Enantioseparation of Dansylated Amino Acids by Ligand-exchange Capillary Electrophoresis Using L-phenylalaninamide , L-lysine or L-threonine as Chiral Selector

In recent years enantioseparation of both active pharmaceutical ingredients and bio molecules such as amino acids became more and more necessary because in most cases the two stereo forms exhibit different pharmacological effects. This article deals with the chiral separation of dansylated amino acids by ligand-exchange capillary electrophoresis using L-phenylalaninamide, L-lysine and L-threonine as chiral selectors. Experiments with different central metal ions such as Cu(II), Co(II), Cd(II), Ni(II) and Zn(II) were carried out. Optimal conditions were found out by studying the effect of the pH and the selector molarity on the chiral resolution. Best separation was obtained for the Cu(II)/L-lysine complex, showing a chiral resolution up to 17 for Dns-DL-Met. (doi: 10.5562/cca1762)


INTRODUCTION
The development of enantiomer separation techniques has attracted great attention since it was investigated that in some cases only one enantiomer of a racemic drug mixture shows the desired pharmacological effect (eutomer).The other form (distomer) may not show any effect e.g.(R)-ibuprofen, 1 unwanted side effects as it is the case with D-thyroxine, or in some cases even toxic effects e.g.D-DOPA.About half of the used drug substances are chiral and about 35 % out of them are administered as pure enantiomers. 2Even if there are not that drastic effects of the distomer, it represents an unnecessary burden for the organism.
The chiral separation of underivatized and Dns-AAs is of special interest not at least since it was postulated that, in contrary to former hypothesis, D-amino acids also occur in higher animals and not only in lower species.The D-forms can have physiological effects such as D-Ser and D-Asp or they can be related to pathophysiological processes, such as Alzheimer's disease, Parkinson's disease, schizophrenia and renal disease. 3AAs and their derivatives are used as building blocks for peptide synthesis or as drugs e.g.3,4dihydroxyphenylalanine (L-DOPA) and L-Trp.Dansyla-tion of AAs is used on the one hand to label the Nterminal end of a peptide and on the other hand to simplify the detection of amino acids.
In recent decades multitudes of direct as well as indirect chiral separation techniques have been developed.In addition to HPLC and GC capillary electrophoresis turned out to be a powerful alternative with some unique advantages e.g.high peak efficiency, good compatibility with biological samples, short analysis times and the low consumption of buffer solutions.Even the flexibility in changing the method conditions played a considerable role in the rise of CE to one of the most spread separation techniques together with HPLC.
In this research, experiments with the three chiral selectors L-phenylalaninamide, L-lysine and L-threonine were performed under different conditions, to resolve Dns-AAs by LECE.For the optimization of the methods the effect of the pH value and the selector concentration on the resolution is shown.For the Cu(II)/L-lysine a validation referring to repeatability was carried out.

EXPERIMENTAL Instrumentation
A fully automated 3D CE system (Agilent Technologies, CA, USA) equipped with a diode array detector was used for the experiments.Measurements were performed in 50 µm ID fused silica capillaries (58.5/50 cm effective length) from Microquartz (Munich, Germany).Detection was performed via on-column measurements of the UV absorption at 208 and 254 nm.Operation temperature was set to 25 °C.Before measurement capillaries were washed with water, 0.2 mol dm −3 NaOH, water and electrolyte.Samples were injected hydrodynamically for 5 s at 10 mbar unless indicated otherwise.
The electrolyte was prepared by dissolving a desired quantity of the chiral selector and the ionic additives in double-distilled water.The pH was adjusted depending on the buffer system with a suitable reagent.The solutions were degassed for 2 minutes by ultrasonification and filtered through a 0.45 µm pore size Teflon filter (Schleicher and Schuell, Dassel, Germany) before use.
Sample solutions were prepared by dissolving the analytes (1 mg/ml) in a mixture of methanol/water (1:1).

RESULTS AND DISCUSSION
Enantioseparation in LECE is based on the formation of diastereomeric ternary mixed metal complexes between the chiral selector ligand and the analyte.Depending on the different complex stability constants of the two mixed complexes enantiomer resolution is reached.The following equilibria should be taken into account: The optimum pH for the complexation differs from chiral selector and the type of analyte.The correlation between the pH and the chiral resolution for different selectors is shown later.

Use of L-phenylalaninamide as Chiral Selector
L-phenyalaninamide was already used for the resolution of Dns-AAs by Chen 34 with Cu(II) and by Qi 28 with Zn(II) as central metal ion.For the following experiments a similar background electrolyte was chosen to guarantee adequate current.Ammonium acetate was replaced by sodium acetate.A chiral selector concentration of 10 mmol dm −3 and a selector to ion ratio of 2:1 was used.
Using Zn(II) as central ion, Dns-DL-NLeu, Dns-DL-Nval, Dns-DL-Leu and Dns-DL-α-ABA were resolved in addition to those Dns-AAs presented in the afore cited articles.With Cu(II) as metal ion no new analytes could be resolved.Table 1 shows the obtained results using Zn(II) as metal ion.In contrast to Cu(II), Zn(II) was used with negative voltage in order to detect the analytes.The change in the polarity was manifested in the enantiomer migration order (EMO).With Zn(II) the D-form migrates faster than the L-form, with Cu(II) it is the opposite.
Copper and zinc are the most frequently used central ions but apart from those, other ions were investigated for their complex building ability. 35We examined the effect of Ni(II), Cd(II) and Co(II) on the resolution of Dns-AAs.Using Ni(II) as metal ion 7 out of 11 tested analytes could be resolved as it is shown in Table 2.For Dns-DL-Met, Dns-DL-Phe and Dns-DL-Trp the resolution was higher than 1.5.
Because Ni(II) was not used before as central ion in combination with phenylalaninamide the optimum pH-value and selector concentration was to be found out. Figure 1 shows the effect of the pH on the resolution of Dns-DL-Trp.The higher the resolution the more stable is the complex at the specific pH value.The best resolution turned out to occur at a pH of 8.2 as it was used with Cu(II) and Zn(II).
The effect of the selector concentration is shown in Figure 2. The selector to metal ion ratio was set to 2:1 and the total molarity of selector plus metal ion was changed.Best results were obtained at a total molarity of 15 mmol dm −3 , which means that 10 mmol dm −3 selector and 5mmol dm −3 Zn(II) were used.The electronic source was set to positive mode and so the EMO   was D before L. Therefore this method is applicable for purity checking of Dns-L-AAs.The determination of the EMO is shown in Figure 3 for Dns-DL-Met.The L-phenylalaninamide-Cd(II)-system showed chiral separation for Dns-DL-NVal, Dns-DL-NLeu and Dns-DL-Asp.Obviously it was the first time resolution of a racemic mixture of Dns-AAs was obtained with Cd(II) as central metal ion.With the metal ion Co(II) no analytes were separated.

Use of L-lysine as Chiral Selector
The suitability of lysine complexes for the chiral separation of underivatized amino acids was introduced by Lu 24 for Cu(II) as central metal ion and by Qi 25 for Zn(II) complexes.To our knowledge there is no publication that deals with the enantioseparation of derivatized amino acids using this selector-ion system.
The background electrolyte consisting of 25 mmol dm −3 ammonium acetate was adopted from previous unpublished experiments with L-ornithin as chiral selector showing similar structure.
Copper(II) was used as central metal ion in a 1 to 2 ratio to the selector.
To optimize the conditions pH values from 5.1 to 9 were tested.Figure 4 shows the relation between the pH and the chiral resolution for the model substance Dns-DL-Trp.The use of L-Lys/Cu(II) showed a high resolution among the whole pH spectrum.Figure 4 and  5 show that even the poorest resolution for Dns-DL-Trp was higher than 10.Further the relation between the total molarity of selector plus copper(II) and the resolution was investigated as it is shown in Figure 5.
Resolution increased with higher concentration of selector-copper(II) complex.With respect to migration time an electrolyte of 10 mmol dm −3 L-lysine and 5 mmol dm −3 Cu(II) at pH 8.0 was to screen a set of Dns-AAs in further experiments.The repeatability of the   method for the analyte Dns-DL-tryptophan is shown in Table 3.
With this method 10 out of 11 tested Dns-amino acids were separated.Results are shown in Table 4.The resolution values range from 3.6 for Dns-DL-Asp and 17.0 for Dns-DL-Met.A simultaneous chiral resolution of a mixture of Dns-DL-Asp, Dns-DL-Met, Dns-DL-Asp, Dns-DL--ABA and Dns-DL-Leu was achieved within 30 minutes.(Figure 6) Due to the faster migration of the L enantiomer this method is not suitable for purity studies of Dns-L-AAs because most of the time the smaller peak of the D- impurity may be overlapped by the L-peak.Simple change of the selector from L-to D-lysine inverts the EMO and purity check can be performed for L- enantiomers.(Figure 7)

Use of L-threonine as Chiral Selector
The last chiral selector tested in this study was L-Thr which was used successfully for enantioseparation of sympathomimetics and β-blockers by Hödl et al. 36 In this work L-Thr is shown to be applicable for the chiral resolution of Dns-AAs as well.In this case no testing of different metal ions was performed.An electrolyte consisting of 90 mmol dm -3 L-Thr and 45 mmol dm −3 Cu(II)-sulfate was used at pH 8.2.The previous results with the other two chiral selectors show that a pH of about 8.0 is suitable for the complex formation of Dns-   AAs and additionally the EOF is relatively strong which results in shorter retention times.In total 4 out of 11 tested Dns-AAs were separated with this selector as it is shown in Table 5. Noticeable are the long migration times of the glutamic acid enantiomers.Because of the two carboxylic groups the molecule seems to be slightly negatively charged and tends to migrate opposite to the EMO to the anode.The EMO turned out to be L before D. The optimization of the method including pH value and selector-concentration will be subject to further investigations.

CONCLUSION
The suitability of L-phenylalaninamide, L-lysine and Lthreonine as chiral selectors in LECE with different central metal ions was shown.The L-phenylalanina- mide/Ni(II) complex was applicable for the enantioseparation of 8 Dns-AAs and even with Cd(II) 3 Dns-AAs were resolved.With L-Lys/Cu(II) 10 out of 11 Dns-AAs were separated with high resolution compared to the other selector/metal complexes.Additionally 5 Dns-AAs were separated simultaneously.L-Thr which up to now has been used only for the chiral separation of sympathomimetics and β-blockers showed an effect on the chiral resolution of Dns-AAs with Cu(II) as metal ion as well.

Figure 4 .
Figure 4. Correlation between the pH value and the resolution for the Cu(II)-L-lysine complex.Conditions: 10 mM L-lysine, 5 mM copper(II) sulfate, 25 mM ammonium acetate; pH adjusted with ammonia; applied voltage: 27 kV to cathode; Injection: 15 mbar for 5 s.

Table 3 .
Repeatability data for retention time and resolution by means of Dns-DL-tryptophanConditions: 10 mM L-lysine, 5mM copper(II) sulfate, 25 mM ammonium acetate; adjusted with ammonia to pH 5.5; applied voltage: 27 kV to cathode; Injection: 15 mbar for 5 s.