Amino acid enantiomers complex

Steric hindrance cannot be directly calculated, but a lower MI energy value indicates lower steric hindrance in a complex. This phenomenon ean be observed in enantiomer recognition and protein-ligand interactions, with the latter also being known as affinity. Such phenomena can be studied via amino acid enantiomer complexes. The optimized energy values of (I )- and (S)-alanine are the same, as summarized in Table 2.4. 

The principle of this method depends on the formation of a reversible diastereomeric complex between amino acid enantiomers and chiral addends, by coordination to metal, hydrogen bonding, or ion—ion mutual action, in the presence of metal ion if necessary. L-Proline (60), T.-phenylalanine (61),... 

One of the most useful applications of chiral derivatization chromatography is the quantification of free amino acid enantiomers. Using this indirect method, it is possible to quantify very small amounts of enantiomeric amino acids in parallel and in highly complex natural matrices. While direct determination of free amino acids is in itself not trivial, direct methods often fail completely when the enantiomeric ratio of amino acid from protein hydrolysis must be monitored in complex matrices. 

Now, GC-IRMS can be used to measure the nitrogen isotopic composition of individual compounds [657]. Measurement of nitrogen isotope ratios was described by Merritt and Hayes [639], who modified a GC-C-IRMS system by including a reduction reactor (Cu wire) between the combustion furnace and the IRMS, for reduction of nitrogen oxides and removal of oxygen. Preston and Slater [658] have described a less complex approach which provides useful data at lower precision. Similar approaches have been described by Brand et al. [657] and Metges et al. [659]. More recently Macko et al. [660] have described a procedure, which permits GC-IRMS determination of 15N/14N ratios in nanomole quantities of amino acid enantiomers with precision of 0.3-0.4%o. A key step was optimization of the acylation step with minimal nitrogen isotope fractionation [660]. 

Analysis using a CMPA is usually resolved on a nonchiral column. A transient diastereomeric complex is formed between the enantiomer and the chiral component in the mobile phase, similar to the complexes formed with chiral stationary phases. A review by Liu and Liu (2002) cites several papers where addition of CPMAs has been used in analyzing amphetamine-related compounds. Some CPMAs include amino acid enantiomers, metal ions, proteins, and cyclodextrins. Advantages of this method of analysis include the use of less expensive columns and more flexibility in the optimization of chiral separation (Misl anova and Hutta, 2003). 

Enantioselective metal chelation is a technique that has been applied to the separation of amino acid enantiomers. In the method, a transition metal-amino acid complex, such as copper(II)-aspartame, in which the full coordination of the complex has not been reached, is added to the buffer. The amino acid enantiomers are able to form ternary diastereomeric complexes with the metal-amino acid additive if there are differences in stability between the two complexes, enantioselective recognition can be achieved. 

Type IV includes chiral phases that usually interact with the enantiomeric analytes through the formation of metal complexes. There are usually used to separate amino acid enantiomers. These types of phases are also called ligand exchange phases. The transient diastereomeric complexes are ternary metal complexes between a transitional metal (usually Cu +), an amino acid enantiomeric analyte, and another compound immobilized on the CSP which is able to undergo complexation with the transitional metal (see also the ligand exchange section. Section 22.5). The two enantiomers are separated based on the difference in the stability constant of the two diastereomeric species. The mobile phases used to separate such enantiomeric analytes are usually aqueous solutions of copper (II) salts such as copper sulfate or copper acetate. To modulate the retention, several parameters—such as the pH of the mobile phase, the concentration of the copper ion, or the addition of an organic modifier such as acetonitrile or methanol in the mobile phase—can be varied. 

An alternative technique for the resolution of enantiomers which lack polar functional groups is a diastereomeric complex formation via non-ionic mechanisms. These complexes may be of the external or the inclusion type. Chiral amino acids, metal complexes or host compounds may serve as useful complexing agents (Table 2) [49-64]. 

In 1960, a patent was granted for the separation of D,L-prollne on a lactose column (56). Davankov s laboratory was the first to report separation of amino acid Isomers on polymeric resins derlvatlzed with optically active amino acids (57). However, separation of amino acid enantiomers by these techniques has been hampered by long separation times (ca. 10 hr) and the difficulty In synthesizing supports of sufficient quality for modern HPLC (spherical particles, small size, uniform chemical modification). Separation of amino acid Isomers on a column consisting of silica bonded with L-amlno acids and complexed with copper (II) has been reported by Gubltz and Jellenz (42). Short analysis times for separation of mixtures of single D,L-amlno acids were reported (ca. 30 min), but complex mixtures have not been separated. 

Mie, A., Jomten-Karlsson, M., Axelsson, B.O., Ray, A., Reimann, C.T., Enantiomer separation of amino acids by complexation with chiral reference compounds and high-field asymmetric waveform ion mobility spectrometry preliminary results and possible limitations. Anal Chem. 2007, 79, 2850. 

The calculated energy values of the amino acid enantiomers derivatized with (+)-l-(9-fluorenyl)ethyl chloroformate using the MM2 program are listed Tables 8.12 and 8.13 with chromatographic data measured in reversed-phase from ref. 18. The energy values of individual complexes between the model phase and an enantiomer are listed Table 8.13. 

Bearing this in mind, we designed and synthesized a number of P-CD derivatives [27-34] which could i) bind copper(II) forming a multisite recognition system ii) show thermodynamic stereoselectivity in copper(II) ternary complexes iii) perform chiral separation of unmodified amino acid enantiomers. Among the monofunctionalized P-CD derivatives, only those functionalized in position 6 with diamines show chiral molecular recognition [29,32,35-37]. On the contrary, the P-CDs both functionalized in position 3 and those where a triamine was attached to the narrower rim of the toroid do not act as chiral receptors. 2-(aminomethyl)pyridine, histamine and NH3 molecules were used to obtain the three isomers of P-CDs (Figure 3), but only the A,BCD-NH2 molecule, coordinated to the copper(II) ion, is seen to have enatioselective effects on aromatic amino acids [38]. 

Further support for this hypothesis comes fi om the thermodynamic parameters for copper(II) complexes with CDen and some amino acid enantiomers [32]. As our hypothesis suggests, we would not expect to find any significant thermodynamic stereoselectivity as the primary and secondary amine groups of the aliphatic diamine, being similar, cannot display significantly different behaviour... 

Summarizing as follows, we could say that we learned the following from the thermodynamic studies (1) The different stability of the copper(H) ternary complexes with functionalized P-cyclodextrins is enthalpy driven, in accordance with what was previously found for the ternary system without the CD cavity (2) The cis-disposition of the amine groups of the two ligands seems to be the discriminating factor, in some cases allowing the interaction of the aromatic residues of one the two isomers with the cavity (3) The difference in AH° values between the two copper(II) diastereoisomeric complexes reflects the different interactions of the aromatic moieties of the two amino acid enantiomers. 

Furthermore, it is also unsuitable for preparative purposes. However, it is suitable for the trace analysis of amino acid enantiomers in complex matrices such as biological samples because of the introduction of a highly sensitive ultraviolet-visible or fluorescence tag. 

Many chiral derivatization reagents have been developed for the enantioseparation of amino acids wherein ultraviolet-visible or fluorescence tags are introduced. The fluorescence derivatization is more effective for the determination of amino acid enantiomers in complex matrices in terms of sensitivity and/or selectivity. Table 1 shows the chiral derivatization reagents, whose structures are shown in Figure 2, used for the enantioseparation of amino acids. 

Chiral ligand-exchange chromatography is based on the formation of diastereomeric ternary complexes that involve a transition metal ion, chiral ligand, and the amino acid enantiomers. Among transition metals, Cu(II) formed the most stable complexes... 

Conversion of amino acids to produce a diaster-eoisomeric peptide by reaction with an N-carboxy-anhydride, e.g., L-Phe-N-carboxy-anhydride, has been used for determination of optical purity using RPC. Alternatively, for analysis of amino acid enantiomers without derivatization, two options are available (1) chiral mobile phases such as the N,N-di- -propyl-L-alanine-copper(n) complex can be used with reversed-phase columns, and (2) stationary phases with a covalently bound ligand capable of stereo recognition can be used. Such ligands include cyclodextrins, albumins, glycoproteins, and copper(II) complexes. 

Functionalized polystyrene resins have been employed for ligand-exchange chromatography of racemates of amino-acids. > The resins carry asymmetric sorbent groups, for example of oc-amino-acids complexed with Cu > or Ni , capable of selective formation of mixed ligands with amino-acid enantiomers. Structures (10) and (11) are examples of reactive sites bonded to polystyrene resins for this purpose. The second type of structure is produced by direct... 

The teicoplanin CSPs are recommended for the separation native amino acid enantiomers in reversed-phase mode [11]. It was established that the carbohydrate units on the teicoplanin selector (Fig. 3) were hindering the enantiomer approach making the transient selector-selectand complex more difficult to form [15]. This mechanistic point will be studied in detail later. 

Berthod et al. also tried copper complexation with teicoplanin and TAG CSPs [19]. Similar results were obtained. Amino acid enantiomers perfectly separated by both teicoplanin and TAG CSP could no longer be separated as soon as copper was present in the mobile phase. The copper-teicoplanin complex is also formed with the primary amine group on the peptidic teicoplanin basket (Figs. 1 and 3). However, unlike the vancomycin-copper complex which was very stable [18], the teicoplanin-copper complex was found to be reversible. Indeed, amino acid enan-tioselectivity was mostly restored after washing the chiral column with several column volumes of copper-free clean mobile phase [19]. 

Cyclodextrins have been used mainly as mobile phase additives for chiral resolutions of a number of racemates, including amino acids and their derivatives, but Alak and Armstrong [25] used fi-CD bounded to silica gel for resolution of dansyl derivatives of DL-amino acid enantiomers, by forming areversible inclusion complex of different stability. Dansyl DL-amino acids were better separated on fi-CDs layers than nonderivatized amino acids, because they have additionally two or more carbohydrogen rings. 

---