formed extremely long and straight rod-shaped crystals,
which were also identified by the polarization microscope
observation (Figure 3b). With a tint plate, the color appearance
along the long and short axes of the rod crystals is reversed to
the case of CP5ES. Probably, the contribution of naphthyl groups
to the short axis in the total refractive index should become larger
in CP3 than CP5ES because of the reduction of the amide number
of CP3 from CP5ES.
Even though CP5ES and CP3 yielded the rod-like crystals, CP3
grew into longer crystals than CP5ES. Because CP3 is a small-
number cyclic peptide, a flat conformation required for cyclic
peptide stacking into the PNT is easy to take place. On the other
hand, CP5ES can take various conformations including bend
shapes which should prevent the cyclic peptide from growing into
a long PNT.
2
Figure 2. FT-IR spectra of (a) CP5ES and (b) CP3 crystals on a CaF plate.
environment is highly homogeneous. The IR analysis therefore sup-
ports the PNT formation [6,9–11].
The reference cyclic tri-β-peptide, CP3, also shows amide I and II
bands suggesting parallel β-sheet like hydrogen bond and a sharp
amide A band indicating a homogeneous structure (Figure 2b).
Polarization Microscope Observation
The rod-like crystals of CP5ES were grown from a methanol solution
under methanol atmosphere and subjected to polarization micro-
scope observation. The crystals were confirmed by visualization un-
der the cross Nicol configuration, and they are considered to be
bundles of PNTs aligning the long axis of the nanotubes along
the long axis of the rods. Further with a tint plate between the
polarizers a blue color was observed when the long axis of the
rod-like crystals coincided with the z′-axis of the tint plate, and a
yellow–orange color appeared when they were in a perpendicular
arrangement (Figure 3a). The color differentiation according to
the rod axes indicates that the long axis of the crystal has a larger
refractive index than that of the short axis. This result coincides with
those in the previous reports of PNTs prepared from cyclic tri-β-
Electron Diffraction Analysis
The small crystals of CP5ES and CP3 were subjected to transmission
electron microscopy (TEM) observations (Figure 4). The TEM images
show the needle-like molecular assemblies were formed. The elec-
tron diffraction patterns were obtained with these crystals, indicat-
ing the spacings of 4.9Å and 4.8 Å respectively for CP5ES and CP3
along the long axis of the needle-like molecular assemblies (Figure
S2). These spacings are agreeable with the distance between cyclic
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J. Pept. Sci. 2016; 22: 391–396

Peptide Nanotube Aligning Side Chains onto One Side
Figure 4. TEM images of (a) CP5ES formed from ethanol and (b) CP3
formed from formic acid–diisopropyl ether. Scale bar = 2 μm.
β-peptides in PNTs as reported previously [6,9–11]. CP5ES and CP3
can therefore self-assemble into PNTs, which bundle together to
grow into the needle- and rod-like crystals.
Circular Dichroism Spectroscopy
Molecular assemblies of CP5ES and CP3 in solutions were studied
by CD measurements. TFE is a good solvent for these cyclic pep-
tides because DLS measurements did not show any evidence for
molecular assemblies. On the other hand, both peptides in metha-
nol showed light scattering because of the presence of molecular
assemblies. CP5ES in methanol shows the exciton coupling in the
absorption region at 274 nm, while that in TFE gives a negative
broad Cotton effect (Figure 5a). CP3 in either solvent does not show
any exciton couplings. It is therefore suggested that the spatial ar-
rangement of the naphthyl groups should be helical along the
CP5ES PNT and random along the CP3 PNT.
Figure 5. (a) CD spectra of CP5ES and CP3 in a 0.05 mM methanol or TFE
solution. (b) UV and (c) fluorescence spectra of the crystals dispersed in
0.05 mM methanol solution.
the ground state and the excited state of the naphthyl groups in
the single PNT.
On the other hand, bathochromic shifts in the absorption spectra
were observed with both cyclic peptides in crystals because of the
strong interaction at the ground state of the naphthyl groups (Fig-
ure 5b). The naphthyl groups should be so close in the crystals ow-
ing to bundle formation of PNTs. A striking difference was observed
between the fluorescence spectra of CP5ES and CP3 crystals (Figure
5c). The maximum wavelength of the emission from CP5ES crystals
was similar to that in solution, but that from CP3 crystals shifted to a
longer wavelength than in solution. This difference may be related
with the straight alignment of the naphthyl groups along the CP5ES
UV Absorption and Fluorescence Spectroscopy
UV spectra of CP5ES and CP3 in TFE and methanol similarly showed
the absorption of the naphthyl groups around 274 nm. Fluores-
cence spectra of these cyclic peptides in methanol were the same
to show the monomer emission. It is considered that single PNTs
should be formed in methanol, and there are no interactions at
J. Pept. Sci. 2016; 22: 391–396
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
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PNT and the random alignment along the CP3 PNT. In the regular
arrangement of the naphthyl groups in CP5ES, the photo-excited
energy should migrate in a large area of the PNT bundles to reach
a monomer-like naphthyl group, which emitted the monomer
emission. On the other hand, there should be various states of
the overlapping naphthyl groups in CP3 crystals to allow the
photo-energy migration into trapping sites of the stacking naphthyl
groups, which made the emission shift to a longer wavelength. The
photo-energy migration distance in CP5ES is estimated as follows.
The rate of photo-migration among the naphthyl groups is calcu-
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Acknowledgements
The authors appreciate associate professor T. Imai (Research in- Supporting Information
stitution for Sustainable Humanosphere, Kyoto University) for
their help in the TEM measurements and electron diffraction
analysis.
Additional supporting information may be found in the online
version of this article at the publisher’s web site.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2016; 22: 391–396