Toward the rational design of molecular rotors ion sensors based on α,γ-cyclic peptide dimers

Article abstract of DOI:10.1007/s00726-011-0886-2

A dimer-forming self-assembling cyclic hexapeptide with a control register and a large association constant in water is described. The self-assembly process is followed by pyrene-excimer emission and the main diastereomeric dimer present in solution is sw

Full text of DOI:10.1007/s00726-011-0886-2

Amino Acids (2011) 41:621–628
DOI 10.1007/s00726-011-0886-2
ORIGINAL ARTICLE
Toward the rational design of molecular rotors ion sensors based
on a,c-cyclic peptide dimers
•
Marıa Jesus Perez-Alvite Manuel Mosquera
•
´
´
´
Luis Castedo Juan R. Granja
•
Received: 11 January 2011 / Accepted: 8 March 2011 / Published online: 31 March 2011
Ó Springer-Verlag 2011
Abstract A dimer-forming self-assembling cyclic hexa-
peptide with a control register and a large association
constant in water is described. The self-assembly process is
followed by pyrene-excimer emission and the main dia-
stereomeric dimer present in solution is switched by con-
trolled addition of divalent cations (e.g., Ca, Mg) or oxalic
acid.
PCBA
SPN
[6,6]-phenyl-C61-butyric acid
Self-assembling Peptide Nanotubes
Tetrabutylamonium chloride
TBAF
TBTU
O-Benzotriazol-1-yl-N,N,N0,N0-
tetramethyluronium tetrafluoroborate
Introduction
Keywords Self-assembling ꢀ Cyclic peptide ꢀ
Molecular rotor ꢀ c-Amino acid ꢀ Dimer ꢀ Excimer
One of the most fundamental and pressing problems for the
implementation of supramolecular synthetic methods in
nanotechnological applications concerns the control of
self-assembly processes through the design of molecular
components and their practical application at the macro-
molecular level of the supramolecular entities (Zhang
2003). In addition, the use of tools such as fluorescence
(Roy et al. 2008) or single molecule detection (Neuman
and Nagy 2008) to follow the formation of supramolecular
entities and assess their functions is also demanded. In
this context, peptides are very important supramolecular
building blocks because of their straightforward synthesis
and the potential to introduce chemical diversity, as well as
the large variety and easy modulation of their 3D structures
Abbreviations
Acp
3-Aminocyclopentanecarboxylic Acid
Cyclic peptides
CPs
a,c-D
DIEA
Dimer of a a,c-cyclic peptide
Diisopropylethylamine
4-DPPBA 4-(Diphenylphosphino)benzoic Acid
ext-TTF
Pap
2-[9-(1,3-dithiol-2-ylidene)anthracen-10(9H)-
ylidene]-1,3-dithiole
5-(pyren-1-yl)pentanoic acid
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-011-0886-2) contains supplementary
material, which is available to authorized users.
¨
(Matsui and Gao 2005; Ashkenasy et al. 2006; Konig and
Kilbinger 2007; Ulijn and Smith 2008 and Pazos et al.
2009). b-sheet-forming peptides are particularly interest-
ing, not only because of their relevance to pathological
disorders, such as HIV, cancer and neurodegenerative
diseases (Stefani and Dobson 2003; Knowles et al. 2007;
Hamley 2007; Kolstoe et al. 2009), but also because of
their potential use in the manufacture of nanotapes
(Smeenk et al. 2005; Whitehouse et al. 2005) or nanotubes
(Brea et al. 2010; Bong et al. 2001). The properties of
b-sheet structures not only depend on their amino acid
´
M. J. Perez-Alvite ꢀ L. Castedo ꢀ J. R. Granja (&)
´
´
Departamento de Quımica Organica, Unidad Asociada al CSIC
´
´
´
y Centro Singular de Investigacion en Quımica Biologica
y Materiales Moleculares, Universidad de Santiago de
Compostela, Campus Vida, 15782 Santiago, Spain
e-mail: juanr.granja@usc.es
M. Mosquera
Departamento de Quımica Fısica y Centro Singular
de Investigacion en Quımica Biologica y Materiales
Moleculares, Universidad de Santiago de Compostela,
Campus Vida, 15782 Santiago, Spain
´
´
´
´
´
123

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M. J. Perez-Alvite et al.
622
composition but also on their inter-chain relative orienta-
tion (Khakshoor and Nowick 2008; Remaut and Waksman
2006 and Searle and Ciani 2004). Control of b-sheet for-
mation and register structure is highly desirable.
fully understand and control self-assembling CP structures.
We describe here a new a,c-CP system with a large asso-
ciation constant, both in organic and aqueous media, and in
which the isomeric dimer formed is under control and can
be switched on demand.
In the last few years, we have been working on the
design and synthesis of self-assembling peptide nanotubes
(SPN) using cyclic peptides (CPs) that contain cyclic
c-amino acids in which the interactions that link the CPs
Materials and methods
´
together are b-sheet-type interactions (Amorın et al. 2003,
2005, 2008; Brea et al. 2007a, 2005). In this context, we
have found that the b-sheet register plays an important role
in electron and energy transfer processes (a,c-CPs) (Brea
et al. 2007b, c, 2011). For example, we estimated the
association constant by dimer-induced excimer formation
of a,c-CPs modified with a pyrene on one of the side chains
(a,c-CP2). Only one of the three-topoisomeric dimers (a,c-
D2Z) allows the pyrene to stack in the appropriate way to
form the excimer while the other two emit as single
monomers (Scheme 1). Thus, the association constant
could only be estimated by considering that the three
nonequivalent dimers that were formed in an equimolecu-
lar ratio (Brea et al. 2007c). An even more important
limitation concerns the electron transfer process between
a,c-CP3 and a,c-CP4, in which the highly efficient inter-
space electron transfer only takes place in dimer a,c-D3-4Z,
where the fullerene acceptor (PCBA) and the donor (ext-
TTF) are oriented in the same direction, while the other
two dimers are not active because of the long distance
between the two electroactive components (Brea et al.
2007b). So b-sheet register control is highly demanded to
(L)-2-(tert-butoxycarbonylamino)-5-(pyren-1-yl)pent-4-
ynoic acid A solution of 4-DPPBA (145 mg, 0.47 mmol),
Cs2CO3 (1.93 g, 5.93 mmol) in a H2O/DMF mixture (5:1,
120 mL) was degassed for 15 min under argon and then CuI
(321 mg, 1.68 mmol), 10% Pd/C (126 mg, 0.12 mmol) and
bromopyrene (1 g, 3.6 mmol) were added and the sample
was degassed for another 30 min. (L)-2-(tert-butoxycar-
bonylamino)pent-4-ynoic acid (506 mg, 2.37 mmol) was
added and the mixture heated at 80°C under argon for 4 h.
After cooling at room temperature, the reaction mixture was
filtered through a Celite pad, washed with H2O/DMF mix-
ture. The resulting solution was washed with CH2Cl2 and the
aqueous phase was lyophilized. The resulting powder was
purified by flash chromatography (2% EtOH/CH2Cl2 with
0.5% AcOH) to give 783 mg of the (L)-2-(tert-butoxycar-
bonylamino)-5-(pyren-1-yl)pent-4-ynoic acid as a yellow
1
foam [80%, Rf = 0.15 (5% MeOH in CH2Cl2)]. H NMR
(CD3OD, 500.14 MHz, d): 8.54 (d, J = 9.1 Hz, 1H), 8.22 (t,
J = 7.3 Hz, 3H), 8.15 (d, J = 9.1 Hz, 1H), 8.13–8.00 (m,
4H), 4.54 (t, J = 6.0 Hz, 1H), 3.25–3.10 (dq, J = 17.0 and
5.2 Hz, 2H), 1.46 (s, 9H); 13C NMR (CD3OD, 125.76 MHz,
Scheme 1 a,c-Cyclic peptide
(a,c-CP) and dimer (a,c-D)
structures used in previous
studies (top) and model of the
equilibrium of the three dimers
of a,c-CP2 (a,c-D2x, a,c-D2y,
a,c-D2z) in which only the
former is able to form the
pyrene excimer (bottom)
123

Toward the rational design of molecular rotors ion sensors based on a,c-cyclic peptide dimers
623
d): 174.3 (CO), 157.8 (CO), 133.1 (C), 132.6 (C), 132.4 (C),
130.8 (CH), 129.3 (CH), 129.0 (CH), 128.2 (CH), 127.4
(CH), 126.6 (CH), 125.5 (CH), 125.4 (CH), 119.3 (C), 92.0
(C), 82.6 (C), 80.8 (C), 62.7 (CH2), 54.2 (CH), 28.7 (CH3),
24.2 (CH2); FTIR (293K, CHCl3): 3433, 2983, 2929, 2854,
1722, 1515 cm-1; MS (ES1) [m/z (%)]: 436 ([M ? Na]?,
36), 414 ([MH]?, 16), 314 ([MH-Boc]?, 100); HRMS
[MH]? calculated for C26H24NO4 414.1700, found
414.1703.
dissolved in CH2Cl2 (20 mL) and the solution washed with
HCl (5%), dried over Na2SO4, filtered and concentrated. The
resulting residue was dissolved in a TFA/CH2Cl2 mixture
(1:1, 6.9 mL) and stirred at room temperature for 15 min.
After removal of the solvents, the residue was dried under
high vacuum and used without further purification. The linear
peptide was dissolved in CH2Cl2 (688 mL) and treated with
HATU (288 mg, 0.76 mmol), followed by dropwise addition
of DIEA (720 lL, 4.19 mmol). The resulting mixture was
stirred for 10 h at room temperature to complete the reaction
and then the solvent was removed under reduced pressure.
The resulting residue was purified by HPLC, affording
500 mg of a,c-CP5 as a white solid [63%, Rt = 13 min
(Phenomenex Maxsil-10 silica semipreparative column,
7–12% MeOH in CH2Cl2, 25 min)]. 1H NMR (CDCl3,
500.14 MHz, d): 8.40–7.68 (m, 12H, 3NH, Pyr), 7.37-7.27
(m, 8H, Bn), 7.25–7.20 (m, 2H, Bn), 5.32-4.92 (m, 7H, CH2-
Bn, a-Prg, Glu and Lys), 4.86–4.67 (m, 3H, Hc Acp),
3.42–3.22 (m, 2H, CH2-Pyr), 3.17–3.08 (m, 2H, CH2-Lys),
3.05–2.96 (m, 9H, CH3), 2.96–2.87 (m, 3H, Ha Acp),
2.46–2.21 (m, 6H, Acp), 2.22–1.98 (m, 4H), 1.97–1.11 (m,
30H);13C NMR (CDCl3, 125.76 MHz, d): 175.4(CO), 175.3
(CO), 173.1 (CO), 172.5 (CO), 156.5 (CO), 136.5 (C), 136.2
(C), 135.9 (C), 135.6 (C), 131.3 (CH), 130.8 (CH), 129.7
(CH), 128.5 (CH), 128.4 (CH), 128.2 (CH), 128.0 (CH),
128.0 (CH), 127.4 (CH), 127.2 (CH), 126.6 (CH), 125.8
(CH), 124.7 (CH), 123.3 (CH), 123.1 (CH), 66.5 (CH2), 54.8
(CH), 50.2 (CH), 48.3 (CH), 47.8 (CH), 42.5 (CH2), 42.3
(CH), 40.6 (CH2), 35.9 (CH2), 33.0 (CH2), 30.2 (CH3), 29.9
(CH3), 29.8 (CH3), 28.3 (CH2), 27.6 (CH2), 27.1 (CH2), 26.9
(CH2), 22.5 (CH2); FTIR (293K, CHCl3): 3435, 3302, 2943,
2873, 2359, 1718, 1620, 1525 cm-1; MS (ES1) [m/z (%)]:
1156 ([MH]?, 100), 578 ([MH]2?, 23); HRMS [MH]? cal-
culated for C68H82N7O10 1156.6118, found 1156.6111.
(L)-2-(tert-butoxycarbonylamino)-5-(pyren-1-yl)penta-
noic acid (L-Boc-Pap-OH) A solution of (L)-2-(tert-
butoxycarbonylamino)-5-(pyren-1-yl)pent-4-ynoic
acid
(110 mg, 0.27 mmol) in ethanol (9 mL) and acetic acid
(45 lL) was degassed for 15 min and then treated with Pd/
C (10%, 56 mg) and stirred overnight at room temperature
under balloon pressure of hydrogen. The resulting mixture
was filtered through a Celite pad, the residue was washed
with ethanol, and the filtrates were concentrated under
reduced pressure. The reaction crude was purified by flash
chromatography (2%EtOH/CH2Cl2 with 0.5% AcOH)
affording 98 mg of L-Boc-Pap-OH as a yellow foam
[88%, Rf = 0.25 (5% MeOH in CH2Cl2)]. 1H NMR
(CD3OD, 500.14 MHz, d): 8.24 (d, J = 9.2 Hz, 1H), 8.10
(d, J = 7.6 Hz, 2H), 8.07–8.02 (m, 2H), 7.99–7.90 (m,
3H), 7.82 (d, J = 7.6 Hz, 1H), 4.27–4.07 (m, 1H),
3.43–3.15 (m, 2H), 2.08-1.66 (m, 4H), 1.39 (s, 9H); 13C
NMR (CD3OD, 125.76 MHz, d): 158.2 (CO), 137.6 (CO),
132.8 (CO), 132.4 (CH), 132.3 (CH), 131.2 (CO), 129.8
(CH), 128.5 (CH), 128.4 (CH), 128.3 (CH), 127.6 (CH),
126.9 (CH), 126.2 (C), 126.1 (C), 125.9 (CH), 125.7 (CH),
124.4 (CH), 80.5 (C), 69.1 (CH2), 40.1 (C), 33.8 (CH2),
32.8 (CH2), 31.6 (CH2), 30.1 (CH2), 29.3 (CH2), 28.7
(CH), 24.9 (CH2), 24.0 (CH2); FTIR (293 K, CHCl3):
3422, 2975, 2928, 2863, 2367, 1722, 1515 cm-1; MS
(ES1) [m/z (%)]: 440 ([M ? Na]?, 100), 418 ([MH]?, 11),
318 ([MH–Boc]?, 58); HRMS [MH]? calculated for
C26H28NO4 418.2013 found 418.2013.
c-[L-Pap-D-MeN-c-Acp-L-Glu-D-MeN-c-Acp-L-Lys-D-Me
N-c-Acp-] (a,c-CP6) To a mixture of c-[L-Pap-D-MeN-c-
Acp-L-Glu(OBn)-D-MeN-c-Acp-L-Lys(Z)-D-MeN-c-Acp-]
(a,c-CP5) (429 mg, 0.37 mmol), pentamethylbenzene
(429 mg, 2.89 mmol) and anisole (430 lL, 4 mmol) in
TFA (43 mL) was treated with HBr/AcOH (33%, 8.6 mL,
45 mmol). After stirring 4 h at room temperature, the
solvent was removed under reduced pressure, and the crude
was purified by HPLC, affording 215 mg of a,c-CP6 as a
white solid [50%, Rt = 29 min (Sugelabor Inertsil C18
column, 60–85% MeOH in H2O)]. 1H NMR (DMSO:
CDCl3 (20:80), 500.14 MHz, d): 8.31–7.63 (m, 12H), 4.93-
4.65 (m, 3H), 2.96–2.82 (m, 4H), 2.78–2.61 (m, 3H),
2.55–2.46 (m, 6H), 2.22–2.12 (m, 1H), 1.99–0.97 (m,
18H); 13C NMR (DMSO: CDCl3 (30:70), 125.76 MHz, d):
173.1 (CO), 172.4 (CO), 170.3 (CO), 170.0 (CO), 134.8
(C), 129.3 (C), 128.8 (C), 127.7 (C), 126.5 (CH), 125.7
(CH), 125.4 (CH), 124.8 (CH), 124.2 (CH), 123.1 (CH),
Peptide synthesis
Linear peptides Boc-[L-Pap-D-MeN-c-Acp-L-Glu(OBn)-
D-MeN-c-Acp-L-Lys(Z)-D-MeN-c-Acp-]OFm was prepared
following the synthetic strategy previously described (see
´
also Supplementary materials, Amorın et al. 2005 and Brea
et al. 2005).
c-[L-Pap-D-MeN-c-Acp-L-Glu(OBn)-D-MeN-c-Acp-L-Lys
(Z)-D-MeN-c-Acp-] (a,c-CP5) A solution of Boc-[L-Pap-
D-MeN-c-Acp-L-Glu(OBn)-D-MeN-c-Acp-L-Lys(Z)-D-MeN-
c-Acp-]-OFm (988 mg, 0.68 mmol) in a piperidine/CH2Cl2
solution (1:4, 6.9 mL) was stirred at room temperature for
20 min. After removal of the solvent, the residue was
123

´
M. J. Perez-Alvite et al.
624
122.9 (CH), 122.8 (CH), 121.7 (CH), 52.3 (CH), 46.7
(CH), 46.4 (CH), 40.3 (CH2), 32.3 (CH2), 31.0 (CH3), 30.3
(CH2), 29.8 (CH2), 28.2 (CH2), 27.4 (CH2), 26.0 (CH2),
25.6 (CH2), 25.3 (CH2), 25.2 (CH2), 20.6 (CH2); FTIR
(293K, CHCl3): 3429, 3294, 2956, 2866, 1684, 1620,
1543 cm-1; MS (ES1) [m/z (%)]: 972 ([M ? K]?, 7), 955
([M ? Na]?, 100), 933 ([MH]?, 34); HRMS [MH]? cal-
culated for C53H70N7O8 932.5280, found 932.5265.
pairing type is not present in any of the other dimers
(Scheme 1). We envisaged that the presence of attractive
interactions between the R1 and R2 side chains would favor
the formation of a,c-DZ, thus inducing the approximation
of the remaining side chain. Based on these considerations,
we designed a new CP (a,c-CP6) in which R1 could be a
side chain bearing a carboxylic acid group (Glu), meaning
that R2 should be a side chain containing a basic group
such us Lys (Scheme 2). To follow the self-assembly
process and dimer control, we included in the third amino
acid a pyrene group.
Time-resolved fluorescence
Fluorescence lifetimes were determined by time-correlated
single-photon counting on an Edinburgh Instruments CD-
900 spectrometer equipped with a hydrogen-filled nano-
second flash lamp. The instrumental response width of the
system is 1.0 ns. We measured usually until 10,000 counts
were reached in (2 9 103 channels). The emission band-
pass for the lifetime measurements was usually 20 nm. The
experiments were performed at room temperature, and
samples were purged with argon prior to measurement.
The pyrenylamino acid (L-Boc-Pap-OH) used for this
study was prepared from Boc-propargylglycine by means
of a Sonogashira cross-coupling reaction with 1-bromop-
yrene and 10% Pd on carbon in an aqueous medium
´
(Lopez-Deber et al. 2001; Brea et al. 2006), followed by
hydrogenation (10% Pd on carbon) in 1% acetic acid in
ethanol (Scheme 2). The resulting (L)-2-(tert-butoxycar-
bonylamino)-5-(pyren-1-yl)pentanoic acid has a higher
fluorescence quantum yield than the pyreneacetic esters
used in previous studies (Brea et al. 2007c, 2010; Masuko
et al. 2000, Nakamura et al. 2008; Kashida et al. 2010 and
Valeur 2002). The solution-phase synthesis of a linear
peptide, using a strategy similar to that previously reported
Results and discussion
´
(see Scheme 1 SI in supporting information) (Amorın et al.
In order to control the dimeric species formed in solution,
we considered that the inter-strand side-chain/side-chain
interactions are very different in the three possible dimers
(a,c-D2X, a,c-D2Y and a,c-D2Z) (Reiriz et al. 2009 and
Garcia-Fandin˜o et al. 2009). For example, the R1 side
chains (in red) of the excimer-forming dimer (a,c-D2Z) are
cross-strand closed to R2 side chains (in green), and this
2005 and Brea et al. 2005), and cyclization with TBTU of
the resulting linear peptide provided a,c-CP5. Treatment of
this peptide with HBr in acetic acid gave unprotected a,c-
CP6. Fully protected a,c-CP5 has similar self-assembly
properties to previously reported CPs, with a Ka value in
chloroform of 1.6 9 106 M-1, which was determined by
Scheme 2 Structure of a,c-CP5
and a,c-CP6 and proposed
model for the structure of
a,c-D6Z; the strategy for the
synthesis of N-Boc-5-pyrenyl-2-
aminopentanoic acid is shown at
the bottom
123

Toward the rational design of molecular rotors ion sensors based on a,c-cyclic peptide dimers
625
least-squares analysis fitting to appropriate equations using
Kaleidagraph 3.5 (Synergy Software, Reading, PA, USA),
(Figure 1 SI in supporting information) (Park et al. 2003
and Martin 1996). The ratio between the three nonequiv-
alent dimers (a,c-D5x, a,c-D5y, a,c-D5z) could not be
established by NMR studies but assumed to be equimolar.
On the other hand, the low solubility of unprotected pep-
tide a,c-CP6 precluded the measurement of its Ka in
chloroform. However, this compound has an associa-
tion constant of 4.5 9 105 M-1 in 20% DMSO/CHCl3
(Fig. 1b) and shows, as expected, an increased dimeriza-
tion constant (association constant in similar conditions
(20% DMSO/CHCl3) of a,c-CP5 was estimated to be
4.8 9 104 M-1, although this was measured at peptide
concentrations that are above the region in which rigorous
quantitative analysis is possible) that can be attributed to
the establishment of the salt bridge interactions. It should
be pointed out that the association constant of a,c-CP6 is
pH dependent, so the 0.12 mM solution (20% DMSO/
CHCl3) of the reverse phase purified a,c-CP6, in which
amino and carboxylic acid groups are protonated, presents
a low excimer signal that increases markedly on addition of
small amounts of base (DIEA) (Fig. 1a). The presence of a
large excess of DIEA leads to a reduction in the excimer
signal.
Additionally, a,c-CP6 was soluble in water and excimer
emission (Shiraishi et al. 2006) was detected even at a low
micromolar concentration, with an association constant at
pH 5.4 of 1.4 9 104 M-1 (Fig. 1d and Figure 2 SI in
supporting information) that it is slightly more acidic than
the espected considering the pKa of Lys (10.5) and Glu
(4.55) in the dimeric structure (Hui et al. 2005; Delphine
et al. 2008). Once again, the self-assembly process is pH-
dependent but there is a more pronounced reduction under
Fig. 1 a Pyrene fluorescence emission (337 nm excitation wavelength)
of 1.2 lM a,c-CP6 in 20% DMSO/CHCl3 (red lines) and upon addition
of 1.8 (dark blue circles), 3.5 (green squares), 5.3 (black diamonds), 7.1
(orange lines) and 28.3 (light blue lines) equiv of DIEA. b Emission of
a,c-CP6 in 20% DMSO/CHCl3 (340 nm excitation wavelength), from
1.2 lM (red lines) to 45 lM (grey inverted triangles), denoting dimer
formation. Insert shows titration for Ka calculation. c pH dependence of
the emission of excimer of a,c-D6 (40 lM) in water solution (35 mM
NaCl) regulated by the addition of NaOH (1 M) and HCl (1 M) to the
neutral solution. d Emission of a,c-CP6 in 10 mM phosphate buffer,
100 mM NaCl at pH 5.6 (340 nm excitation wavelength), from 7.1 lM
(red lines) to 83.0 lM (light green inverted triangles)
123

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M. J. Perez-Alvite et al.
626
basic conditions than in acidic media, with the optimal
conditions identified at pH 5.4 (Fig. 1c).
solvents (the excimer/monomer intensity ratio (IE/IM),
which was calculated from the fluorescence intensity of
the monomer (376 nm) and excimer (470 nm), takes a
value of 18 in water and 2.5 in CHCl3). A monoexpo-
nential decay was also observed for the pyrene emission
wavelength (380 nm) with a long life-time factor (100 ns)
compared to organic solutions (Figure 4 SI in support-
ing information), confirming that there is no intercon-
version between the monomer emitting form to the
excimer one.
Time-resolved fluorescence techniques in nonpolar sol-
vents (CHCl3) showed a triexponential fluorescence decay
at the excimer emission wavelength (475 nm, see Fig. 2a
and Figure 3 SI in supporting information) (Brea et al.
2007c, 2011). The shorter lifetimes (2.0 and 7.6 ns) show
negative amplitudes, which must be assigned to the for-
mation of the excimer. The longer lifetime (22.8 ns) shows
positive amplitude and must correspond therefore to the
excimer decay. We interpret the fact that two different rate
constants contribute to the excimer formation process as
indicative of the geometrical rearrangements that are nee-
ded for excimer formation (Brea et al. 2007c, 2011). This
result suggests that in nonpolar media the pyrene side
chains are not pre-organized in a stacked structure, which
is only formed in the excited state due to the greater
interaction between the molecules of pyrene when one of
them is in the first excited singlet state (excimer interac-
tion). Based on this, we assume that the p-p stacking
interaction of the aromatic moieties is undetectable in
organic solvents and is therefore not contributing to sta-
bilization of the dimer structure. A completely different
behavior is found in aqueous solution, as deduced from the
monoexponential decay of the excimer fluorescence
(65.0 ns, Fig. 2a and Figure 4 SI in supporting informa-
tion). The non-observation of a rise-time in the fluores-
cence decay emission of the excimer in water implies that
the pyrene moieties are already stacked in the electronic
ground state, i.e., the hydrophilic solvent favors the
stacking interaction of the pyrene moieties. This fact cau-
ses the much greater contribution of the excimer band to
the fluorescence spectrum in water as compared to organic
Finally, we decided to study the control and switching of
the dimer register by altering external signals (Zhang et al.
2009). At neutral pH, a,c-CP6 (2.5 10-5 M) self-assembles
into the corresponding excimer-emitting dimer (a,c-D6Z),
as discussed above (Scheme 3). Addition of divalent cat-
ions, such as Ca2? (CaCl2), Ba2? [Ba(CF3SO3)2] or Mg2?
(MgCl2), led to the disappearance of the excimer signal
(Fig. 2b) and an increase in the monomer band. These
changes are attributed to the formation of a,c-D6X.Ca21 as
a result of the coordination of the two carboxylic side
chains with the divalent cations. Addition of monovalent
cations, such as Na? or K?, or even divalent ions such like
Zn2?, did not cause any noteworthy change in either the
excimer emission or the dimer register. The calcium-
coordinated dimer (a,c-D6X.Ca21) can revert to the
Z–form (a,c-D6Z) by treatment with tetrabuylammonium
fluoride, which induces the precipitation of CaF2 and
restores the excimer emission. On the other hand, the
addition of oxalic acid or its sodium salt to the a,c-D6Z
solution (1 9 10-5 M) again caused a reduction in the
emission of the 470 nm band (Figure 5SI in supporting
information). In this case, the double salt bridge interaction
between Lys side chains with the carboxylates of oxalate
Fig. 2 a Fluorescence decay of 5 lM a,c-D6 in chloroform (475 nm,
black) and in water (480 nm, grey), with excitation at 333 nm.
b Pyrene fluorescence emission (337 nm excitation wavelength) of
2.5 9 10-5 M a,c-CP6 in 30% DMSO:CHCl3 (red lines) upon
addition of 1.1 (blue circles), 3.4 (green squares) equiv of calcium
chloride and 1.1 (black diamonds) and 10 (orange inverted triangles)
equivalent of TBAF, suggesting switching between dimer a,c-D6z and
a,c-D6x (see Scheme 3)
123

Toward the rational design of molecular rotors ion sensors based on a,c-cyclic peptide dimers
627
Scheme 3 Representation of
inter-dimer interconversion of
a,c-D6x, a,c-D6y and a,c-D6z
through the addition of different
chemical signals (Ca2?/TBAF
or oxalate/morpholine)
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