HEPES (N-(2-hydroxyethyl) piperazine-N’-2-ethane-sulfonic acid) buffer (0.01 M, pH 7.4, with 0.15 M NaCl) was purchased from GE Healthcare. The following compounds were obtained from commercial sources as guaranteed reagents and used without further purification: potassium 6-p-toluidinonaphthalene-2-sulfonate [K(TNS)] (from NacalaiTesque, Inc., Kyoto, Japan), 6-anilinonaphthalene-2-sulfonic acid (2,6-ANS) (from Molecular Probes, Eugene, OR). ¹H and ¹³C spectra were taken on Bruker NMR AVANCE III 400 spectrometer. Fluorescence spectra, IR spectra, and MALDI TOF MS were recorded on JASCO FP-750, Perkin-Elmer spectrum one, and BrukerAutoflex Speed spectrometers, respectively.

Acetic anhydride (0.5 mL) was added to a solution of cyclophane derivative

bearing three Boc-β-Ala residues 3 (105 mg, 0.103 mmol) [15] in dry dichloromethane (DCM, 3 mL) and resulting mixture was stirred for 2 hours at room temperature. Chloroform (100 mL) was added to the mixture, and then washed with 5% citric acid solution (20 mL). After being dried (MgSO₄), the solution was evaporated to dryness under reduced pressure. The residue was chromatographed on a column of silica gel (SiO₂) with ethyl acetate. Evaporation of the product fraction under reduced pressure gave a white solid (75 mg, 69%): ¹H NMR (400 MHz, CDCl₃, 298 K) δ 1.42 (m, 35 H), 1.74 (s, 3 H), 2.09 (m, 6 H), 3.25 (m, 6 H), 3.63 (m, 8 H), 3.96 (s, 4 H), 5.33 (s, 3 H), 6.95 (m, 8 H), and 7.19 (d, 8 H). ¹³C NMR (100 MHz, CDCl₃, 298 K) δ 22.8, 24.8, 28.4, 34.8, 36.4, 41.0, 48.6, 79.0, 128, 130, 140, 141, 156, 170, and 171. IR 1645 cm⁻¹ (C=O). Found: C, 67.68; H, 7.76; N, 9.19.Calcd for C₆₀H₈₁N₇O₁₀: C, 67.96; H, 7.70; N, 9.25. MALDI-TOF MS: m/z 1083.4 [M + Na]⁺, where M shows C₆₀H₈₁N₇O₁₀.

Trifluoroacetic acid (TFA, 1.0 mL) was added to a solution of 4 (75 mg, 0.071mmol) in dry DCM (3 mL), and the mixture was stirred for 2 hours at room temperature. Evaporation of the solvent under reduced pressure and the residue was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluent. Evaporation of the product fraction under reduced pressure gave a white solid (78 mg, quantitative): ¹H NMR (400 MHz, CD₃OD, 298 K) δ 1.45 (m, 8H), 1.68 (s, 3H), 2.29 - 2.41 (m, 6 H), 3.09 (m, 6 H), 3.66 (m, 8 H), 4.02 (s, 4 H), 7.01 - 7.16 (m, 8 H), and 7.34 (m, 8 H). ¹³C NMR (100 MHz, CD₃OD, 298 K) δ 22.7, 25.7, 32.4, 36.9, 41.7, 49.7, 130, 132, 141, 143, 171, and 173. IR 1634 cm⁻¹ (C=O). Found: C, 53.99; H, 5.81; N, 8.65. Calcd for C₅₁H₆₀F₉N₇O₁₀∙2H₂O: C, 53.82; H, 5.67; N, 8.62. MALDI-TOF MS: m/z 760.9 [M + H]⁺, 782.9 [M + Na]⁺, where M shows C₄₅H₅₇N₇O₄ as a free amine.

To a solution of cyclophane 2a (83 mg, 0.075 mmol) and triethylamine (TEA, 1 mL) in dry DCM (3 mL) was added succinic anhydride (45 mg, 0.45 mmol). The mixture was stirred for 4 days at room temperature. The solution was distilled off under reduced pressure to give a white solid. The residue was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluent. The resulting salt was converted into the sodium salt by ion-exchange chromatography on a column of Amberlite IR-120 with methanol as an eluent. Evaporation of the product fraction under reduced pressure gave a white solid. (53 mg, 63%): ¹H NMR (400 MHz, CD₃OD, 298 K) δ 1.41 (m, 8 H), 1.66 (s, 3 H), 2.17 (t, 6 H), 2.39 (m, 12 H), 3.07 (m, 6 H), 3.68 (m, 8 H), 4.02 (m, 4 H), 6.97 (m, 8 H), and 7.30 (m, 8 H). ¹³C NMR (100 MHz, CD₃OD, 298 K) δ 22.7, 25.6, 33.7 - 35.2, 36.6, 41.7, 49.5, 129, 131, 141, 142, 172 - 175, and 180. IR 1634 cm⁻¹ (C=O). Found: C, 57.73; H, 5.99; N, 8.61. Calcd for C₅₇H₆₆N₇Na₃O₁₃∙3H₂O: C, 58.01; H, 6.15; N, 8.31. MALDI-TOF MS: m/z 1059.3 [M − H]⁻,1083.1 [M + Na]⁺ where M shows C₅₇H₆₉N₇O₁₃.

Dicyclohexylcarbodiimide (DCC, 72 mg, 0.35 mmol) was added to a solution of Boc-protected cyclophane having a carboxylic acid 5 [15] (378 mg, 0.34 mmol) in dry DCM (3 mL) at 0˚C, and the mixture was allowed to stand at the same temperature while being stirred for 20 min. The mixture was added to a solution of 2a (102 mg, 0.093 mmol) in dry DCM (2 mL), and the resulting mixture was stirred for 5 days at room temperature. Precipitates that formed (N,N’-dicyclohexylurea) were removed by filtration, the solvent was eliminated under reduced pressure, and the residue was dissolved in ethyl acetate (EtOAc, 10 mL). Insoluble materials were removed by filtration, and the filtrate was evaporated to dryness under reduced pressure. The residue was chromatographed on a column of silica gel (SiO₂) with chloroform-methanol = 9:1 v/v as an eluent. Evaporation of the product fraction under reduced pressure and the residue was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluent. Evaporation of the product fraction under reduced pressure gave a white solid (123 mg, 33%): ¹H NMR (400 MHz, CDCl₃, 298 K) δ 1.43 (m, 113 H), 1.76 (s, 3 H), 2.09 (m, 24 H), 2.24 (m, 6 H), 2.38 (m, 6 H), 3.31 (m, 24 H), 3.63 (m, 32 H), 3.96 (s, 16 H), 5.33 (s, 9 H), 6.59 (s, 3 H), 7.01 (m, 32 H), and 7.21 (m, 32 H). ¹³C NMR (100 MHz, CDCl₃, 298 K) δ 22.8, 24.9, 28.4, 29.9, 31.3, 34.8, 41.0, 48.7, 78.9, 128, 130, 140, 156, and 172. IR 1644 cm⁻¹ (C=O). Found: C, 67.56; H, 7.59; N, 9.54. Calcd for C₂₃₁H₃₀₀N₂₈O₃₇∙3H₂O: C, 67.42; H, 7.50; N, 9.53. MALDI-TOF MS: m/z 4084.5 [M + Na]⁺, where M shows C₂₃₁H₃₀₀N₂₈O₃₇.

TFA (1.0 mL) was added to a solution of 6 (60 mg, 0.015 mmol) in dry DCM (3 mL), and the mixture was stirred for 2 hours at room temperature. Evaporation of the solvent under reduced pressure and the residue was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluent. Evaporation of the product fraction under reduced pressure gave a white solid (50 mg, 81%): ¹H NMR (400 MHz, CD₃OD, 298 K) δ 1.42 (m, 32 H), 1.62 (s, 3 H), 2.01 - 2.24 (m, 24 H), 2.39 (m, 12 H), 3.03 (m, 18 H), 3.25 (m, 6 H), 3.67 (m, 32 H), 4.01 (s, 16 H), 6.92-7.16 (m, 32 H), and 7.32 (m, 32 H). ¹³C NMR (100 MHz, CD₃OD, 298 K) δ 22.6, 25.4, 30.8, 31.7, 32.5, 35.1, 36.8, 41.7, 49.2, 129, 131, 140, 141, 142, 143, 171, 173, and 174. IR 1644 cm⁻¹ (C=O). Found: C, 57.43; H, 6.00; N, 9.11. Calcd for C₂₀₄H₂₃₇F₂₇N₂₈O₃₇∙5H₂O: C, 57.30; H, 5.82; N, 9.17. MALDI-TOF MS: m/z 3161.5 [M + H]⁺, 3183.5 [M + Na]⁺, where M shows C₁₈₆H₂₂₈N₂₈O₁₉ as a free amine.

To a solution of cyclophane 1a (81 mg, 0.019 mmol) and triethylamine (TEA, 1 mL) in dry DCM (3 mL) was added succinic anhydride (35 mg, 0.346 mmol). The mixture was stirred for 4 days at room temperature. The solution was distilled off under reduced pressure to give a white solid. The residue was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluent. The resulting salt was converted into the sodium salt by ion-exchange chromatography on a column of Amberlite IR-120 with methanol as eluent. Evaporation of the product fraction under reduced pressure gave a white solid. (50 mg, 61%): ¹H NMR (400 MHz, CD₃OD, 298 K) δ 1.42 (m, 32 H), 1.65 (s, 3 H), 2.12 - 2.28 (m, 36 H), 2.39 (m, 18 H), 2.53 (m, 18 H), 3.31 (m, 24 H), 3.66 (m, 32 H), 3.99 (m, 16 H), 6.99 (m, 32 H), and 7.29 (m, 32 H). ¹³C NMR (100 MHz, CD₃OD, 298 K) δ 23.0, 25.5, 30.5 - 31.6, 35.1, 36.6, 41.7, 49.9, 129, 131, 141, 142, 172 - 174, and 176. IR 1633 cm⁻¹ (C=O). Found: C, 59.83; H, 6.34; N, 8.84. Calcd for C₂₂₂H₂₅₅N₂₈Na₉O₄₆∙10H₂O: C, 60.07; H, 6.25; N, 8.84. MALDI-TOF MS: m/z 4059.1 [M ? H]⁻, 4083.6 [M + Na]⁺ where M shows C₂₂₂H₂₆₄N₂₈O₄₆.

To each solution of TNS and 2,6-ANS (1.0 μM) in HEPES buffer were added increasing amounts of the hosts such as 1a, 1b, 2a, and 2b. The fluorescence spectra were recorded after each addition of the hosts. The binding constants were calculated on the basis of the Benesi-Hildebrand method for titration data.

We designed cationic and anionic cyclophane tetramers (1a and 1b) as a branch-type host (Figure 1). The cyclophane tetramers are composed of four tetraaza[6.1.6.1]paracyclophane derivatives. Three external cyclophane derivatives are connected to a core cyclophane through an alkyl spacer having amide linkages. Hydrophilic polar side chains were introduced onto nine nitrogen atoms of the external cyclophanes to afford water-solubility on the resulting hosts, while one of nitrogen atoms on the core cyclophane was acetylated. Cationic and anionic cyclophane tetramers, 1a and 1b, have cationic ammonium and anionic carboxylate groups, respectively. Both cyclophane tetramers are expected to act as a water-soluble host having internal hydrophobic cavities. In addition, in order to compare its guest-binding ability, we also designed cationic and anionic cyclophanes (2a and 2b) as a monomeric host. These hosts were prepared based on Boc chemistry by following the reaction sequence given in Scheme 1. Cationic cyclophane 2a was prepared from acetylation of N,N’,N”-tris(Boc-β-alanyl)-1,6,20,25-tetraaza[6.1.6.1]paracyclophane (3) [16] with acetic anhydride, followed by removal of the Boc-protecting groups with TFA. A precursor of 1a, Boc-protected cyclophane tetramer 6 was prepared by condensation of 2a with three equivalent of Boc-protected cyclophane having a carboxylic acid 5 in the presence of dicyclohexylcarbodiimide (DCC). Cationic cyclophane tetramer 1a was obtained by removal of the protecting groups with TFA. Both anionic cyclophanes 1b and 2b were obtained from 1a and 2a, respectively, by a reaction with succinic anhydride. All new compounds were purified by gel filtration chromatography and identified by ¹H and ¹³C NMR and MALDI-TOF MS spectroscopy as well as by elemental analysis. Cationic cyclophane

Scheme 1. Preparation of cyclophanes 1a, 1b, 2a, and 2b.

tetramer 1a had good water-solubility of 0.4 g/mL. At least at concentrations below 60 μM of 1a, a good linear Beer’s plot of absorbance at 232 nm was obtained, which indicates that 1a was not self-aggregated. A similar property in the water-solubility of 1b, 2a, and 2b was also observed by identical methods.

Cyclophane tetramers provide four hydrophobic internal cavities for organic guest molecules in water, while monomeric cyclophanes haveone hydrophobic internal cavity. Therefore, the guest-binding abilities of cyclophane tetramers are expected to be larger than those of monomeric cyclophanes, reflecting multivalent effect [11] . In addition, it is expected that electrostatic attraction between the hosts and the guests is an effective driving force for molecular recognition. That is, the peripheral side chains such as cationic ammonium and anionic carboxylate groups are expected to play an important role for guest recognition.

In order to evaluate the guest-binding behavior of 1a, fluorescence titration experiments were executed by using anionic and hydrophobic guests such as 6-p-toluidinonaphthalene-2-sulfonate (TNS) and 6-anilinonaphthalene-2-sulfonate (2,6-ANS) in an aqueous HEPES (0.01 M, pH 7.4, with 0.15 M NaCl) buffer (Figure 2). These guests are well-known fluorescent probes, which exhibits intensive fluorescence in non-polar solvents, but with low fluorescence intensity in water [18] . Upon addition of an excess amount of 1a to the HEPES buffer containing TNS, a fluorescence intensity at around 433 nm originating from TNS increased, reflecting formation of the corresponding host-guest complex, as shown in Figure 3(a). Such experimental conditions at an excess amount of 1a are favorable for the formation of 1:1 host:guest complexes of 1a with the guest. The stoichiometry for the complex was actually confirmed to be 1:1 host:guest by Job’s continuous variation method. Binding constant for the formation of 1:1 host:guest complexes (K) of 1a with TNS was evaluated to be 4.8 × 10⁵ M⁻¹ on the basis of the Benesi-Hildebrand relationship [19] , and listed in Table 1. The fluorescence titration experiments toward TNS were executed by using 1b, 2a, and 2b (Figures 3(b)-(d), respectively) and the corresponding K values are summarized in Table 1. In a comparison between the guest-binding abilities of 1a and monomeric host 2a, the saturated fluorescence intensities upon complexation of 1a with TNS were much larger than those by 2a, as shown in Figures 3(a)-(c). The K value of 1a with TNS was about 80-fold larger than that of 2a with the identical guest, reflecting multivalent effect (Table 1).

Comparison between the guest-binding abilities of cationic host 1a and anionic host 1b gave us meaningful information as regards electrostatic interactions between the hosts and the guests. The saturated fluorescence intensities upon complexation of 1b with TNS were much smaller than those by 1a (Figure 3(a) and Figure 3(b)). The K values of 1b toward these guests were 1 order of magnitude weaker than those of 1a due to electrostatic repulsion between the anionic host and anionic guests (Table 1). Similar guest recognition based on electrostatic interactions was observed between monomeric cyclophanes 2a and 2b

^aExcitation: 326 and 318 nm for TNS and 2,6-ANS, respectively. ^bReference [17] .

(Figure 3(c) and Figure 3(d), Table 1). In addition, the above-mentioned multivalent effect on the guest binding was observed between anionic cyclophanes 1b and 2b (Figure 3(b) and Figure 3(d), Table 1). Therefore, 1a was found to be most desired host exhibiting both multivalent effect and favorable electrostatic interactions among the adopted hosts for TNS binding. Similar enhancements of 1b in the guest-binding ability were also observed, when 2,6-ANS was employed as a guest (Table 1).

In addition, we compared the guest-binding behavior between branch-type cyclophane tetramer 1a and pendent-type cyclophane oligomers C_n [17] (Figure 4). The K values of 1a with TNS and 2,6-ANS were larger than those of C₄ and comparable to those of C₅ (Table 1). These results suggested that molecular design of branch-type cyclophane oligomers was more suitable for multivalently enhanced guest-binding exhibition than that of pendent-type cyclophane oligomers.