Design of benzo-15-crown-5 azoprobe/ γ-cyclodextrin complexes for alkali metal ion recognition in water

Novel photosignal transduction systems of benzo-15-crown-5 (B15C5) azoprobe/ γ-cyclodextrin ( γ -CyD) complexes were designed. Four B15C5 azoprobes were synthesized by azo coupling of 4 ’ -aminobenzo-15-crown-5 with phenol (for 15C5-Az-Ph ) and N,N -dialkylanilines (for 15C5-Az-Cn ). These azoprobes exhibited strong induced circular dichroism (ICD) by forming an inclusion complex with γ -CyD. Examination of the spectral responses and 1 H NMR analysis of the B15C5 azoprobe/ γ -CyD complexes in the presence of alkali metal cations revealed that K + binding selectively induced dimer formation of the B15C5 azoprobes inside the γ -CyD cavity, which resulted in distinct changes in their UV-vis and ICD spectra in water. The effect of alkyl chain length of 15C5-Az-Cn  n  γ -CyD complexes was examined in 90% H 2 O/10% CH 3 CN (v/v). The highest K + ion response was noted for the 15C5-Az-C2  γ -CyD complex. The 15C5-Az-C4  γ -CyD complex was found to show abnormally high ICD responses for TMA + and Cs + .


Introduction
The selective transduction of molecular recognition events into physical or chemical signals is a key concept for the design of novel supramolecular sensors. 1 Cyclodextrins (CyDs) are attractive hosts for construction of supramolecular structures since water-soluble CyDs possess nanosize hydrophobic cavities which behave as a "molecular flask" to incorporate various guest molecules in water. 2 We have developed supramolecular benzo-15-crown-5 (B15C5) fluorophore/γcyclodextrin (γ-CyD) complexes for selective potassium (K + ) ion recognition in water. 3,4Dimerization of the fluorophore inside the γ-CyD was found to be selectively promoted by K + binding, which resulted in a K + -selective pyrene dimer emission.This finding led us to design more efficient photosignal transduction systems for alkali metal ion recognition in water.

-CyD
Azobenzene chromophores are known to exhibit strong induced circular dichroism (ICD) by forming an inclusion complex with CyDs based on the chiral nature of the CyD cavities. 5,6This ICD response is expected to be affected by the ion recognition events of the B15C5 modified azoprobe/γ-CyD complexes.In addition, an exciton interaction of the azodimer may induce distinct changes in the UV-vis absorption spectra. 7,8Thus, selective UV-vis and ICD spectral changes of the B15C5 azoprobe/γ-CyD complexes are expected for alkali metal ion recognition in water.
Based upon the above concept, we designed four simple structure of B15C5 azoprobes 15C5-Az-Ph and 15C5-Az-Cn (n = 1, 2, 4).The novel photosignal transduction systems of the B15C5 azoprobe/γ-CyD complexes for alkali metal ion recognition in water were assessed by ICD and UV-vis spectral characteristics as well as 1 H NMR analysis in relation to their supramolecular CyD complex structures.

Response properties of 15C5-Az-Ph/γ-CyD complex
Figure 1 shows (a) ICD and (b) UV-vis spectra of 15C5-Az-Ph (0.050 mM) in 10.0 mM γ-CyD aqueous solution containing 0.10 M tetramethylammonium chloride (TMACl) or alkali metal chlorides.In the presence of TMA + or Na + , the 15C5-Az-Ph/γ-CyD complex exhibited no changes in its UV-vis and ICD spectra.The maximum wavelength (370 nm) observed in the UVvis spectrum can be assigned to a long-axis polarized * transition of the -N=N-group in 15C5-Az-Ph. 10When the long-axis transition of the azo group is parallel to the symmetry axis of the CyD cavity, the ICD spectrum typically exhibits a strong positive Cotton effect at the * transition band.However, the observed positive ICD at n-* transition (440 nm) suggests that 15C5-Az-Ph forms a relatively loose 1:1 complex with γ-CyD, which results in the changes of the azo group angle inside the CyD cavity or locates the n-* transition of the azo group outside the CyD cavity. 5When 0.10 M KCl was added to this solution, the UV-vis spectrum shifted slightly to the shorter wavelength region, and the positive ICD at * transition was intensified with decreasing ICD intensity at longer wavelengths.These spectral changes imply a selective 2:1 complex formation of 15C5-Az-Ph with K + inside the γ-CyD cavity.

Response properties of 15C5-Az-C1/γ-CyD complex
To obtain more evidence of the response mechanism of the azoprobe/γ-CyD complex in water, 15C5-Az-C1 having an azochromophore structure similar to methyl orange (MO) was designed.Since the inclusion behavior of the γ-CyD for MO has been well studied, 11,12 the spectral characteristics of 15C5-Az-C1/γ-CyD can be directly assessed by comparing with those of the MO/γ-CyD system.Although the 15C5-Az-C1/γ-CyD complex formed a precipitate in the presence of 10.0 mM γ-CyD, a clear solution was obtained by reducing γ-CyD concentration to 0.50 mM.This indicates that the interaction with γ-CyD of 15C5-Az-C1 is much stronger than that of 15C5-Az-Ph in water.The spectral responses of the 15C5-Az-C1/γ-CyD complex are depicted in Figure 2. 13 Since the pKa of protonated 15C5-Az-C1 (15C5-Az-C1  H + ) was experimentally determined to be 3.43, the solution pH was adjusted to 7.0 by the addition of 5.0 mM H3PO4/TMAOH buffer to avoid protonation.In the presence of 0.10 M K + , the UV-vis absorption spectrum exhibited a blue shift with reduction of molar absorptivity in the longer wavelength region (Figure 2b).This spectral response is quite similar to that of the MO/γ-CyD complex when the MO dimer is formed inside the γ-CyD cavity. 8In addition, the split-type ICD spectrum observed for the 15C5-Az-C1/γ-CyD complex in the presence of K + (Figure 2a) is characteristic of the exciton interaction induced by azodimer formation inside the chiral environment. 11These results demonstrate that selective 2:1 complex formation of 15C5-Az-C1 with K + is taking place inside the γ-CyD cavity (Scheme 1), though the positioning of crown ether units at secondary rim side of γ-CyD in Scheme 1 has not been proved.

H NMR analysis of 15C5-Az-C1/γ-CyD complex
In order to raise the probe concentration to a 1.0 mM level to obtain sufficient sensitivity in the 1 H NMR signals, the solvent system of 80% D2O/20% CD3CN (v/v) was used.In the presence of 0.10 M KHCO3 (pH = 9.8), it was found that the 1 H NMR signals of the phenyl protons in 15C5-Az-C1 (1.0 mM) exhibited significant upfield shifts upon addition of γ-CyD (Figure 3b); the chemical shifts of the phenyl protons shown in Figure 3 were 7.66 (H a ), 7.37 (H b ), 7.26 (H c ), 6.99 (H d ), 6.76 (H e ) in the absence of γ-CyD, and 7.38 (H a ).7.26 (H b ), 6.95 (H c ), 6.93 (H d ), 6.55 (H e ) in the presence of 2.0 mM γ-CyD.It is evident that the upfield shifts of all phenyl protons are much larger for KHCO3 system (Figure 3b) than those for NaHCO3 system (Figure 3a).When azo dyes such as MO form a 1:1 inclusion complex with CyDs, their phenyl proton signals generally shift to a lower magnetic field due to van der Waals shift and steric perturbation. 12hus, the upfield shift observed for the 15C5-Az-C1/γ-CyD complex can be explained by the large ring current effect of the benzene ring from the corresponding probe in the azodimer.
For the 15C5-Az-C1/γ-CyD complex, a Job's plot analysis by 1 H NMR spectra was also carried out (Figure 4).The  in Figure 4

Binding efficiency and selectivity of 15C5-Az-C1/γ-CyD complex for K +
To evaluate the binding efficiency of the 15C5-Az-C1/γ-CyD complex for K + , the ICD and UVvis spectral responses of the 15C5-Az-C1/γ-CyD complex upon addition of K + in water were examined (Figure 5).The results revealed a clear crossover point in both the UV-vis and ICD spectra, indicating the presence of monomer/dimer equilibrium of 15C5-Az-C1 with the γ-CyD.
When the changes in the ICD spectra are induced only by the 2:1 complex formation of 15C5-Az-C1 with K + inside the γ-CyD cavity, the molar ellipticity () is expressed by eqn.1: where [L]t is the initial concentration of the probe and m and d are the molar ellipticities of the monomer and the dimer, respectively.Under constant γ-CyD concentration of 0.50 mM, the apparent 2:1 binding constant (K21 app ) of 15C5-Az-C1 with K + as calculated by a nonlinear program using the changes in  at 458 nm was (2.0 0.3) x10 5 M -2 .Similarly the changes in the ratio of the UV-vis absorbances at 418 nm and 449 nm (A1/A2) can be expressed as: where m and d are the molar absorptivities of monomer and dimer, respectively.From eqn. 2, the K21 app value for K + was determined to be (1.4 0.7) x10 5 M -2 .It is worth noting that the observed K21 app values of 15C5-Az-C1/γ-CyD complex are 10 4 -fold lower than the value reported for the B15C5 fluorophore/γ-CyD complex with K + (3.8 x10 9 M -2 ). 3 This is ascribed to the difference in the binding ability of probes with γ-CyD in water.In fact, a large difference in the K21 app value is also noted for the B15C5 fluorophores possessing different alkyl spacer chains. 3Therefore the binding efficiency is expected to be dramatically altered by proper modification of the probe structure such as changing the alkyl chain length of the dimethylamine unit in 15C5-Az-C1.The binding selectivity of the 15C5-Az-C1/γ-CyD complex for alkali metal ions was assessed from the ICD and UV-vis spectra (Figure 6).Based upon the 2:1 binding ability of B15C5 moiety with K + , 3,14 the 15C5-Az-C1/γ-CyD complex was found to exhibit selective response for K + over other alkali metal ions in water.

Effect of alkyl chain length in 15C5-Az-Cn upon the response function
To assess the effect of alkyl chain length, azoprobes with diethyl chains (15C5-Az-C2) and dibutyl chains (15C5-Az-C4) were additionally prepared, and their response functions as γ-CyD complexes examined.Due to the low solubility of 15C5-Az-C2 and 15C5-Az-C4/γ-CyD complexes in water, the response behavior of all of the 15C5-Az-Cn/γ-CyD complexes were evaluated in 90% H2O/10% CH3CN (v/v).The resultant UV-Vis and ICD spectra are summarized in Figure 7.It is evident that the response function of 15C5-Az-C1/γ-CyD complex decreased significantly due to low binding ability of 15C5-Az-C1 with γ-CyD in 10% CH3CN solution.For all 15C5-Az-Cn probes, the UV-Vis spectra exhibited a K + -selective blue shift with reduction of molar absorptivity in the longer wavelength (Figures 7b, d, f), indicating a selective 2:1 complex formation of 15C5-Az-Cn with K + .In the ICD spectra (Figures 7a, c, e), different spectral responses were observed.Thus for 15C5-Az-C1 and 15C5-Az-C2, the K + -selective ICD response was clearly intensified by increasing the alkyl chain length from methyl to ethyl.However for 15C5-Az-C4 possessing longer dibutyl chains, abnormally large ICD responses were noted for TMA + (Figure 7e) or Cs + (data was not shown).These different responses in the UV-Vis and ICD spectra may be ascribed to the difference in their binding mode with γ-CyD.Figure 8 shows the ICD response selectivity of 15C5-Az-Cn/γ-CyD complexes for alkali metal ions.It is evident that 15C5-Az-C1 and 15C5-Az-C2 exhibited K + selectivity based on the selective dimer formation of azoprobes inside the γ-CyD.However 15C5-Az-C4 showed the abnormally large ICD response toward Cs + .For ICD response, the relative positioning of 15C5-Az-Cn inside the γ-CyD strongly affects the shape of ICD spectra.As seen in Figure 7e, the strong ICD intensities for 15C5-Az-C4 are noted not only in the * and n-* regions of azo moiety but also in the  region of benzocrown ether unit.Thus the abnormal response observed for 15C5-Az-C4 may indicate the formation of different mode of CyD inclusion complex such as head-to-tail type 2:2 complex of 15C5-Az-C4 with γ-CyD (Figure 9).These are unique functions of supramomolecular CyD complexes based on the difference in inclusion ability of γ-CyD for 15C5-Az-Cn in the presence of alkali metal salts.

Conclusions
In this study, novel photosignal transduction systems of B15C5 azoprobe/γ-CyD complexes were designed.Four structures of the B15C5 azoprobes were synthesized by azo coupling 4'aminobenzo-15-crown-5 with phenol (for 15C5-Az-Ph) and N,N-dialkylaniline (for 15C5-Az-Cn).It was found that these azoprobes exhibited strong induced circular dichroism (ICD) by forming an inclusion complex with γ-CyD.Examination of the spectral responses and 1 H NMR analysis of the B15C5 azoprobe/γ-CyD complexes in the presence of alkali metal ions revealed that K + binding selectively induced dimer formation of the B15C5 azoprobes inside the γ-CyD cavity, which resulted in distinct changes in their UV-vis and ICD spectra.Thus the dynamic molecular recognition events of the B15C5 azoprobe/γ-CyD complexes induced by K + ion binding are successfully transduced into the UV-vis and ICD spectral responses in water.The apparent 2:1 binding constants of the 15C5-Az-C1/γ-CyD complex for K + determined by ICD and UV-vis analyses were (2.0 0.3) x10 5 M -2 and (1.4 0.7) x10 5 M -2 , respectively.To assess the effect of alkyl chain length in 15C5-Az-Cn n as γ-CyD complex, the response functions for alkali metal ions were examined in 90% H2O/10% CH3CN (v/v).The highest K + ion response was noted for 15C5-Az-C2 γ-CyD complex.For 15C5-Az-C4 γ-CyD complex, abnormally large ICD responses were observed for TMA + and Cs + .These results demonstrate that the binding efficiency can be tuned by proper modification of the azoprobe structure.The present photosignal transduction systems are expected to be useful in the design of more sophisticated supramolecular CyD complexes, 15 as well as CyD-based rotaxane sensors. 16

Experimental Section
General.UV-vis absorption spectra were recorded on a Hitachi U-3000 spectrophotometer with a 1-cm quartz cell.The absorption spectra of each sample were obtained by subtraction of the spectra of γ-CyD solution containing 1% or 10% CH3CN in the absence of probe.ICD spectra were obtained on a JASCO J-820 spectrophotometer with a 1-cm quartz cell. 1 H NMR spectra were obtained using a JEOL-GSX270 (270 MHz; JEOL DATUM).Water was doubly distilled and deionized by a Milli-Q Labo system (Millipore) before use.The γ-CyD (Wako Pure Chemical Industries) was used without further purification.Acetonitrile (fluorescence reagent, Nakalai Tesque) was used as-received.All other solvents and reagents were obtained at the highest commercial quality and used without further purification.