Enantiomers amino acid

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),... 

Erank H, G. J. Nicholson and E. Bayer, Rapid gas cliromatographic separation of amino acid enantiomers with a novel cliiral stationary phase , J. Chromatogr. Sci. 15 174-176(1977). 

C. Wang, E. Haitmut, G. Wang, L. Zhou, E. Bayer and P. Lu, Determination of amino acid enantiomers by two-column gas cliromatography with valveless column switching , J. Chromatogr. 262 352-359 (1983). 

Early examples of enantioselective extractions are the resolution of a-aminoalco-hol salts, such as norephedrine, with lipophilic anions (hexafluorophosphate ion) [184-186] by partition between aqueous and lipophilic phases containing esters of tartaric acid [184-188]. Alkyl derivatives of proline and hydroxyproline with cupric ions showed chiral discrimination abilities for the resolution of neutral amino acid enantiomers in n-butanol/water systems [121, 178, 189-192]. On the other hand, chiral crown ethers are classical selectors utilized for enantioseparations, due to their interesting recognition abilities [171, 178]. However, the large number of steps often required for their synthesis [182] and, consequently, their cost as well as their limited loadability makes them not very suitable for preparative purposes. Examples of ligand-exchange [193] or anion-exchange selectors [183] able to discriminate amino acid derivatives have also been described. 

Separation of Amino Acid Enantiomers after Derivatization with Or/ho-Phthaldialdehyde (OPA) and a Unichiral Tliiol Compound... 

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. 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

Figure 5.7 Only one of the two amino acid enantiomers shown can achieve three-point binding with the hypothetical binding site (e.g., in an enzyme). 

Macko, S. A., Uhle, M. E., Engel, M. H. and Andrusevich, V. (1997) Stable nitrogen isotope analysis of amino acid enantiomers by gas chromatography combustion/isotope ratio mass spectrometry. Analytical Chemistry 69, 926 929. 

FIGURE 4.19 Amino acid enantiomers are determined by reaction (A) with l- or D-amino-acid oxidase at pH 7-8.75 Added catalase decomposes the hydrogen peroxide (B), which would otherwise oxidize the a-oxoacid. Quantitation is achieved by measuring oxygen consumption, which is 0.5 mol/mol of substrate. 

S Einarsson, B Josefsson, P Moller, D Sanchez. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chlo-roformate and reversed phase liquid chromatography. Anal Chem 59, 1191, 1987. 

A study was made of the effects of derivatization on the 13C analysis of amino acid enantiomers. Conventional isotope ratio MS and GC-isotope ratio MS were used. The latter method requires volatilization of the analytes, which was accomplished by introducing O-isopropyl and A-trifluoroacetyl groups, causing a change in the 13C analysis of the original analytes. It was proposed to use a set of known standards for such analyses, which are applied in geological studies68. 

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]. 

This 2D-method was validated for the concentration range between 0.005 and 0.5 pmol for D-amino acids and 0.05-5 pmol for L-amino acids. Within-day and interday precisions were always better than 8% relative standard deviation (RSD) and the accuracies for spiked rat plasma samples were between 95.5% and 100.2%. Limit of detections (LCDs) and limit of quantitations (LOQs) were reported to be as low as 3 fmol (S/N = 3-5 corresponding to 0.15 nmolg wet tissue) and 5 fmol (corresponding to 0.25 nmolg wet tissue). It was concluded that this assay is supposed to be one of the most sensitive analysis method for amino acid enantiomers in mammalian samples. 

Ilisz, I., Berkecz, R., and Peter, A., HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibiotic-based chiral stationary phases a review, J. Sep. ScL, 29, 1305, 2006. 

Peyrin, E. et al., Dansyl amino acid enantiomer separation on a teicoplanin chiral stationary phase effect of eluent pH, J. Chromatogr. A, 923, 37, 2001. 

Amino acid enantiomers can be separated on a chiral stationary phase after derivatization with chloroformates (Abe et al., 1996). The derivatization procedure is quite simple and rapid, but the derivatizing reagent must be synthesized, which complicates the assay. Another method for the analysis of amino acid enantiomers uses N,0-pentafluoropropionyl isopropyl derivatives and a chiral column with NPD detection (Hashimoto et al., 1992). 

Abe I, Fujimoto N, Nishiyama T, Terada K, Nakahara T. 1996. Rapid analysis of amino acid enantiomers by chiral-phase capillary gas chromatography. J Chromatogr A 722 221. Ahuja S. 1976. Derivatization in gas chromatography. J Pharm Sci 65 163. 

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). 

OPA in combination with chiral thiols is one method used to determine amino acid enantiomers. A highly fluorescent diastereomeric isoindole is formed and can be separated on a reverse-phase column. Some of these chiral thiols include N-acetyl-L-cysteine (NAC), N-tert-butyloxy-carbonyl- L-cysteine (Boc-L-Cys), N-isobutyryl- L-cysteine (IBLC), and N-isobutyryl- D -cysteine (IBDC). Replacing OPA-IBLC with OPA-IBDC causes a reversal in the elution order of the derivatives of D- and L-amino acids on an ODS column (Hamase et al., 2002). Nimura and colleagues (2003) developed a novel, optically active thiol compound, N-(tert-butylthiocarbamoyl)- L-cysteine ethyl ester (BTCC). This reagent was applied to the measurement of D-Asp with a detection limit of approximately 1 pmol, even in the presence of large quantities of L-ASP. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

Chen, Z., Ozawa, H., Uchiyama, K., and Hobo, T. (2003). Cyclodextrin-modified monolithic columns for resolving dansyl amino acid enantiomers and positional isomers by capillary electrochromatography. Electrophoresis 24, 2550-2558. 

CL Copper, JB Davis, RO Cole, MJ Sepaniak. Separations of derivatized amino acid enantiomers by cyclodextrin-modified capillary electrophoresis Mechanistic and molecular modeling studies. Electrophoresis 15 785-792,... 

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