Synthesis of Branch-Type Cyclophane Tetramers Having a Multivalently Enhanced Guest-Binding Ability

A cationic branch-type cyclophane tetramer (1a) was synthesized by introducing three Boc-protected cyclophane derivatives into a N-acetylated tetraaza[6.1.6.1]paracy-clophane derivative as a core skeleton through DCC condensation, followed by removal of the external Boc-protecting groups. Cationic cyclophane tetramer 1a exhibited a high affinity toward an anionic and hydrophobic fluorescent guest, TNS, with binding constant of 4.8 × 10 M. This value of 1a was about 80-fold larger than that of the corresponding monomeric cyclophane for the identical guest, reflecting multivalent effect on the guest binding. As for electrostatic recognition, the obtained binding constant of 1a was one order of magnitude larger than that of an analogous anionic cyclophane tetramer (1b) for the identical guest. These enhanced guest-binding abilities of 1a were easily evaluated by fluorescence titration experiments.


Introduction
Cyclophane having an internal cavity is an inherent host that binds guest molecules to form host-guest complexes [1] [2] [3].Much effort has been paid to develop water-soluble cyclophane derivatives to act as a host in water.Their guest-binding behaviors such as binding affinity and molecular recognition have been widely investigated [4] [5] [6].In the process of forming host-guest complexes in water, hydrophobic interaction is supposed to be a major driving force [7].Additionally, non-covalent interactions such as electrostatic and hydrogen bonding interactions become operative for molecular recognition [8] [9] [10].
O. Hayashida  However, hydrophobicity of the internal cavity provided by the simple-monomeric cyclophanes is not always enough to form stable host-guest complexes [11].On the other hand, naturally occurring multivalent receptors on cell-surfaces are claimed to exhibit enhanced binding abilities toward their ligands [12] [13] [14].
Certainly, multiplying of the binding sites is a reasonable approach to increase the binding affinities toward guest molecules [15].Recently, we have developed a liner-type cyclophane trimer, which was constructed with three macrocyclic skeletons and two connecting spacers [16].Alternatively, we have also developed pendent-type cyclophane oligomers such as dimer, trimer, tetramer, and pentamer that were constructed with several macrocyclic skeletons and a peptide backbone [17].Such liner-and pendent-type cyclophane oligomers show increased guest-binding abilities due to multivalent effects of macrocycles.In the course of our ongoing research on water-soluble hosts, we became interested in developing branch-type cyclophane oligomers, which are constructed with several macrocyclic skeletons in a branching fashion.Especially, it is interesting to compare the guest-binding behavior between branch-and pendent-type cyclophane oligomers.In addition, electrostatic interactions between these host and the guest molecules are regarded as an effective recognition factor.On these grounds, we designed branch-type cyclophane tetramers, 1a and 1b, as cationic and anionic water-soluble hosts, respectively (Figure 1).In addition, we also designed cationic and anionic cyclophanes, 2a and 2b, respectively, as a monomeric host (Figure 1).In this context, we report synthesis of 1a and 1b and their host-guest binding behavior from a viewpoint of multivalency effect and electrostatic recognition.This work has been directed toward the development of a new class of cyclophanes capable of performing sophisticated molecular recognition.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 (MgSO 4 ), the solution was evaporated to dryness under reduced pressure.The residue was chromatographed on a column of silica gel (SiO 2 ) with ethyl acetate.Evaporation of the product fraction under reduced pressure gave a white solid (75 mg, 69%):

Cationic Cyclophane 2a
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):

Anionic Cyclophane 2b
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.

A precursor of 1, Boc-Protected Cyclophane Tetramer 6
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 (SiO 2 ) 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%):
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.

Binding Constants of Cyclophane with Fluorescence Guests
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.

Synthesis of Branch-Type Cyclophane Tetramers
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]paracyclophanederivatives.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 Advances in Chemical Engineering and Science 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 1 H and

Binding Behavior for Fluorescence Guests
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 × 10 5 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 Advances in Chemical Engineering and Science
et al.

2.1. General Methods
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 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.