Synthesis of a Rhodamine-Appended Cyclophane as a Fluorescence Host in Water

A cationic water-soluble cyclophane (1a) having a rhodamine moiety as a red-fluorescence fluorophore was prepared by reaction of a monoamine derivative of tetraaza[6.1.6.1]paracyclophane having three N-t-butoxycarbonyl-β-alanine residues with rhodamine B isothiocyanate, followed by removal of the protecting groups. The guest-binding behavior of 1a toward anionic guests such as dabsyl derivative and 4-(1-pyrene)butanoate was investigated by fluorescence spectroscopy. The results suggested the formation of host-guest complexes with a stoichiometric ratio of 1:1 and the binding constants (K) of the host-guest complexes were evaluated.


Introduction
In recent years, much attention has been focused on development of fluorescent host sensor systems, which are able to detect small organic compounds [1].Many types of fluorophore-appended macrocyclic hosts based on cyclodextrins [2], calixarenes [3], and cyclophanes [4] were widely investigated.Numerous successful studies of fluorophore-appended hosts based on these macrocyclic compounds were reported [5].Among them, azacyclophanes [6] having a hydrophobic internal cavity are favorable candidates as the framework of macrocyclic host, because shape and size of the cavity can be easily designed for the capture of target guest molecules.In addition, exterior modifications of azacyclophanes can be achieved by the introduction of various functional groups such as polar side chains for water-solubility and fluorophores for fluorescent sensing onto the nitrogen atoms through an appropriate spacer [7].In the preceding paper, we have developed water-soluble blue fluorescent cyclophanes [8], which are composed of a tetraaza[6.1.6.1]paracyclophaneskeleton, three polar side chains for water-solu-bility, and a pyrene fluorophore.The pyrene-appended cyclophanes showed characteristic fluorescence spectra originated from pyrene moiety in aqueous media upon irradiation with UV light [8].A fluorescence intensity originated from the pyrene-appended host decreased upon addition of 8-anilino-naphthalene-1-sulfonate (ANS) as a guest [9], reflecting the formation of host-guest complexes [8].
On the other hand, many types of fluorescent dyes such as fluorescein isothiocyanate [10], rhodamine derivatives [11], and molecular beacons [12] have been designed and developed in order to investigate interactions of biomolecular complexes and assemblies.Among them, rhodamine derivatives emitting in the red region of visible spectrum are widely used as fluorescent labels for lipids, proteins, peptides, nucleic acids, and other biomolecules [13].They display high absorption coefficients and emission in the visible region, high fluorescence quantum yields, and high chemical stability and photostability [11].In the course of our ongoing research on cyclophanes capable of performing guest-inclusion and fluorescent sensing, we became interested in developing fluorescent cyclophanes emitting in longer wavelength ranges than UV waves.As a water-soluble red fluorescent cyclophane, we have now designed cationic cyclophane bearing a rhodamine moiety (1a) and analogous anionic cyclophane (1b) (Figure 1).We report here the synthesis of water-soluble cyclophane having a rhodamine moiety and its guest-binding abilities.

Precursor of 1a (3)
Piperidine (1.0 mL) was added to a solution of cyclophane derivative bearing N-protected amines (2) (179 mg, 0.14 mmol) in dry dichloromethane (DCM, 5 mL), and the mixture was stirred for 5 h at room temperature.Then the solvent was evaporated off under reduced pressure to give a pale yellow solid (monoamine of cyclophane).The monoamine of cyclophane was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluant.The precursor fraction was evaporated to dryness under reduced pressure to give a pale yellow solid (cyclophane monoamine, 152 mg).Triethylamine was added to a solution of the monoamine of cyclophane (140 mg, 0.13 mmol) in dry DCM (8 mL) at room temperature, and the mixture was allowed to stand at same temperature.The mixture was added to a solution of rhodamine B isothiocyanate (91 mg, 0.17 mmol) in dry DCM (2 mL), and the resulting mixture was stirred for 1 day at the same temperature.After being dried (Na 2 SO 4 ), the solution was evaporated to dryness under reduced pressure to give a dark purple solid.The crude product was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluant.Evaporation of the product fraction under reduced pressure gave a dark purple solid (151 mg, 73%): mp 144˚C -145˚C. 1 H NMR (400 MHz, CDCl 3 , 293 K) δ 1.3 (m, 12H), 1.4 (m, 35H), 2.1 (m, 8H), 3.3 (m, 8H), 3.5 (m, 8H), 3.6 (m, 8H), 3.9 (m, 4H), 5.3 (m, 5H), 6.6 (m, 4H), 7.0 (m, 10H), 7.1 (m, 10H) and 7.5 (m, 1H). 13

Computational Procedure
The calculations were carried out on a Pentium 4 3.2 GHz × 2 machine using Macro Model 9.1 molecular modeling software on a Red Hat Enterprise Linux WS 4.3 operating system.The geometry of 1a and 1b was optimized using molecular mechanics employing the OPLS_2005 force field for the simulation of the hosts.The geometry was optimized without any constraints allowing all atoms, bonds, and dihedral angles to change simultaneously.

Binding Constants of Cyclophanes with the Guests
To each solution of fluorescent cyclophane (0.5 μM) in HEPES buffer were added increasing amounts of 5 and 6, and the fluorescence intensity was monitored after each addition by excitation at 558 nm.Aqueous stock solution of 5 was prepared after addition of NaOH.The binding constants were calculated on the basis of the Benesi-Hildebrand method for titration data.

Design and Synthesis of Rhodamine-Appended Cyclophanes
From a viewpoint of development of cyclophanes emitting in the red region of visible spectrum, we have de-signed water-soluble cyclophanes having a rhodamine moiety.Actually, we have adopted a simple strategy to prepare rhodamine-appended cyclophanes by introducing a rhodamine moiety into tetraaza[6.1.6.1]paracyclophane[15] through a β-alanine spacer.Rhodamine-appended cyclophanes bearing cationic and anionic polar side chains 1a and 1b, respectively, were synthesized by following the reaction sequence shown in Scheme 1.In the preceding paper, we have synthesized a cyclophane derivative bearing N-protected amines 2 as a key intermediate [14].A precursor (3) of 1a was synthesized by a reaction of rhodamine B isothiocyanate (RITC) [16] with a monoamine derivative of cyclophane, which was easily prepared from 2 by removal of the Fmoc protecting group with piperidine, in a 73% yield.Cationic cyclophane bearing a rhodamine moiety 1a was derived from 3 by a treatment with trifluoroacetic acid (TFA).Then, 1a was converted to a cyclophane having carboxylic acid residues 1b by a reaction with succinic anhydride.New compounds were fully characterized by means of spectroscopy ( 1 H and 13 C NMR, and TOF-MS) and elemental analysis.Even though compounds 1a and 1b contain a hydrophobic cavity, both compounds were soluble in aqueous neutral media at biological pH owing to three polar side chains.From a practical standpoint, cyclophanes 1a and 1b had good H 2 O-solubility of 0.27 and 0.38 g/mL, respectively.Judging from molecular mechanics studies of cyclophanes 1a and 1b, both compounds provide a rigid internal cavity and the peripheral polar side chains with reasonably separated distances from the cavity (Figure 2).These results indicate that 1a and 1b having hydrophobic cavities were expected to act as water-soluble hosts.

Guest-Binding Behavior of Cyclophanes
As mentioned above, rhodamine derivatives have an intense visible absorption.Actually, rhodamine-appended water-soluble cyclophanes 1a and 1b had high absorption coefficients and absorption in the visible region owing to the rhodamine moieties.In addition, they showed fluorescence emission spectra originated rhodamine moieties with a fluorescence maximum at 579 nm in aqueous media 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 (Figure 3 for 1a).First, the guest-binding behavior of 1a toward anionic dabsyl derivative 5 as a dark quencher guest, was examined by fluorescence spectroscopy in aqueous HEPES buffer (0.01 M, pH 7.4, 0.15 M with NaCl).The fluorescence intensity originated from 1a at 579 nm decreased upon addition of 5, reflecting formation of 1a•5 complexes, as shown in Figure 3(a).The stoichiometry for the complex was confirmed to be 1:1 1a:5 by a Job plot (Figure 4(a)).The 1:1 binding constant (K) of 1a toward 5 was calculated to be 2.7 × 10 4 M −1 on the basis of the Benesi-Hildebrand relationship.On the other hand, the K value of anionic cyclophane 1b with the identical guest 5 was not determined due to the low affinity in HEPES buffer by the identical method.These results indicate that the electrostatic interaction between host and guest molecules is effective recognition factor for the Scheme 1. Preparation of rhodamine-appended cyclophanes 1a and 1b.host-guest complexation.A similar fluorescence feature was observed when 4-(1-pyrene)butanoate ( 6) was employed as an anionic florescence guest.That is, upon addition of 6 to an aqueous solution containing 1a, fluorescence intensity originated from 1a decreased, as shown in Figure 3(b), reflecting the formation of host-guest complexes.Such fluorescence quenching of 1a at 579 nm seems to be caused by the interactions between rhodamine group of 1a and entrapped pyrene molecule.The stoichiometry for the complex was also confirmed to be 1:1 1a:6 by a Job plot (Figure 4(b)).The K value of 1a with 6 was calculated to be 2.9 × 10 4 M −1 , which was almost comparable to that of 1a with 5.

Conclusion
Rhodamine-appended cyclophanes bearing three cationic polar side chains 1a were successfully prepared by reaction of RITC with a monoamine derivative of cyclophane, followed by removal of the protecting groups in a fairly good yield.1a showed fluorescence bands with a fluorescence maximum at 579 nm in an aqueous HEPES buffer.Formation of the host-guest complexes of the present cyclophane with anionic guests was demonstrated by fluorescence quenching experiments.The fluorescence intensity originating from 1a was subjected to decrease, upon complexation with anionic guests such as 5 and 6.

Figure 2 .
Figure 2. Computer-generated CPK models for 1a (a) and 1b (b).Carbon, hydrogen, oxygen, nitrogen, and sulfur atoms are shown in green, white, red, blue, and yellow respectively.