Estimation of Formation Constants of Ternary Cu(II) Complexes with Mixed Amino Acid Enantiomers Based on Ligand Exchange by Capillary Electrophoresis

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1 2000 The Japan Society for Analytical Chemistry 837 Estimation of Formation Constants of Ternary Cu(II) Complexes with Mixed Amino Acid Enantiomers Based on Ligand Exchange by Capillary Electrophoresis Zilin CHEN, Katsumi UCHIYAMA, and Toshiyuki HOBO Department of Applied Chemistry, Graduate School of Engineering, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, Hachioji, Tokyo , Japan Based on the principle of ligand exchange, a new method for estimating the formation constants of ternary Cu(II) complexes with mixed amino acid enantiomers has been proposed by capillary electrophoresis. As examples, the formation constants of four complexes, namely (L-OH-Pro)Cu(II)(D-Phe), (L-OH-Pro)Cu(II)(L-Phe), (L-OH-Pro)Cu(II)(D- Trp) and (L-OH-Pro)Cu(II)(L-Trp), were estimated by this method. The dependence of stability of complexes on the stereo-selectivity and the possibility of predicting the enantiomer migration order from complex stability has been discussed. (Received April 26, 2000; Accepted May 31, 2000) Capillary electrophoresis (CE) has been developed to be a powerful separation and analysis technique in recent decades. Many separation modes, for examples, micellar electrokinetic chromatography (MEKC) 1,2 and ligand exchange-mekc (LE- MEKC) 3 6 have been proposed. They offer wider applications for CE. Recently, not only has CE been reported as a separation technique, but also as an important tool for determining the physical parameters of chemical compounds, such as the critical micelle concentration (CMC) of surfactants 7 9 and the formation constants of complexes The principle of ligand exchange (LE) was used in liquid chromatography for the chiral separation. 13,14 Zare and his coworkers reported its application on the separation of dansyl amino acid enantiomers by CE. 15,16 Our previous work has demonstrated its successful application for simultaneous separation of positional and optical isomers of amino acids when it is combined with MEKC. 3,4 This work focuses on its application for estimating the formation constants of ternary copper(ii) complexes with mixed amino acid enantiomers. Although some methods like chromatographic 17 and CE methods were used for the determination of formation constants of binary complexes, very few papers dealt with the determination of formation constants of the ternary Cu(II) complexes with mixed amino acid enantiomers. A probable reason is that the difference in formation constants of copper(ii) complexes with amino acid enantiomers is so small that the determination becomes very difficult. The traditionally employed methods are potentiometric titration, 18 circulardichroism (CD) and electronic spectra. 19 To our knowledge, no paper has been reported yet for the determination of formation constants using the LE principle. To whom correspondence should be addressed. zlchen@ecomp.metro-u.ac.jp Presented at the 22 nd International Symposium on Capillary Chromatography & Electrophoresis, Nov. 8 12, 1999, Gifu, Japan. As ligand exchange is used in chiral CE, complex stability is an important parameter, for example, complex stabilities can be used for the prediction of enantiomer migration order (EMO). 6 Therefore, it is meaningful to develop a method for the estimation of formation constants. The chiral separation using LE principle is based on the difference in the stabilities of ternary complexes. In other words, complex stability controls the EMO; i.e. the electropherograms of enantiomers provide the information about complex stability. Therefore, the LE principle can be used for estimating the formation constant of complexes. In this work, theoretical equations for this purpose have been proposed and applied to several copper(ii) complexes. The dependence of EMO on complex stability has further been discussed. Experimental Instrumentation The CE instrumental setup involves a HEL5-30P2-TTu high voltage power supply (Matsusada Precision Devices. Inc., Japan), a CE-971 UV detector (Jasco Corporation, Japan) and a C-R6A Chromatopac Recorder (Shimadzu, Japan). The separations were carried out in fused-silica capillaries (0.050 mm i.d mm o.d.) with a total length of 56 cm and an effective length of 40 cm obtained from GL Sciences Inc., Japan. Chemicals All chemicals were of reagent grade and were used as received. Cupric sulfate and L-hydroxyproline (L-OH-Pro) were obtained from Wako Pure Chemical Industries. Ltd. (Tokyo, Japan). Ammonia solution was from Kanto Chemical (Tokyo, Japan). All amino acid enantiomers were obtained from Sigma (USA) and Wako (Japan). Samples were prepared by dissolving amino acids in an electrolyte at the concentration range of and 1.0

2 838 ANALYTICAL SCIENCES AUGUST 2000, VOL M. For the identification of the peaks of enantiomers, D- (or L-) enantiomer was spiked to racemic amino acids. The running electrolytes used for the measurement of mobilities of anantytes were prepared by the use of same concentration of CuSO 4 and L-hydroxyproline at the concentration range of 5 to 40 mm adjusted to ph 4.5 with ammonia. For the mobilities of free analytes, 20 mm CuSO 4 solution adjusted at ph 4.5 with ammonia was used. All solutions were filtered through 0.45 µm membrane (Nihon Millipore Ltd. Japan) and degassed by vacuum and ultrasonication. Water was purified by distillation apparatus (Advantec Toyo, Japan). Capillary electrophoresis Before use, the capillaries were washed with 0.1 M sodium hydroxide, water and running electrolyte for 15, 30 and min, respectively. When the electrolyte composition was changed, the capillary was washed and equilibrated with new electrolytes. The samples were injected electrokinetically for 3 5 s at 10 kv. UV detection was at 208 nm. Electroosmotic flow (EOF) was determined by the migration time of acetone. Effective electrophoretic mobility of analytes was calculated with the equation: L 1 1 µ eff = µ obs µ eof = dl t (1) V tr teof Here, µ eff, µ obs and µ eof are the effective electrophoretic mobility, observed electrophoretic mobility of analytes and electroosmotic mobility, respectively. t r is the migration time measured directly from the electropherogram. t eof is the migration time of neutral marker for determining EOF. L t and L d are the total length of capillary and the length of capillary from injection to detection. V is the applied voltage. The electrophoretic mobility of free analyte, µ 0, was calculated by µ 0 = µ obs 0 µ eof, where µ obs 0 is the mobility without chiral selector complex in electrolyte. All experiments were carried out at room temperature (25 C). Results and Discussion Theory for estimating the formation constants of complexes In aqueous solution, Cu(II)(d 9 ) is tetragonally coordinated by four water molecules with the two axial water molecules at longer distances from the copper. Amino acids are bidentate ligands with a high affinity to Cu(II). In the presence of amino acids as ligands, water molecules located in the square planar position are replaced by either one or two molecules of amino acids, forming copper-amino acid complexes as chiral selectors at ratios varying from 1:1 to 1:2 as a function of amino acid concentrations. 20 Based on the LE principle, when analytes, DLamino acids (DL-AA), are introduced, they will exchange ligands (either an amino acid for 1:2 selector complex or two water molecules for 1:1 selector complex) to form a ternary copper(ii) complex with mixed ligands. The resolution of DLamino acids is based on the difference in the stability of ternary complexes, i.e. the formation constants. The possible equilibria of ligand exchange could be expressed as follows, where water molecules are omitted: 2Cu(II)(L*) + D,L-AA (L*)Cu(II)(D-AA) +(L*)Cu(II)(L-AA) (1) 2Cu(II)(L*) 2 + D,L-AA (L*)Cu(II)(D-AA) + (L*)Cu(II)(L-AA) + 2(L*) (2) 2Cu(II)(L*) 2 +2D,L-AA Cu(II)(D-AA) 2 + Cu(II)(L-AA) 2 + 4(L*) (3) 2Cu(II)(L*) + D,L-AA Cu(II)(D-AA) + Cu(II)(L-AA) + 2(L*) (4) Cu(II)(L*) 2 + D,L-AA Cu(II)(D-AA)(L-AA) + 2(L*) (5) Here L* stands for L-ligand in chiral selectors. When the L- ligands (L*) are used, the formation constants of ternary Cu(II) complexes like (L*)Cu(II)(D-AA) and (L*)Cu(II)(L-AA), K L* -D and K L* -L, can be expressed as: K L* -D = [(L*)Cu(II)(D-AA)] (6) [(L*)Cu(II)][D-AA] [(L*)Cu(II)(L-AA)] K L* -L = (7) [(L*)Cu(II)][L-AA] K 1D, K 2D and K DL, the formation constants of complexes of Cu(II)(D-AA), Cu(II)(D-AA) 2 and (D-AA)Cu(II)(L-AA) are expressed: K 1D = [(Cu(II)(D-AA)] (8) [Cu(II)][D-AA] K 2D = [Cu(II)(D-AA)] (9) [Cu(II)(D-AA)][D-AA] K DL = [(D-AA)Cu(II)(L-AA)] (10) [Cu(II)][D-AA][L-AA] When analytes (DL-AA) were introduced into the background electrolyte, they will exchange the ligands with the chiral selectors of Cu(II)(L*) complex. After ligand exchange, D- amino acids exist in five possible species: free amino acid, (L*)Cu(II)(D-AA), Cu(II)(D-AA), (D-AA)Cu(II)(L-AA) and Cu(II)(D-AA) 2. Because the concentration of chiral selector is very high, and the concentration of D-AA (analyte) is quite low, Cu(II)(D-AA) will be further bonded with the chiral selectors to form complex (L*)Cu(II)(D-AA). Thus, the existence of Cu(II)(D-AA) will not be taken into account. The effective electrophoretic mobility of the D-analyte (D-AA), µ d, which is contributed to by the statistical weight quantity of the mobilities of free amino acid, (L*)Cu(II)(D-AA), (D-AA)Cu(II)(L-AA) and Cu(II)(D-AA) 2, can be expressed as Eq. (11), [D-AA] µ d = µ 0 + µ 1 + µ 2 [(L*)Cu(II)(D-AA)] [(D-AA)Cu(II)(L-AA)] [(Cu(II)(D-AA) + µ 3 2] (11) where, µ 0, µ 1, µ 2 and µ 3 are the effective electrophoretic mobilities of free and complexed analytes: (L*)Cu(II)(D-AA),

3 839 Table 1 Linear regression equations and formation constants Complex Linear regression equation Formation constant/ M 1 [L-OH-Pro]Cu(II)[D-Phe] y= x (r 2 =0.9881) 21.3 [L-OH-Pro]Cu(II)[L-Phe] y= x (r 2 =0.9823) 28.3 [L-OH-Pro]Cu(II)[D-Trp] y= x (r 2 =0.9717) 34.3 [L-OH-Pro]Cu(II)[L-Trp] y= x (r 2 =0.9557) 45.1 Fig. 1 Variation of (µ 0 µ)/µ versus concentrations of 1:1 complexes of Cu(II) to L-hydroxyproline. (D-AA)Cu(II)(L-AA) and Cu(II)(D-AA) 2, respectively. Since the complexed analytes, Cu(II) complexes, in present experimental conditions are neutrally charged, µ 1, µ 2 and µ 3 are regarded as zero. Therefore, Eq. (11) can be rewritten as: [D-AA] µ d = µ 0 (12) Combination of Eqs. (6) and (8) (12) gives, µ 0 µ d µ d Likewise, µ 0 µ l µl = K L* -D[Cu(II)(L*)] + (K 2DK 1D[Cu(II)][D-AA] + K DL[Cu(II)][L-AA]) (13) = K L* -L[Cu(II)(L*)] + (K 2LK 1L[Cu(II)][L-AA] + K DL[Cu(II)][D-AA]) (14) When (µ 0 µ d)/µ d and (µ 0 µ l)/µ l are plotted as the function of [Cu(II)(L*)], the slopes of lines give K L* -D and K L* -L, and the intercepts the values of (K 2DK 1D[Cu(II)][D-AA] + K DL[Cu(II)][L- AA]) and (K 1LK 2L[Cu(II)][L-AA] + K DL[Cu(II)][D-AA]). Therefore, based on Eqs. (13) and (14), the formation constants of ternary Cu(II) complexes: K L* -D, K L* -L can be estimated. Estimation of formation constants of ternary complexes To estimate the formation constants of ternary complexes by the above theory, L-hydroxyproline (L-OH-Pro) was used as the L-ligand (L*) of selector complex, and DL-Phe and DL-Trp were used as test analytes. If we assume that the binary complexes (chiral selector) of Cu(II)(L-OH-Pro) are formed when the added concentration ratio of Cu(II) and L-OH-Pro is kept at 1:1, the equilibrium concentration of Cu(II)(L-OH-Pro) complex, [Cu(II)(L*)], can be calculated as follows. (2CK [Cu(II)(L*)] = 1 + 1)± 4CK (15) 2K 1 Here C is the concentration of Cu(II) and L-OH-Pro added, K 1 the constant of binary complex: Cu(II)(L-OH-Pro). The log K 1 of Cu(II)(OH-Pro) was reported to be The C used in our experiment is in the range of M to M. If the K 1 and C are put into Eq. (15), the calculated result of [Cu(II)(L*)] can be approximately regarded to be C. Besides, the influence of the consumption of selector by solutes will be negligible, because the concentration of selector (about 10 2 M) is much greater than that of solutes (about 10 4 M). Therefore, the equilibrium concentrations [Cu(II)(L*)] in Eqs. (13) and (14) can be approximated as the added complex concentration C. The values of item (µ 0 µ)/µ of four ternary copper(ii) complexes: (L-OH-Pro)Cu(II)(D-Phe), (L-OH-Pro)Cu(II)(L-Phe), (L-OH-Pro)Cu(II)(D-Trp), (L-OH-Pro)Cu(II)(L-Trp) are plotted against C, as shown in Fig. 1. The variations of (µ 0 µ)/µ versus the concentration of selector complexes show excellent linearity, which agree well with the theoretical Eqs. (13) and (14). The linear regression equations and the estimated formation constants are listed in Table 1. The results in Table 1 indicate that the difference in the formation constants between D-enantiomer and L-enantiomer is very small, which probably makes the determination by use of other methods very difficult. We tried to compare our results with other methods, but unfortunately, we failed to determine the formation constants obtained by the spectrometric method. 18 Resolution of amino acid enantiomers at different concentration ratio of Cu(II) and L-hydroxyproline Some papers have focused on the influence of the ratio of Cu(II) and ligand on the enantiomeric separation. Sundin et al. 20 reported that an excess of N,N-di-decyl-D-alanine (DDA) beyond a 1:2 ratio of [Cu(II)]:[DDA] does not affect the separation. Gozel et al., 16 using aspartame as a ligand, found that an excess of aspartame enhances the resolution of the dansyl amino acids. In order to investigate the effect of ratio of Cu(II) and ligand in the proline system on enantioseparation, the separation of D,L-phenylalanine was performed by varying the ratio of [Cu(II)]:[L-OH-Pro] between 2:1 to 1:4 mm/mm. Results shown in Fig. 2 indicated that separation can be carried out not only at the ratio of 1:2, but also at the ratio of 1:1, even with higher selectivity factor. As the amount of ligands increased, the selectivity factor decreases. When the ratio is 1:4, the enantioseparation cannot be observed. This suggests that the presence of an excess amount of ligand probably make analytes lose the chance of ligand exchange between analyte and the complex of chiral selector. The fact of separation with high resolution at the ratio of 1:1 suggests that water molecules of 1:1 Cu(II) complex are easily exchanged by the ligands. Effect of ammonia used in adjusting ph of running electrolytes To investigate the effect of ammonia in adjusting the ph of running electrolytes, NaOH solution, instead of ammonia, was used to adjust the ph, when the running electrolytes were

4 840 ANALYTICAL SCIENCES AUGUST 2000, VOL. 16 Fig. 2 Effect of electrolyte composition (the ratio of Cu(II):ligand) on separation factor. The concentration of Cu(II) was kept at 20 mm, the concentration of L-hydroxyproline was changed from 10 to 80 mm, ph 4.0. Fig. 4 Electropherograms of D,L-Phe (A) andd,l-trp (B), in which the concentration of D-enantiomer is higher than that of L- enantiomer. Running electrolytes contained 30 mm CuSO 4, 30 mm L-hydroxyproline at ph 4.5. Fig. 3 Proposed chemical structures of ternary Cu(II) complexes with mixed amino acids. prepared. UV-Vis spectra (not shown here) of both electrolytes containing 25 mm 1:1 complexes of Cu(II)(L-OH-Pro) adjusted with ammonia or NaOH showed that they had very similar absorbance and maximum absorbance wavelength at ph 4.5. The agreement indicated that ammonia affects the formation of complex of Cu(II)(L-OH-Pro) at weak acidic conditions very little. However, at very high ph (>10), the maximum absorbance wavelength shifted toward ultraviolet band quite drastically; this shift suggests the formation of a complex of Cu(II)(NH 3) 4. Complex stability and enantiomer migration order The results in Table 1 show that the ternary Cu(II) complexes with two L-enantiomers are more stable than that with mixed D- and L-ligands. Based on the discussions about complex structure, 20,22 26 we show a proposed structural model of complexes in Fig. 3, where D,L-Phe is used as an example of analyte, and L-hydroxyproline as the ligand. The complex with a L-Phe (bottom) offers a more stable conformation than the one with D-Phe (top), because it can take the trans-conformation of lowest energy with proline ring around the copper coordination plane, although the phenyl ring in phenylalanine rotates freely. However, the free rotation of phenyl substituent in complex with a D-Phe makes either a cis conformation between phenyl and proline rings around the copper coordination plane or a sterically hindered interaction between phenyl ring and axial water molecule. This stereo-conformation suggests that the ternary complex of (L-OH-Pro)Cu(II)(D-Phe) has lesser stability than the (L-OH-Pro)Cu(II)(L-Phe). The fact that D-enantiomer is faster than L-analogue, as shown in Fig. 4, supports the results obtained. When L-hydroxyproline is used as the ligand of chiral selector complex, L-analyte will preferentially interact with chiral selector of Cu(II) complex since it can form a more stable ternary complex than D-analyte, which agrees with the estimated results K L* -L > K L* -D. EMO shows D-enantiomers are faster than L-ones. Likewise, if D- ligands are used, the stabilities of the ternary complexes show K D* -D > K D* -L, resulting in the EMO that L-enantiomers are faster than D-analogues. 6 Acknowledgements Z. Chen is grateful to the Ministry of Education, Science, Culture, and Sports of Japan (Monbusho), and the Japan Society for the Promotion of Science (JSPS) for supporting his doctorate and postdoctorate studies. This work was partially supported by

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