Amino Acids 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 Marı´a Jesu´s Pe´rez-Alvite • Manuel Mosquera Luis Castedo • Juan R. Granja

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Received: 11 January 2011 / Accepted: 8 March 2011 Ó Springer-Verlag 2011

Abstract 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 switched by controlled addition of divalent cations (e.g., Ca, Mg) or oxalic acid.

PCBA SPN TBAF TBTU

Keywords Self-assembling  Cyclic peptide  Molecular rotor  c-Amino acid  Dimer  Excimer

Introduction

Abbreviations Acp 3-Aminocyclopentanecarboxylic Acid CPs Cyclic peptides a,c-D Dimer of a a,c-cyclic peptide DIEA Diisopropylethylamine 4-DPPBA 4-(Diphenylphosphino)benzoic Acid ext-TTF 2-[9-(1,3-dithiol-2-ylidene)anthracen-10(9H)ylidene]-1,3-dithiole Pap 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. M. J. Pe´rez-Alvite  L. Castedo  J. R. Granja (&) Departamento de Quı´mica Orga´nica, Unidad Asociada al CSIC y Centro Singular de Investigacio´n en Quı´mica Biolo´gica y Materiales Moleculares, Universidad de Santiago de Compostela, Campus Vida, 15782 Santiago, Spain e-mail: [email protected] M. Mosquera Departamento de Quı´mica Fı´sica y Centro Singular de Investigacio´n en Quı´mica Biolo´gica y Materiales Moleculares, Universidad de Santiago de Compostela, Campus Vida, 15782 Santiago, Spain

[6,6]-phenyl-C61-butyric acid Self-assembling Peptide Nanotubes Tetrabutylamonium chloride O-Benzotriazol-1-yl-N,N,N0 ,N0 tetramethyluronium tetrafluoroborate

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 macromolecular 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 (Matsui and Gao 2005; Ashkenasy et al. 2006; Ko¨nig and Kilbinger 2007; Ulijn and Smith 2008 and Pazos et al. 2009). b-sheet-forming peptides are particularly interesting, 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

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composition but also on their inter-chain relative orientation (Khakshoor and Nowick 2008; Remaut and Waksman 2006 and Searle and Ciani 2004). Control of b-sheet formation and register structure is highly desirable. 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 together are b-sheet-type interactions (Amorı´n 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,cD2Z) 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 equimolecular 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 interspace electron transfer only takes place in dimer a,c-D3-4Z, where the fullerene acceptor (PCBA) and the donor (extTTF) 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 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)

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fully understand and control self-assembling CP structures. We describe here a new a,c-CP system with a large association constant, both in organic and aqueous media, and in which the isomeric dimer formed is under control and can be switched on demand.

Materials and methods (L)-2-(tert-butoxycarbonylamino)-5-(pyren-1-yl)pent-4ynoic 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-butoxycarbonylamino)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 mixture. 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-butoxycarbonylamino)-5-(pyren-1-yl)pent-4-ynoic acid as a yellow foam [80%, Rf = 0.15 (5% MeOH in CH2Cl2)]. 1H 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,

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

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. (L)-2-(t e r t-butoxycarbonylamino)-5-(pyren-1-yl)pentan o i c a c id (L-Boc-Pap-OH) A solution of (L)-2-(tertbutoxycarbonylamino)-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. 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, Amorı´n 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-PapD-MeN-c-Acp-L-Glu(OBn)-D-MeN-c-Acp-L-Lys(Z)-D-MeNc-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

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, CH2Bn, 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]? calculated for C68H82N7O10 1156.6118, found 1156.6111. 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-cAcp-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), 4.934.65 (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),

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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]? calculated for C53H70N7O8 932.5280, found 932.5265. Time-resolved fluorescence Fluorescence lifetimes were determined by time-correlated single-photon counting on an Edinburgh Instruments CD900 spectrometer equipped with a hydrogen-filled nanosecond 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 bandpass for the lifetime measurements was usually 20 nm. The experiments were performed at room temperature, and samples were purged with argon prior to measurement.

Results and discussion 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

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-2aminopentanoic acid is shown at the bottom

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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. 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-bromopyrene and 10% Pd on carbon in an aqueous medium (Lo´pez-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-butoxycarbonylamino)-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 (see Scheme 1 SI in supporting information) (Amorı´n et al. 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,cCP6. 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

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

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 nonequivalent 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 peptide a,c-CP6 precluded the measurement of its Ka in chloroform. However, this compound has an association constant of 4.5 9 105 M-1 in 20% DMSO/CHCl3 (Fig. 1b) and shows, as expected, an increased dimerization 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 pHdependent 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)

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basic conditions than in acidic media, with the optimal conditions identified at pH 5.4 (Fig. 1c). Time-resolved fluorescence techniques in nonpolar solvents (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 formation 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 needed 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 interaction). 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 stabilization 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 information). The non-observation of a rise-time in the fluorescence 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 causes the much greater contribution of the excimer band to the fluorescence spectrum in water as compared to organic

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 monoexponential 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 supporting information), confirming that there is no interconversion between the monomer emitting form to the excimer one. 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 cations, 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 calciumcoordinated 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)

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Toward the rational design of molecular rotors ion sensors based on a,c-cyclic peptide dimers 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)

must be responsible for the swap to the Y-form dimer [a,cD6y.(CO2)22]. Addition of similar amounts of acetic acid or sodium acetate to the a,c-D6Z dimer did not induce any significant change in the excimer emission, suggesting that the previously observed dimer switching is neither due to the ionic strain of the media nor due to pH changes but to the interaction between oxalate and Lys side-chains being stronger than those existing in a,c-D6Z. Finally, dimer a,cD6y.(CO2)22 could be switched back to the excimeremitting form by the addition of morpholine (Dermer and Dermer 1937).

Conclusion In summary, we have carried out a new self-assembly process based on an a,c-CP that has precise control of the supramolecular ensemble and, at the same time, the three non-equivalent dimers can be inter-switched by addition of chemical signals that compete with the inter-strand salt bridge interaction present in the excimer-emitting dimer. The process is followed by the characteristic emission of a pyrene moiety linked to the CP. The design process could have applications in the development of sensors or molecular rotors through the introduction of appropriated substituents on amino acid side chains. Work is in progress to investigate this possibility further. Acknowledgments This work was supported by the Spanish Ministry of Science and Innovation (MICINN) and the ERDF [SAF200761015, CTQ2007-68057-C02-01/BQU and Consolider Ingenio 2010 (CSD2007-00006)], the Xunta de Galicia (PGIDIT08CSA047209PR and GRC2010/012) and European project Magnifyco (NMP4-SL2009-228622). MJPA thanks the Spanish MICINN for her PhD contract (FPI).

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Toward the Rational Design of Molecular Rotors Ion Sensors María Jesús Pérez-Alvite,† Manuel Mosquera,‡ Luis Castedo,† Juan R. Granja†*

†

Departamento de Química Orgánica y Unidad Asociada al C.S.I.C. Centro Singular de Investigación en Química Biológica y Materiales Moleculares, Campus Vida, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, (Spain). ‡

Departamento de Química Física y Centro Singular de Investigación en Química Biológica y Materiales Moleculares, Campus Vida, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, (Spain). [email protected]

SUPPORTING INFORMATION

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Scheme 1SI: Synthetic strategy for preparation of α,γ-cyclic peptides (α,γ-CP5 and α,γ-CP6)

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Figura 1SI: Fluorescence emission spectra (excitation wavelength 337 nm) of α,γ- CP5 [0.17 µM (green) to 7.28 µM (orange)] in CHCl3. Spectra were normalized to a value of one at the 0–0 transition for clear representation. Titration for KA (1.6 x 106 M-1) calculation in chloroform.

Figura 2SI: Fluorescence emission spectra (excitation wavelength 337 nm) of α,γ- CP6 [7.1 µM (red) to 82.7 µM (light green)] in phosphate buffer 10 mM, 100mM NaCl and titration for KA (1 x 104 M-1) calculation at pH 5.2.

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Figure 3SI. Time resolved 390 nm (blue) and 475 nm emission (green) of 5 µM α,γ-D6 in cloroform, with excitation at 333 nm. Red lines are the best fit to the experimental data. Time-resolved fluorescence techniques in nonpolar solvents (CHCl3) showed a fluorescence decay at 390 nm fitted to a three exponential model with three different times of 25.1, 6.5 and 0.16 ns. The longer time (25.1 ns) corresponds to the normal pyrene emission decay in a nondeoxygenated solvent. The shorter lifetime of 6.5 ns and the 0.16 ns time are assigned to the process of conformational change that gives the required geometrical arrangement for excimer formation – i.e. the dimer with pyrene side chains in register (α,γ-D2Z, Figure 1).

Figure 4SI. Time resolved 380 nm (green) and 480 nm emission (blue) of 5 µM α,γ-D6 in water, with excitation at 333 nm. Red lines are the best fit to the experimental data.

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Figura 5SI: Fluorescence emission spectrum (excitation wavelength 337 nm) of 1 x 10-5 M (20% DMSO/CHCl3) of α,γ-CP6 (red) upon addition of 5.5 (dark blue), 33 (dark green) equiv of sodium oxalate; followed by addition of 1.36 (black), 45.6 (orange), 137 (light blue) equiv of morpholine. This suggests the switching between dimer α,γ-D6z and α,γ-D6x (see Scheme 3).

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GENERAL METHODS, INSTRUMENT DETAILS AND MATERIALS ►General. Commercially available N-Boc amino acids, O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU) and O-Benzotriazol-1-yl-N,N,N′,N′tetramethyluronium tetrafluoroborate (TBTU) were all used as obtained from Novabiochem, Applied Biosystems or Bachem. Deuterated chloroform (CDCl3), 1-Bromopyrene, Perylene, N,N´Diisopropylcarbodiimide (DIC) and 4-Dimethylaminopyridine (DMAP) were obtained from Aldrich. All other reagents obtained from commercial suppliers were used without further purification unless otherwise noted. Dichloromethane (DCM) and piperidine were dried and distilled over calcium hydride1,2. DIEA was dried and distilled over calcium hydride, and then redistilled over nynhidrin121,2. DMF used in coupling steps was dried over 4 Å molecular sieves. Analytical thin-layer chromatography was performed on E. Merck silica gel 60 F254 plates. Compounds, which were not UV active, were visualized by dipping the plates in a nynhidrin solution and heating. Silica gel flash chromatography was performed using E. Merck silica gel (type 60SDS, 230-400 mesh). Solvent mixtures for chromatography are reported as v/v ratios. HPLC purification was carried out on Phenomenex Maxsil-10 silica column with CH2Cl2/MeOH gradients between 100 and 85:15 or Sugerlabor Inertsil C18 with CH3CN (0.1%TFA)/H2O (0.1%TFA) gradients between 5:95 and 95:5. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Varian-Inova 750 Mhz, Bruker AMX-500 MHz or Bruker WM-250 MHz spectrometers. Chemical shifts were reported in parts per million (ppm, δ) relative to tetramethylsilane (δ 0.00). 1H NMR splitting patterns are designated as singlet (s), doublet (d), triplet (t) or quartet (q). All first-order splitting patterns were assigned on the basis of the appearance of the multiplet. Splitting patterns that could not be easily interpreted are designated as multiplet (m) or broad (br). Carbon nuclear magnetic resonance (13C NMR) spectra were recorded on Bruker AMX-500 MHz or Bruker WM-250 MHz spectrometers. Carbon resonances were assigned using distortionless enhancement by polarization transfer (DEPT) spectra obtained with phase angles of 135. Electronic Impact (EI) mass spectra were recorded on a Hewlett-Packard HP5988A mass spectrometer. Fast Atom Bombardement (FAB) mass spectra were recorded on a Micromass Autospec mass spectrometer. Electrospray (ESI) mass spectra were recorded on a Bruker BIOTOF II mass spectrometer. Mass Spectrometry of Laser Desorption/Ionization-Time of Flight (MALDI-TOF) was obtained on a Bruker Autoflex mass spectrometer. UV/Vis measurements were made on a Cary 100 Bio UV/Visible spectrophotometer, using NaCl solution UV cells. FTIR measurements were made on a JASCO FT/IR-400 spectrophotometer using 5-10 mM in CHCl3 and placed in a NaCl solution IR cell. Fluorescence measurements were obtained on a SPEX FluoroMax-3 spectrofluorometer. ► 1H-NMR Assignments of Cyclic Peptides (CPs). The signals of the 1H NMR spectra of the peptides in CDCl3 were identified from the corresponding double-quantum-filled 2D COSY (2QFCOSY), TOCSY and/or NOESY and ROESY spectra acquired at concentration and temperature indicated. Mixing times (~250 ms or 400 ms) were not optimized. Spectra were typically acquired (1) Brown, H. C. “Organic Synthesis via Boranes”, Ed. John Wiley & Sons, 1975 (2) Perrin, D. D.; Armarego, W. I. F. “Purification of Laboratory Chemicals”, Ed. Pergamon Press, 1988

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using Bruker standard pulse sequences on 500 MHz apparatuses, and were referenced relative to residual proton resonances in CDCl3 (at 7.26 ppm). 1H-NMR spectra also were obtained on a Varian Inova-750 MHz spectrometer. ► 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 nanosecond flash lamp. The instrumental response width of the system is 1.0 ns. We measured usually until 10,000 counts were reached in (2 x 103 channels). The emission bandpass for the lifetime measurements was usually 20 nm. The experiments were performed at room temperature, and samples were purged with argon prior to measurement.

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EXPERIMENTAL SECTION Boc-[L-Pap-D-MeN-γ-Acp-]-OFm. A solution of L-Boc-MeN-γ-Acp-OFm3 (1.6g, 3.84 mmol) in a TFA/DCM mixture (1:1, 38 mL) was stirred at rt for 15 min. After removal of the solvent, the residue was dried under high vacuum for 3 h. The resulting TFA salt was dissolved in dry DCM (38 mL), and L-Boc-Pap-OH (1.6g, 3.84 mmol), HATU (1.61g, 4.22 mmol) and DIEA (2.68 mL, 23.04 mmol) were successively added. After 1 h stirring at rt, the solution was poured into a separation funnel and washed with HCl (5 %) and NaHCO3 (sat). The organic layers were dried over Na2SO4, filtered and concentrated under reduce pressure, providing a yellow oil that was purified by flash chromatography (40% AcOEt/hexane) to give 1.94 g of the wished dipeptide as a yellow foam [70%, Rf = 0.53 (40% AcOEt in hexane)]. 1H NMR (CD3OD, 500.14 MHz, δ): 8.287.76 (several m, 9H, pyrene), 7.72-7.17 (several m, 8H), 5.55-5.45 (dd, 1H, NH), 4.98-4.83 (m, 1H,), 4.72-4.65 (m, 1H), 4.57-4.40 (m, 2H), 4.22-4.12 (m, 1H), 3.53-3.30 (m, 2H), 2.86-2.74 (m, 1H), 2.72 (s, 2H), 2.65 (s, 1H), 2.05-1.50 (m, 9H), 1.47 (s, 9H); FTIR (293 K, CHCl3): 3425, 2970, 2945, 2870, 2359, 2324, 1705, 1635, 1447 cm-1; MS (ES+) [m/z (%)]: 760 ([M + K]+, 4), 743 ([M + Na]+, 100), 721 ([MH]+, 44); HRMS [MH]+ calculated for C47H49N2O5 721.3636, found 721.3647. Boc-[L-Glu(OBn)-D-MeN-γ-Acp-L-Lys(Z)-D-MeN-γ-Acp-]-OFm. A solution of Boc-[LGlu(OBn)-D-MeN-γ-Acp]-OFm (2.31 g, 3.6 mmol) in piperidine/DCM mixture (4:1, 38 mL) was stirred at rt for 20 min, and then the solvent removed under vacuum. The resulting residue was dissolved in DCM (40 mL) and washed with HCl (5%), dried over Na2SO4, filtered and concentrated, to give Boc-[L-Glu(OBn)-D-MeN-γ-Acp-L-Lys(Z)-D-MeN-γ-Acp-]-OH, which was used without further purification. A solution of Boc-[L-Lys(Z)-D-MeN-γ-Acp-]-OFm (2.46 g, 3.6 mmol) in a TFA/DCM mixture (1:1, 38 mL) was stirred at rt for 15 min. After removal of the solvents, the residue was dried under high vacuum and then dissolved in dry DCM (76 mL), and Boc-[L-Glu(OBn)-D-MeN-γ-Acp]-OH, HATU (1.59 g, 4.17 mmol) and DIEA (3.99 mL, 22.74 mmol) were successively added. After 1 h stirring at rt, the solution was washed with HCl (5%), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (80% AcOEt/hexanes) gave 3 g of wished tetrapeptide as a white foam [81%, Rf = 0.38 (AcOEt)]. 1H NMR (CDCl3, 500.14 MHz, δ): 7.68 (d, J = 7.5 Hz, 2H), 7.49 (d, J =7.5 Hz, 2H), 6.66-6.37 (m, 10H), 5.51-5.32 (m, 2H), 5.12-4.95 (m, 1H), 4.90-4.74 (m, 4H), 4.72-4.64 (m, 3H), 4.72-4.64 (m, 1H), 4.60-4.52 (m, 2H), 4.49-4.35 (m, 1H), 3.18-3.00 (m, 2H), 2.92-2.84 (m, 2H), 2.79-2.70 (m, 4H), 2.68-2.63 (m, 1H), 2.62-2.52 (m, 1H), 2.50-2.26 (m, 1H), 2.00-1.37 (m, 19H), 1.36-1.30 (m, 9H, CH3), 1.30-1.20 (m, 3H); MS (ES+) [m/z (%)]: 1050 ([M + Na]+, 100), 1028 ([MH]+, 27) HRMS [MH]+ calculated for C59H74N5O11 1028.5379, found 418.5350 Boc-[L-Pap-D-MeN-γ-Acp-L-Glu(OBn)-D-MeN-γ-Acp-L-Lys(Z)-D-MeN-γ-Acp-]-OFm. A solution of the dipeptide Boc-[L-Pap-D-MeN-γ-Acp-]-OFm (793 mg, 1.1 mmol) in a piperidine/DCM mixure (1:4, 11 mL) was stirred at rt for 20 min and then the solvent removed under vacuum. The resulting residue was dissolved in DCM (40 mL) and the solution washed with HCl (5%), dried over Na2SO4,

(3) Brea, R. J.; Amorín, M.; Castedo, L.; Granja, J. R Angew. Chem., Int. Ed. 2005, 44, 5710

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filtered and concentrated, to give Boc-[L-Pap-D-MeN-γ-Acp-]-OH, which was used without further purification. A solution of Boc-[L-Glu(OBn)-D-MeN-γ-Acp-L-Lys(Z)-D-MeN-γ-Acp-]-OFm (1.13 g, 1.1 mmol) in a TFA/DCM miture (1:1, 11 mL) was stirred at rt for 15 min. After removal of the solvents, the resulting residue was dried under high vacuum. After dissolving the resulting residue in dry DCM (22 mL), Boc-[L-Pap-D-MeN-γ-Acp-]-OH, HATU (460 mg, 1.21 mmol) and DIEA (1.15 mL, 6.6 mmol) were successively added. After stirring at rt for 1 h, the solution was washed with HCl (5%), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (80% AcOEt in hexane) gave 1 g of the hexapeptide as a white foam [63%, Rf = 0.41 (2% MeOH in DCM)]. 1H NMR (CDCl3, 500.14 MHz, δ): 8.32-8.14 (m, 1H), 8.11-7.85 (m, 7H), 7.83-7.74 (m, 1H), 7.71-7.64 (m, 2H), 7.54-7.45 (m, 2H), 7.37-7.20 (m, 14H), 6.75-6.07 (m, 3H), 5.53-5.37 (m, 1H), 5.08-4.95 (m, 4H), 4.94-4.74 (m, 3H), 4.67-4.55 (m, 2H), 4.51-4.33 (m, 2H), 4.19-4.05 (m, 1H), 3.46-3.17 (m, 3H), 3.17-2.97 (m, 2H), 2.94-2.70 (m, 9H), 2.68-2.49 (m, 1H), 2.42-2.21 (m, 2H), 2.04-1.37 (m, 25H), 1.38-1.31 (m, 9H, CH3), 1.30-1.01 (m, 5H); FTIR (293 K, CHCl3): 3419, 3321, 2966, 2938, 2866, 2361, 2332, 1718, 1626, 1450 cm-1; MS (ES+) [m/z (%)]: 1475 ([M + Na]+, 100), 1452 ([MH]+, 9), 1375 ([M + Na - Boc]+, 16), 1352 ([MH - Boc]+, 14); HRMS [MH]+ calculated for C87H102N7O13 1452.7530, found 1452.7521.

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NMR, IR SPECTRA (L)-2-(tert-butoxycarbonylamino)-5-(pyren-1-yl)pent-4-ynoic acid

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(L)-2-(tert-butoxycarbonylamino)-5-(pyren-1-yl)pentanoic acid (L-Boc-Pap-OH)

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c-[L-Pap-D-MeN-γ-Acp-L-Glu(OBn)-D-MeN-γ-Acp-L-Lys(Z)-D-MeN-γ-Acp-] (α,γ-CP5).

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HPLC of α,γ-CP5 in a gradient between 2-12% of MeOH in DCM in 30 min in a Kromasil silica 5 µm column.

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c-[L-Pap-D-MeN-γ-Acp-L-Glu-D-MeN-γ-Acp-L-Lys-D-MeN-γ-Acp-] (α,γ-CP6).

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Reverse phase HPLC of α,γ-CP6 (A: H2O 0.1%TFA B: CH3CN 0.1%TFA) with a gradient between 25-75% in 30 min in a Sugelabor Inertsil C18 analytical column (A monitored at 210 nm and B at 250 nm).

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