Synthesis and Guest-Binding Behavior of Water-Soluble Cyclophanes Bearing PEG Residues

A water-soluble cyclophane (1) having poly(ethylene glycol) (PEG) moieties has been prepared from a tetraazide-functionalized cyclophane derivative and four poly(ethylene glycol) methyl ether acetylenes by Cu(I)-catalyzed 1,3 dipolar cycloadditions (click chemistry). An analogous derivative having a pyrene moiety (2) was also prepared in a similar manner. The guest-binding behavior of 1 and 2 toward anionic guests such as 6-p-toluidinonaphthalene-2-sulfonate was investigated by fluorescence spectroscopy. The binding constants (K) of the host-guest complexes were evaluated.


Introduction
Cyclophanes are macrocyclic compounds that form host-guest complexes with various organic compounds [1].Among macrocyclic hosts, cyclophanes play a wider and prominent role in host-guest chemistry and supramolecular chemistry [2].In aqueous media, hydrophobic interaction is the major driving force for host-guest complexation [3].It is desirable for water-soluble cyclophanes as hosts to provide hydrophobic binding sites that can be well desolvated upon guest incorporation.Therefore, the hydrophobic internal cavity must be reasonably separated from hydrophilic groups that are required for giving water solubility to cyclophanes.In our previous study, we developed various water-soluble cyclophanes having a hydrophobic internal cavity by introducing polar side chains such as saccharide residues, ammonium, and carboxylate groups into the cyclophane skeleton through an alkyl spacer having amide linkage [4].
On the other hand, chemical modification of poly(ethylene glycol) (PEG) to peptides or proteins is currently used to offer improved water solubility and stability [5].In addition, PEG is generally considered biologically inert and safe [6].In the course of our ongoing research on water-soluble cyclophanes capable of performing guest-inclusion, we became interested in developing nonionic and water-soluble cyclophanes having PEG residues.Specifically, we have designed cyclophane 1 by introducing four PEG moieties into tetraaza [6.1.6.1]paracyclophane [7] through a spacer (Figure 1).We expected that PEG could improve the surface hydrophilicity of cyclophanes.In addition, we also designed pyrene-appended cyclophane bearing three PEG residues 2 (Figure 1).Cyclophane 2 is expected to act as water-soluble fluorescent host capable of binding and sensing guest molecules.We report here the synthesis of water-soluble cyclophanes having PEG residues and their guest-binding abilities.

Binding Constants of Cyclophanes with the Guests
To each solution of a fluorescent guests (1.0 μM) in HEPES buffer were added increasing amounts of the 1 at 298K, and the guest fluorescence intensity was monitored after each addition by excitation at 326 and 318 nm for TNS and 2,6-ANS, respectively.
To a solution of fluorescent cyclophane 2 (1.0 μM) in HEPES buffer were added increasing amounts of TNS and 2,6-ANS, and the fluorescence intensity was monitored after each addition by excitation at 322 nm.The binding constants were calculated on the basis of the Benesi-Hildebrand method for titration data.

Design and Synthesis of Pyrene-Appended Cyclophanes
Cu(I)-catalyzed Huisgen's 1,3-dipolar "click" cycloaddition of an azide and an alkyne has been increasingly used in many fields such as biological [10] and materials [11].We adopted the click reaction to synthesize the cyclophanes due to the advantages of its high efficiency, regioselectivity, and compatibility with reaction conditions.Water-soluble cyclophane bearing PEG residues 1 was synthesized by following the reaction sequence shown in Scheme 1.In the preceding paper, we have synthesized a tetraazide derivative of cyclophane 3 [8].Then, cyclophane 1 was synthesized by click reaction between 3 and poly(ethylene glycol)methyl ether acetylene (average Mn = 2000) in a good yield of 88 %.On the other hand, triazide derivative of cyclophane 5 was synthesized by a reaction of triamine derivative of cyclophane 4 [9] with α-azidoisobutyric acid in heptane in the Scheme 1. Preparation of cyclophanes 1 and 2.
presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (Scheme 1).Similarly, pyrene-appended water-soluble cyclophane bearing PEG residues 2 was synthesized by Cu(I)-catalyzed Huisgen's 1,3-dipolar cycloaddition of 5 with poly(ethylene glycol)methyl ether acetylene (average Mn = 2,000) in a good yield of 95 %.Even though compounds 1 and 2 contain a hydrophobic cavity, both compounds were soluble in aqueous neutral media at biological pH owing to three PEG side chains.From a practical standpoint, cyclophanes 1 and 2 had good H 2 O-solubility of 0.3 and 0.2 g/mL, respectively.Both 1 and 2 having hydrophobic cavities were expected to act as water-soluble hosts.

Guest-Binding Behavior of Cyclophanes
In order to evaluate guest-binding ability of 1 and 2, we adopted well-known fluorescent probes such as 6-p-toluidinonaphthalene-2-sulfonate (TNS) and 6-anilinonaphthalene-2-sulfonate (2,6-ANS), whose emission is extremely sensitive to the microenvironmental polarity of the surrounding medium in both intensity and wavelength [12].In addition, binding constants (K) for the formation of inclusion complexes of 1 with TNS and 2,6-ANS in a 1:1 molar ratio were evaluated on the basis of the Benesi-Hildebrand relationship [13] and summarized in Table 1.
On the other hand, pyrene-appended water-soluble cyclophane 2 showed characteristic fluorescence spectra originated the pyrene moiety in aqueous media (Figure 3).The guest-binding behavior of 2 toward TNS and 2,6-ANS, was also examined by fluorescence spectroscopy in aqueous HEPES buffer (0.01 M, pH 7.4, 0.15 M with NaCl).Upon addition of TNS to an aqueous HEPES buffer containing 2, a fluorescence intensity originated from the pyrene moiety of 1 at 376 and 396 nm decreased along with a concomitant increase of the fluorescence intensity of entrapped TNS molecules at around 500 nm, as shown in Figure 3  were also calculated on the basis of Benesi-Hildebrand relationship and summarized in Table 1.The K values of 2 with TNS and 2,6-ANS were larger than those of 1 with the identical guests.As mentioned above, the most important driving force for host-guest comeplexation of cyclophane in aqueous media is the hydrophobic interaction.These results indicated that effective hydrophobic microenviroments were provided by 2 due to cyclophane cavity as well as a pyrene moiety.

Conclusion
We found that Cu(I)-catalyzed 1,3 dipolar cycloaddition was useful to synthesize cyclophanes bearing PEG residues 1 and 2 with high efficiency.As a host, the K values for 1 with TNS and 2,6-ANS were 1.0 × 10 4 and 9.4 × 10 3 M −1 , respectively.On the other hand, 2 showed fluorescence bands with fluorescence maximum at 376 nm in an aqueous HEPES buffer.Formation of the host-guest complexes of 2 with the identical guests was demonstrated by fluorescence quenching experiments.The K values for 2 with TNS and 2,6-ANS were 2.3 × 10 4 and 2.3 × 10 4 M −1 , respectively, which were larger than those of 1 with the identical guests, reflecting the effective hydrophobic interactions.
First, the guest-binding behavior of cyclophane 1 toward TNS and 2,6-ANS was examined by fluorescence spectroscopy in aqueous HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer (0.01 M, pH 7.4, 0.15 M with NaCl) at 298 K.The fluorescence intensity originating from TNS increased along with a concomitant blue shift of the fluorescence maximum upon addition of a large excess amount of 1 as shown in Figure 2(a), showing that the guest molecules are incorporated into the hydrophobic cavity provided