Molecular Recognition of Diazine Isomers and Purine Bases by the

Akira Ohashi,† Satoshi Tsukahara, and Hitoshi Watarai*. Department of Chemistry, Graduate School of Science, Osaka University,. Toyonaka, Osaka 560-...
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Langmuir 2003, 19, 4645-4651

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Molecular Recognition of Diazine Isomers and Purine Bases by the Aggregation of Palladium(II)-Pyridylazo Complex at the Toluene/Water Interface Akira Ohashi,† Satoshi Tsukahara, and Hitoshi Watarai* Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received November 14, 2002. In Final Form: February 18, 2003 We discovered a new type of molecular recognizing reaction of the interfacial aggregation of monocationic palladium(II)-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complex (PdL) with neutral diazine (Dz) or purine bases at the toluene-water system by use of centrifugal liquid membrane (CLM) spectrometry. The formation of the interfacial aggregates of PdL-Dz isomer complexes was investigated by CLM/UVvis spectroscopy, CLM/resonance Raman spectroscopy and optical microscopic images. The high molar absorptivity of the PdLCl complex (4.33 × 104 M-1 cm-1 at 564 nm) was drastically reduced by the formation of the interfacial aggregates with diazine. This suggested that the electric resonance structure (charged quinone structure) in Pd(II)-5-Br-PADAP was prevented by the interaction with ClO4-. The composition of the interfacial aggregates depended on the structure of Dz isomers; PdL-1,2-Dz formed fine domains with the molar ratio of 1,2-Dz/PdL ) 1:1, whereas PdL-1,4-Dz was as a very thin membrane with a molar ratio of 1,4-Dz/PdL ) 1:2. The ability for the formation of the interfacial aggregate of the PdL-Dz isomer complexes increased in the order 1,3-Dz < 1,2-Dz < 1,4-Dz. The formation of the interfacial aggregate of the PdL-Dz isomer was greatly dependent on the structure of Dz, not on the basicity of Dz. The interfacial aggregation was sensitively detected even in a diluted concentration lower than 10-5 M for 1,4-Dz. Moreover, it was demonstrated that the molecular recognition by the aggregation of PdL was applicable for the bases of nucleic acids. PdL could form the interfacial aggregates preferentially with purine bases (adenine and guanine).

Introduction Chemical reactions at the liquid-liquid interface have played an important role in various fields such as analytical separation chemistry, electrochemistry, biochemistry, and photochemistry.1-3 In particular, the formation of aggregate or assembly is one of the most specific reactions at the interface, because the concentration of monomer reactants at the interface can be increased by adsorption much higher than those in bulk phases. Recently, various spectroscopic methods have been developed to measure directly the liquid-liquid interfacial reaction: for example, attenuated total internal reflection (ATR) spectroscopy,4,5 total internal reflection fluorometry (TIRF),6-9 total internal reflected resonance light scattering (TIR-RLS),10 external reflection (ER) spectros* Corresponding author. E-mail: [email protected]. † Present address: Department of Environmental Sciences, Faculty of Science, Ibaraki University, Mito 310-8512, Japan. (1) Liquid-Liquid Interfaces. Theory and Methods; Volkov, A. G., Deamer, D. W., Eds.; CRC Press: Boca Raton, FL, 1996. (2) Liquid Interfaces in Chemical, Biological, and Pharmaceutical Applications; Volkov, A. G., Ed.; Marcel Dekker: New York, 2001. (3) Liquid Interfaces in Chemistry and Biology; Volkov, A. G., Deamer, D. W., Tanelian, D. L., Markin, V. S., Eds.; John Wiley & Sons CRC Press: Chicester, England, 1998. (4) Perera, J. M.; Stevens, G. W.; Grieser, F. Colloids Surf. A 1995, 95, 185-192. (5) Tsukahara, S.; Watarai, H. Chem. Lett. 1999, 89-90. (6) Fujiwara, M.; Tsukahara, S.; Watarai, H. Phys. Chem. Chem. Phys. 1999, 1, 2949-2951. (7) Fujiwara, N.; Tsukahara, S.; Watarai, H. Langmuir 2001, 17, 5337-5342. (8) Tsukahara, S.; Yamada, Y.; Watarai, H. Langmuir 2000, 16, 6787-6794. (9) Bessho, K.; Uchida, T.; Yamauchi, A.; Shioya, T.; Teramae, N. Chem. Phys. Lett. 1997, 264, 381-386. (10) Feng, P.; Shu, W. Q.; Huang, C. Z.; Li, Y. F. Anal. Chem. 2001, 73, 4307-4312.

copy,11,12 and second harmonic generation (SHG) spectroscopy.13,14 Besides these methods, the centrifugal liquid membrane (CLM) method is a very useful technique to measure interfacial reactions. This method utilizes a very thin two-phase liquid membrane with an extremely high specific interfacial area produced in a rotating cell, and it can directly measure the interfacial species by a transmission spectrometry15-17 or other spectrometries.18,19 2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol (5-BrPADAP) has been utilized as a colorimetric reagent for trace amounts of metal ions, for it can form intensely colored and stable complexes with various metal ions.20,21 Since 5-Br-PADAP is a tridentate ligand, it forms a 1:1 square-planar complex with palladium(II) leaving one site of palladium(II) for the coordination of another ligand. The palladium(II)-5-Br-PADAP (PdL) complex has specific characteristics, such as an extremely high molar absorptivity (4.33 × 104 M-1 cm-1 at 564 nm in toluene)22 and an interfacial adsorptivity at the toluene/water interface, and is a soft Lewis acid. Previously, we clarified (11) Moriya, Y.; Amano, R.; Sato, T.; Nakata, S.; Ogawa, N. Chem. Lett. 2000, 556-557. (12) Fujiwara, K.; Watarai, H. Bull. Chem. Soc. Jpn. 2001, 74, 18851890. (13) Corn, R. M.; Higgins, D. A. Chem. Rev. 1994, 94, 107-125. (14) Conboy, J. C.; Daschbach, J. L.; Richmond, G. L. J. Phys. Chem. 1994, 98, 9688-9692. (15) Nagatani, H.; Watarai, H. Anal. Chem. 1998, 70, 2860-2865. (16) Yulizar, Y.; Ohashi, A.; Watarai, H. Anal. Chim. Acta 2001, 447, 247-254. (17) Ohashi, A.; Watarai, H. Anal. Sci. 2001, 17, 1313-1319. (18) Nagatani, H.; Watarai, H. Chem. Lett. 1999, 701-702. (19) Ohashi, A.; Watarai, H. Chem. Lett. 2001, 1238-1239. (20) Johnson, D. A.; Florence, T. M. Talanta 1975, 22, 253-265. (21) Busev, S. I.; Vin’kova, V. A. Zh. Anal. Khim. 1967, 22, 552-556. (22) Ohashi, A.; Tsukahara, S.; Watarai, H. Anal. Chim. Acta 1998, 364, 53-62.

10.1021/la0268451 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/02/2003

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its molecular recognition ability for diazine (Dz) isomers at the toluene/water interface. The interfacial formation constants of 1:1 complex between PdL and Dz isomers increased in the order of the basicity of Dz: pyridazine (1,2-Dz, pKa 2.33) . pyrimidine (1,3-Dz, pKa 1.34) > pyrazine (1,4-Dz, pKa 0.59).23 In the present study, we studied the aggregation of the ternary PdL-Dz complexes at the toluene/water interface by the CLM method, and demonstrated the new principle of the interfacial detection of Dz isomer by the molecular recognizing aggregation. Moreover, it was proposed that the interfacial aggregation could be applied to the molecular recognition of purine bases of nucleic acids. Experimental Section Reagents. 5-Br-PADAP (Dojindo Lab.) was used as purchased and dissolved in toluene. Toluene (G. R., Nacalai Tesque) was purified with the same means as the literature.22 A PdLCl toluene solution was prepared by the solvent extraction in the following way; aqueous solution (20 mL) of 1.0 × 10-3 M palladium(II) chloride (99.99%, Wako Pure Chemicals) in 0.1 M hydrochloric acid and toluene solution (60 mL) of 5.6 × 10-5 M 5-Br-PADAP were introduced into the Erlenmeyer flask with a stopper, and the mixture was stirred overnight. Pyridazine (G. R., Nacalai Tesque), pyrimidine, and pyrazine (G. R., Tokyo Kasei) were used as purchased and dissolved in water. Adenine, guanine, cytosine, thymine, and adenosine were purchased from Nacalai Tesque. These compounds except guanine were dissolved in water, and guanine was dissolved in 0.01 M HClO4 and 0.09 M NaClO4 aqueous solution in order to increase its solubility. These stock solutions were stored at 4 °C in a refrigerator. Water was distilled and deionized with a Milli-Q system (Milli-Q SP.TOC., Millipore). Batch Examinations. Preliminary experiments for the formation of the aggregates of PdL-Dz and PdL-base complexes were carried out by the batch method. Equal volumes (5 cm3) of 1.2 × 10-5 M PdLCl toluene solution and Dz or base aqueous solution were shaken in a glass tube for 2 h. The concentrations of 1,2-Dz, 1,3-Dz, and 1,4-Dz were 4.0 × 10-4, 1.0 × 10-2, and 4.0 × 10-4 M, respectively, and the concentrations of adenine, guanine, cytosine, and thymine were 9.7 × 10-4, 1.1 × 10-4, 1.2 × 10-3, and 9.0 × 10-4 M, respectively. After the phase separation, the absorption spectra of both phases were measured with a UV/vis spectrophotometer (V-570, Jasco). All measurements in this study were carried out under the conditions that the ionic strength and pH were fixed at 0.1 M and 2.0 with (H+, Na+)ClO4-, respectively, in a thermostated room at 298 ( 2 K. UV)Vis Absorption Spectra Measurements of Interfacial Aggregates. The principles of the CLM method were described elsewhere.15 The apparatus of CLM method was essentially the same as the one reported previously.24 An aqueous solution (0.250 cm3) of Dz or base and a toluene solution (0.150 cm3) of 5.6 × 10-5 M PdLCl were introduced into a cylindrical cell, whose height and outer diameter were 3.3 and 2.1 cm, respectively. The cell was rotated at 10 000 rpm. The sum of absorption spectra of bulk phase and interface was measured with a UV/vis spectrophotometer (V-570, Jasco) in a wavelength range of 350-800 nm. The concentration of chloride ion was 0 or 2.0 × 10-3 M. The concentrations of Dz isomers and bases were varied in the ranges 0-0.08 and 0-2.9 × 10-3 M, respectively. Raman Spectra of Interfacial Aggregates. Measurements of Raman spectra for aggregates of ternary PdL-Dz complexes formed at toluene/water interface were carried out by a CLMresonance Raman microprobe spectroscopy, whose apparatus was essentially the same as the one reported previously19 as shown in Figure 1. An aqueous solution (0.300 cm3) of 1.3 × 10-3 M Dz and a toluene solution (0.300 cm3) of 5.1 × 10-5 M PdLCl were introduced into the cylindrical cell, and then the cell was rotated at 10 000 rpm. The Raman spectra were measured in the range (23) Ohashi, A.; Tsukahara, S.; Watarai, H. Anal. Chim. Acta 1999, 394, 23-31. (24) Yulizar, Y.; Ohashi, A.; Nagatani; H.; Watarai, H. Anal. Chim. Acta 2000, 419, 107-114.

Figure 1. Schematic drawing of the apparatus for the centrifugal liquid membrane-Raman microprobe spectroscopy. 1700-800 cm-1 by the use of 514.5 nm line of an Ar+ ion laser with a power of 40 mW. To improve the S/N ratio, the exposure time was set to 50 s. The focal point was moved along the Z-axis, which was perpendicular to the interface, and the spectra were observed at four points of 600, 660, 740, and 810 µm by controlling the height of the XYZ-stage, which were corresponding to the inner surface of the cell, the bulk aqueous phase, the toluene/ water interface, and the bulk toluene phase, respectively.19 The position of Z ) 0 was defined at the outer surface of the cylindrical cell. The observed frequencies were calibrated with respect to the Raman peaks of toluene and were accurate to within (2 cm-1. Microscopic Measurement. The aggregates of PdL-Dz complexes formed at the toluene-water interface were observed with microscopy with a CCD camera. A 6.0 × 10-4 M Dz aqueous solution (15 cm3) was introduced in a laboratory dish, and then 8 cm3 of 5.1 × 10-5 M PdLCl toluene solution was quietly added onto the aqueous phase. After one night, the microscopic images of the aggregates of PdL-Dz complexes formed at the toluene/ water interface were obtained by an objective lens (45×) directly above the toluene phase. A clear microscopic image for the interfacial aggregate of PdL-1,4-Dz was not obtained due to the formation of membranelike species with the homogeneous surface. Therefore, it was carefully transferred onto a glass substrate and then observed.

Results and Discussion Microscopic Images of the Interfacial Aggregates. From the results of batch experiments, we confirmed that PdLCl barely existed in both bulk phases after shaking in the presence of Dz, but in the absence of Dz, PdLCl remained in the toluene phase. Colored compounds were formed at the toluene/water interface for all Dzs; the color was blue in the cases of 1,2-Dz and 1,3-Dz and pink for 1,4-Dz. These interfacial compounds disappeared with the addition of 1.0 × 10-2 M NaCl, as PdLCl returned into the toluene phase. This result showed that the interfacial compound was the aggregates of complexes of Pd(II)-5Br-PADAP with Dz isomers, which were formed by the exchange of chloride ion with Dz isomer at the interface. Microscopic images of the aggregates of PdL-Dz isomer complexes formed at the interface were observed in the laboratory dish as shown in Figure 2. The microscopic observations revealed that the shape of the aggregates of PdL-1,2-Dz and PdL-1,3-Dz was somewhat crystallike. The aggregate of PdL-1,4-Dz transported on the glass substrate was membranelike. These microscopic images indicatd that the shapes of the interfacial aggregates of PdL-Dz complexes were greatly different among Dz isomers.

Molecular Recognition

Figure 2. Microscopic images of the assembly of the PdL1,2-Dz complex at the toluene/water interface (a) and the assembly of the PdL-1,4-Dz complex transferred on the glass substrate (b). Each interfacial aggregate was formed under the conditions of [PdLCl]init ) 5.1 × 10-5 M, [1,2-Dz] ) [1,4-Dz] ) 6.0 × 10-4 M, [ClO4-] ) 0.1 M, and [Cl-] ) 0 M and pH 2.0.

Absorption Spectra of the Interfacial Aggregates of PdL)Dz Isomer Complexes. The absorption spectra of the aggregates of PdL-Dz isomer complexes formed at the interface were directly measured by CLM method. Figure 3a shows the absorption spectra of PdLCl and the aggregates of PdL-Dz complexes. The spectrum of PdLCl in the absence of Dzs was the sum of absorption spectrum of PdLCl in the toluene phase and that adsorbed at the interface, because about 90% of PdLCl still existed in the toluene phase under the experimental condition. When the Dz isomers were added into the aqueous phase, the maximum absorbance at 564 nm of PdLCl in the toluene phase was significantly decreased and a new spectrum appeared for each Dz isomer. In the case of 1,2-Dz, a broad peak appeared around 600-700 nm. Addition of 1,4-Dz produced a peak at 469 nm. The absorbance at the maximum wavelength was proportional to the initial concentration of PdLCl in the range of at least (1.7-6.2) × 10-5 M. The spectrum observed by CLM method in the case of 1,4-Dz coincided with that of the aggregate of PdL1,4-Dz transferred on the glass substrate (Figure 3b), indicating that the interfacial aggregate was stable. The interfacial concentrations of PdL (>3.0 × 10-10 mol cm-2) calculated from the decrease in the absorbance of PdLCl in toluene were four times higher than the saturated concentration of NiL2 (6.5 × 10-11 mol cm-2).25 Moreover, the rates of the absorbance changes upon the addition of Dzs were much slower than those of the formation of 1:1 PdL-Dz monomer complexes at the liquid-liquid interface.23 From these results, it was confirmed that the new spectra were attributable to the aggregates of PdL-Dz complexes formed at the interface. Interference of Chloride Ion on the Aggregation. Figure 4 shows the dependencies of equilibrium absor(25) Watarai, H.; Gotoh, M.; Gotoh, N. Bull. Chem. Soc. Jpn. 1997, 70, 957-964.

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Figure 3. Absorption spectra of PdLCl and the interfacial aggregates of ternary PdL-Dz complexes measured by CLM method (a) and that of the aggregate of the PdL-1,4-Dz complex transferred on the glass substrate (b). (a) [PdLCl]init ) 5.6 × 10-5 M, [1,2-Dz] ) 2.0 × 10-4 M, [1,3-Dz] ) 8.0 × 10-3 M, [1,4Dz] ) 8.0 × 10-5 M, [ClO4-] ) 0.1 M, [Cl-] ) 0 M, pH 2.0. (b) Solid line: aggregate of the PdL-1,4-Dz complex transferred on the glass substrate. Dashed line: the same spectrum shown in part a.

Figure 4. Variation of the absorbance at 564 nm on the total concentration of Dz, [N]T. [PdLCl]init ) 5.6 × 10-5 M, [ClO4-] ) 0.1 M, pH 2.0. Key: circle, 1,2-Dz; square, 1,3-Dz; triangle, 1,4-Dz; open, [Cl-] ) 0 M; closed, [Cl-] ) 2.0 × 10-3 M. Each line in this figure was prepared as a guide for the eyes.

bance at 564 nm measured by the CLM method on the concentration of Dz isomers at [Cl-] ) 0 M and 2.0 × 10-3 M. As the concentration of Dz isomers was higher, the absorbance at 564 nm decreased due to the formation of the interfacial aggregates of PdL-Dz isomers. [N]1/2 was defined as the Dz concentration at which the absorbance at 564 nm reached the half value (A1/2) of the sum of the absorbances in the initial (A0) and the equilibrium (Ae) conditions, A1/2 ) (A0 + Ae)/2, and its value in each system was estimated from Figure 4 and listed in Table 1. In the presence of chloride ion ([Cl-] ) 2.0 × 10-3 M), a higher concentration of Dz isomers was required to form the interfacial aggregates. This interference effect of the chloride ion was attributed to the blocking effect of Cl- for

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Figure 5. Determination of the composition of PdL/Dz in the interfacial aggregate by the mole ratio method. [PdLCl]init ) 5.6 × 10-5 M, [ClO4-] ) 0.1 M, [Cl-] ) 0 M, pH 2.0 Table 1. Values of log[N]1/2 in Each System Estimated from Figure 3 log([N]1/2/M) diazine

[Cl-] ) 0 M

[Cl-] ) 2.0 × 10-3 M

1,2-Dz 1,3-Dz 1,4-Dz

-4.7 -3.3 -5.1

-3.8 -1.9 -3.6

the coordination of Dz isomers to PdL complex. This result also confirmed that chloride ion of PdLCl was exchanged to Dz isomers in the aggregates. Even at lower concentrations (4.0 × 10-6 M) of 1,2-Dz and 1,4-Dz, the aggregates of PdL-Dz were formed at the interface as shown in Figure 4. This phenomenon was caused by the specific concentration effect of the liquid-liquid interface. Absorbance slightly decreased in a lower 1,3-Dz concentration range, and then drastically decreased above 8.0 × 10-4 M. The slight decrease in the absorbance resulted from the formation of the PdL-1,3-Dz monomer complex whose composition was 1:1 at the interface.23 The drastic decrease in the absorbance above 8.0 × 10-4 M was attributable to the formation of aggregate between PdL and 1,3-Dz aggregate. It was reported that 1,3-Dz could form an aggregate of itself in a high concentration range, containing neutral and protonated 1,3-Dz together.26 Interestingly, the ability to form the interfacial aggregate of the PdL-1,4-Dz complex was highest among all Dzs, although the ability to form the interfacial complex of 1:1 PdL1,4-Dz at [Cl-] ) 0.1 M was lowest.23 This result implied that the formation of the interfacial aggregate of the PdLDz isomer was greatly influenced by the structure of Dz, not by the basicity of Dz. Composition of the Interfacial Aggregates. Figure 5 shows the results of the mole ratio method to determine the composition of the interfacial aggregates of PdL-1,2Dz and PdL-1,4-Dz by the CLM method. The mole ratio was defined by the use of the initial concentration added in the system as

mole ratio )

[Dz]iniVa [PdLCl]iniVo

(1)

where the subscript ini means initial, and Va and Vo are the volumes of the aqueous and the organic phases, respectively (Va ) 0.250 cm3 and Vo ) 0.150 cm3). The initial concentration of PdLCl was kept at 5.6 × 10-5 M. Equilibrium absorbance at 564 nm was plotted against (26) Peral, F.; Gallego, E. J. Mol. Struct. 1995, 372, 101-112.

Figure 6. Resonance Raman spectra of PdLCl, (a) in the toluene phase and (b) at the interface, (c) the assemblies of the PdL1,2-Dz complex at the interface, and aggregates of the PdL1,4-Dz complex (d) at the interface and (e) on a glass substrate. [PdLCl]init ) 5.1 × 10-5 M, [ClO4-] ) 0.1 M, [Cl-] ) 0 M, pH 2.0, [1,2-Dz] ) [1,4-Dz] ) 1.3 × 10-3 M.

the concentration of Dz isomers added. The composition ratios of 1,2-Dz/PdL and 1,4-Dz/PdL were found to be 1:1 and 1:2, respectively, from the cross points. Two chloride ions should be released, when one 1,4-Dz was consumed to form one (PdL)2-1,4-Dz complex in the aggregate. Therefore, the interference effect of chloride ion for the formation of the interfacial aggregate was largest in the case of 1,4-Dz. In a large number of papers, 1,4-Dz was used as a framework to synthesize one- and twodimensional metal complex crystals.27-29 Therefore, we assumed that the unit structure of the aggregate of PdL1,4-Dz contained two palladium(II) atoms bound to the nitrogen atoms of 1,4-Dz. This 1:2 complex formed a membranelike aggregate. Resonance Raman Spectra of the Interfacial Aggregates. To discuss the structures of the aggregates in detail, direct Raman spectral measurements of the aggregates of ternary PdL-Dz complexes formed at the toluene/water interface were carried out with a CLMresonance Raman microprobe spectroscopy. The Raman spectra measured at the inner surface of the cell (Z ) 600 µm) and the bulk aqueous phase (Z ) 660 µm) did not show any peaks attributable to PdLCl, confirming that PdLCl scarcely existed in these regions. Because Raman spectrum measured at Z ) 740 µm contained the spectra of the liquid-liquid interface and both bulk phases, the interfacial Raman spectrum was obtained by subtracting the spectrum measured at Z ) 810 µm (the bulk toluene phase) from that at Z ) 740 µm (the interface) after the normalization by using the Raman intensity at 1210.5 cm-1 of toluene. A resonance Raman spectrum of PdLCl in the toluene phase (Z ) 810 µm) was also obtained by the subtracting the spectrum for toluene after the normalization of Raman intensity at 1210.5 cm-1 of toluene. Parts a-d of Figure 6 show the resonance Raman spectra of PdLCl in the toluene phase, PdLCl at the interface, PdL-1,2-Dz aggregate at the interface, and PdL-1,4-Dz aggregate at the interface, respectively. A fine spectrum was not obtained for the aggregate of PdL1,3-Dz, because of the low absorbance at 514.5 nm. Figure 6e shows the resonance Raman spectrum for the aggregate (27) Belford, R. C. E.; Fenton, D. E.; Truter, M. R. J. Chem. Soc., Dalton Trans. 1974, 17-24. (28) Haynes, J. S.; Rettig, S. J.; Sams, J. R.; Thompson, R. C.; Trotter, J. Can. J. Chem. 1987, 65, 420-426. (29) Moreno, J. M.; Suarez-Varela, J.; Colacio, E.; Avila-Ro´son, J. C.; Hidalgo, M. A.; Martin-Ramos, D. Can. J. Chem. 1995, 73, 1591-1595.

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Table 2. Shapes, Raman Shifts, and the Values of the Ratio of Raman Intensities around 1307 and 1284 cm-1 of a Crystal, of Aggregates, and of a Complex of PdLCla Raman shift/cm-1 PdLClcrystal PdLClaggregate (PdL-1,2-Dz)n (PdL-1,4-Dz)n PdLCltoluene/water interface PdLClheptane/water interface PdLCltoluene bulk a

shape

νNdN

νCNNCazo

νCNNCimi

I1307/I1284

crystal crystallike crystallike membranelike

1400 1402 1408 1409 1408 1408 1401

1301 1300 1309 1301 1303 1303 1307

1279 1285 1286 1286 1280 1284 1283

2.19 3.52 2.11 1.16 1.47 1.85 2.87

The absorption spectra measured by CLM method in various base systems.

of PdL-1,4-Dz on the glass substrate. This spectrum was close to the interfacial spectra measured by CLMresonance Raman microprobe spectroscopy. The bands at 1594, 1478, 1401, and 1307 cm-1 in Figure 6a were assigned mainly to ν(CdC) of benzene and pyridine rings, ν(CdC) of pyridine ring, ν(NdN), and ν(CNNC), respectively, by comparison with those reported for pyridylazophenol complexes.30-32 The Raman spectrum of PdLCl adsorbed at the toluene/water interface was basically similar to that of PdLCl in the toluene solution, but was largely different in the region from 1500 to 1450 cm-1 and around 1300 cm-1. In our previous study, a similar spectral change of PdLCl was observed by the change of the dielectric constant of solvent, and it was accounted for by the change of the ratio of azo-imine forms of PdLCl with the change of the hydration of PdLCl.33 Therefore, we thought that the spectral change of the interfacial PdLCl resulted from the hydration from the polar aqueous phase. In the Raman spectra of the interfacial aggregates of PdL1,2-Dz and PdL-1,4-Dz complexes, the band at 1408 cm-1 hardly changed. This result suggested that the formation of the interfacial aggregates of PdL-Dz complexes was not accompanied by a structural change of 5-Br-PADAP such as twisting or cis-isomerization. The complex of Pd(II) with 5-Br-PADAP exists as a mixture of three resonance forms: two imine and one azo tautomers, and it is postulated that the charged quinone resonance structure, which is the intramolecular charge separation structure, in the imine tautomer is the cause of the high molar absorptivity, which is twice as sensitive as that of 4-(pyridylazo)resorcinol (PAR).20,34 In the charged quinone resonance structure, the diethylamino group plays an important role as an electron donor. The significant decrease in the molar absorptivity of the aggregates of PdL-Dz complexes seemed to result from the decrease in the ratio of the charged quinone structure in these aggregates. The hypochromic phenomenon was not observed in the aggregation of the neutral PdLCl itself at the heptane/water interface.35 Therefore, we assumed that the interaction of ClO4- as a counter ion with the positively charged PdL-Dz complexes prevented the electron donation from the diethylamino-group to the nitrogen atoms of the azo group and lowered the possibility of a π-π* transition. Because Raman peaks attributable to the imine structure was still observed in the spectra of the aggregates of PdL-Dz complexes in Figure 6, the resonance in the PdL was not completely ceased. (30) Drozdzewski, P. M. Spectrochim. Acta 1985, 41, 1035-1039. (31) Dines, T. J.; Wu, H. J. Chem. Soc. Faraday Trans. 1995, 91, 463-468. (32) Drozdzewski, P. M. J. Raman Spectrosc. 1988, 19, 111-115. (33) Ohashi, A.; Watarai, H. Langmuir 2002, 18, 10292-10297. (34) Ueno, K.; Imamura, T.; Cheng, K. L. Handbook of Organic Analytical Reagents, 2nd ed.; CRC Press: Boca Raton, FL, 1992; pp 237-243. (35) Ohashi, A.; Watarai, H. Manuscript in preparation.

Table 2 lists the observed Raman shifts and the Raman intensities at the peak positions around 1307 and 1284 cm-1 of PdL in the various situations; the bands at 1307 and 1284 cm-1 were assigned to ν(CNNC) in the azo and imine forms, respectively.33 The ratio of Raman intensities at the peak positions around 1307 and 1284 cm-1, I1307/ I1284, for the aggregate of the PdL-1,2-Dz complex was larger than that for the aggregate of the PdL-1,4-Dz complex. In other words, the azo form was more stable in the aggregate of the PdL-1,2-Dz complex than that in the aggregate of the PdL-1,4-Dz complex. This result suggested that the microenvironment in the interfacial aggregate of the PdL-1,2-Dz complex had lower polarity than that in the interfacial aggregate of the PdL-1,4-Dz complex. The interfacial aggregate of PdL-1,4-Dz in the microscopic image was membranelike, while that of PdL1,2-Dz was crystallike. The structure of the aggregate of the PdL-1,2-Dz complex was thought to be similar to that of the PdLCl crystal,36 because the mole ratio of PdL to 1,2-Dz in the aggregate was 1:1. The values of I1307/I1284 of the PdLCl crystal, PdLCl aggregate and PdL-1,2-Dz aggregate were relatively larger. This may suggest that the crystallike structures of these species made the microenvironment in the PdL complexes less polar. On the other hand, the value of I1307/I1284 of the aggregate of the PdL-1,4-Dz complex was relatively smaller. Figure 7 shows (a) a possible structure of the aggregate unit of the PdL-1,4-Dz complex, (b) a schematic illustration of this aggregate at the toluene/water interface, and (c) the dimer formation in the single-crystal of PdLCl determined by an X-ray structural analysis.36 As shown in Figure 7c, PdLCl formed the dimer by a π-stacking interaction in the crystal. Therefore, it was expected that two PdL complexes bonded to two nitrogen atoms of 1,4-Dz and the one-dimensional aggregate were formed by a π-stacking interaction between the PdL molecules in the mole ratio of 2:1 for PdL to 1,4-Dz as a fundamental structure. Assembly of long one-dimensional flexible aggregates will form a membranelike aggregate. We thought that the polarity around PdL in the aggregate of the PdL-1,4-Dz complex was larger than the crystallike structure, because the membranelike structure easily can contain water molecules in the aggregate. The structure of the interfacial aggregate of PdL-1,3-Dz could not be studied in detail, because its low stability caused difficulty with the formation of the aggregate in various conditions, and furthermore, the aggregate of 1,3-Dz itself prevented measuring that of the PdL-1,3-Dz complex. Application of Molecular Recognition Aggregation to Bases of Nucleic Acid. Because the bases of nucleic acid have the same property of heterocyclic base as Dzs have, a structure recognition aggregation is expected to occur among the bases of nucleic acid. (36) Ohashi, A.; Tsukuda, T.; Watarai, H. Anal. Sci., in press.

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Figure 8. Absorption spectra measured by CLM method in four base systems. [PdLCl]init ) 5.6 × 10-5 M, [adenine] ) 7.7 × 10-5 M, [guanine] ) 4.4 × 10-5 M, [cytosine] ) 8.8 × 10-4 M, [thymine] ) 8.8 × 10-4 M, [ClO4-] ) 0.1 M, [Cl-] ) 0 M, pH 2.0.

Figure 7. Possible structure of (a) the assembly unit of the PdL-1,4-Dz complex, (b) a schematic illustration of the assembly of the PdL-1,4-Dz complex at the interface, and (c) the dimer structure of PdLCl in its crystal that was determined by an X-ray structural analysis.36

Therefore, the interfacial aggregation with PdL was studied for the bases of nucleic acid (adenine, guanine, thymine, and cytosine). When adenine or guanine aqueous solution and PdLCl toluene solution were shaken in a glass tube, PdLCl barely existed in both bulk phases. At this time, blue species were formed at the toluene/water interface and somewhat on the glass wall. The addition of cytosine or thymine decreased PdLCl from the toluene phase; however, PdLCl somewhat remained in the toluene phase (about 58% in all). No aggregate was formed at the toluene/water interface in each system, but the decrease in the concentration of PdLCl indicated the adsorption of PdL-cytosine or PdL-thymine complexes at the interface and on the glass wall. This result indicated the higher stabilities of the interfacial aggregates of PdL-purine bases than those of PdL-pyrimidine bases, reflecting the higher hydrophobicity and the higher basicity for the purine bases. Figure 8 shows the absorption spectra of the two-phase liquid membranes systems contained bases in the aqueous phase. The absorbance at 564 nm of PdLCl in purine base/ toluene system decreased due to the formation of the interfacial aggregates of the PdL-base complexes. On the other hand, the absorbance decreases at 564 nm in pyrimidine base systems were obviously smaller than those in purine base systems, even though the concentrations of pyrimidine bases were about 10 times higher than those of purine bases. From the results of the batch experiments, we found that the PdL-pyrimidine base complex was not aggregated at the interface. Therefore,

Figure 9. Determination of the composition of PdL/base in the interfacial aggregate by the mole ratio method. [PdLCl]init ) 5.6 × 10-5 M, [ClO4-] ) 0.1 M, [Cl-] ) 0 M, pH 2.0

the absorption spectra measured in pyrimidine base systems were the sum of the absorption spectrum of PdLCl in the toluene phase and that of PdL-base complexes at the interface. Figure 9 shows the dependencies of equilibrium absorbance at 564 nm measured by the CLM method on the concentrations of adenine and guanine. The absorbance decreases at 564 nm were proportional to the purine concentrations due to the formation of the interfacial aggregates of PdL-purine base complexes. The absorbance decreased very steeply in even lower concentrations than 5.0 × 10-6 M for the purine base. This result suggests that the interfacial aggregation phenomenon is promising for the sensitive and selective detection of adenine and guanine less than 10-6 M. The compositions of the interfacial aggregates of PdL-adenine and PdLguanine were determined by the mole ratio method as PdL:adenine ) 6:1 and PdL:guanine ) 7:1, respectively. Large numbers of PdL moieties could bind to the purine bases. The structure of these interfacial aggregates will be clarified in future study. However, it might be expected that a PdL molecule could bind to a purine base through Pd-N bonding and the excess PdL molecules might stack with the PdL-purine complex. Conclusion We found out the structure recognizing aggregation of Pd(II)-5-Br-PADAP with Dz isomers at the toluene/ water interface by using the CLM method. The formation of the interfacial aggregate of PdL-Dz was detected by a drastic absorbance decrease of PdLCl at 564 nm with a change in the spectral shape. The composition of the

Molecular Recognition

interfacial aggregate of PdL-Dz depended on the difference in the geometric structure of the Dz isomers; PdL1,2-Dz aggregate was observed as a blue domain at the interface and the composition ratio was 1,2-Dz:PdL ) 1:1, whereas PdL-1,4-Dz aggregate was a membrane at the interface and 1,4-Dz:PdL ) 1:2, and PdL-1,3-Dz aggregate had blue domains. Despite the lowest stability of the 1:1 complex between PdL and 1,4-Dz at the toluene-water interface, the stability for the formation of the interfacial aggregate was highest among the Dz isomers. This result revealed that the formation of the interfacial aggregate of the PdL-Dz isomer was governed by the structure of Dz, not by the basicity of Dz. The resonance Raman spectra of the interfacial aggregates were measured by the use of the CLM method combined with Raman microspectroscopy and showed that the polarity around PdL in the aggregate of the PdL-1,4-Dz complex was larger than that in the crystallike structure of the PdL-1,2-Dz complex. Moreover, the hypochromic effect observed in the PdL-Dz complexes was ascribed to the interaction of 5-Br-PADAP ligand with ClO4- as a counter anion incorporated in these aggregates. The interaction should prevent the resonance

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in the charged quinone structure of the ligand in the Pd(II)-5-Br-PADP. In the present study, the interfacial isomer recognizing aggregation by Pd(II) complex was demonstrated for the first time. The formation of aggregates of PdL-1,2-Dz, -1,4-Dz, -adenine, and -guanine was detected spectrophotometrically at very low concentrations of Dzs and purine bases. The phenomenon of the molecular recognizing aggregation at the liquid-liquid interface will be used as a highly sensitive and selective detection method of various compounds such as nucleic acid bases, DNAs, drugs, and pesticides. Acknowledgment. This study was financially supported by a Grant-in-Aid for Scientific Research (A) (No. 12304045) and a Scientific Research of Priority Area (No. 13129204) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by a grant of JSPS Research Fellows (02308). LA0268451