Separation of D and L enantiomers

Separation of D- and L-Enantiomers. In addition, the absolute configuration of component monosaccharides (i.e. whether D- or L-enantiomers) could be determined by using chiral stationary phases (33) and/or by reacting the monosaccharide with a reagent which will introduce chiral centres (34). 

There are two major approaches to achieve enantiomeric separation of d- and L-amino acids. The first involves precolumn derivatization with a chiral reagent, followed by RP-HPLC [226], while the second involves direct separation of underivatized enantiomers on a chiral bonded phase [227], Weiss et al. [209] determined d- and L-form of amino acids by applying derivatization with OPA and chiral /V-isobutyryl-L-cysteine. 

Owing to the different biological activity of D- and L-enantiomers of seleno-amino acids, the chiral separation of optical isomers has been undertaken in sele-nized yeast and in yeast-based commercial supplements. Both, chiral stationary phase (crown ether) and chiral derivatization prior to reversed-phase HPLC were used [16, 77, 78],... 

Fig. 5.18. Effect of polymerisation and elution temperature on the enantiomer separation factor (a) in the separation of D- and L-PA on L-PA imprinted polymers. Polymers were prepared by thermochemical initiation at either 60 or 40°C using AIBN or ABDV respectively as initiators. The samples consisted of ca. 20 nmol of each of D- and L-PA and BOC-L-PA as void marker. Flow rate 0.5 mL/min. Mobile phase MeCN/acetic acid 95/5 (v/v). The columns were thermostatted by immersing them in a circulating water bath at the indicated temperature. From O Shannessy et al. [8].

When amino acids are synthesized in nature, only the L-enantiomer is formed (Section 5.20). However, when amino acids are synthesized in the laboratory, the product is usually a racemic mixture of D and l enantiomers. If only one isomer is desired, the enantiomers must be separated. They can be separated by means of an enzyme-catalyzed reaction. Because an enzyme is chiral, it will react at a different rate with each of the enantiomers (Section 5.20). For example, pig kidney aminoacylase is an enzyme that catalyzes the hydrolysis of A -acetyl-L-amino acids, but not Ai-acetyl-D-amino acids. Therefore, if the racemic amino acid is converted into a pair of Wacetylamino acids and the A -acetylated mixture is hydrolyzed with pig kidney aminoacylase, the products will be the L-amino acid and A -acetyl-D-amino acid, which are easily separated. Because the resolution (separation) of the enantiomers depends on the difference in the rates of reaction of the enzyme with the two A -acetylated compounds, this technique is known as a kinetic resolution. 

Pyka (1993) developed a new topological index for predicting the separation of D and L optical isomers of amino acids on chiral plates. LeFevre (1993) used reversed-phase TLC to separate dansyl-amino acid enantiomers. Das and... 

FIGURE 5 Effect of chiral eluant on the separation of d- and L-amino acid enantiomers by ligand-exchange chromatography. 

Kovacs-Hadady, K. and Kiss, I.T., Attempts for the chromatographic separation of D- and L-penicillamine enantiomers, Chromatographia, 24, 677,1987. 

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. 

Valko et al. (22) studied the separation of mandelic acid enantiomers with y-CD and calculated very low binding constants 2.8 and 2.4 M x for the d- and L-enantiomers, respectively. The weak binding of mandelic acid to y-CD was explained by the large size of the y-CD torus for such a small molecule as mandelic acid. Unlike the enantiomers of leucovorin and its active metabolite, for which weak binding was associated with low enantio-selective (19), fairly high enantioselectivity was found for weakly bonded enantiomers of mandelic acid with y-CD (KD/KL = 1.17). 

A number of variations on this type of coating have been prepared and offer some improvement over the original phase. Figure 11.11 shows the volatile pentafluoropropionamide-trifluoroethyl ester (PFP-TFE) derivatives of L and D phenylalanine. Figure 11.12 shows the separation of PFP-TFE derivatives of the D and L enantiomers of the amino acids phenylalanine and p-tyrosine on a Chirasil Val column, the D(/ )-enantiomers elute first. Chirasil Val generally performs best for the separation of enantiomers of amino acids, for many other compounds it is not as effective. 

The carbanion257197 was prepared by reacting at -20 °C LDA in situ with the ester 198. d- and L-enantiomers of 14C-labelled tyrosine have been separated by a Daicel Chiralpak WH column258. The enantiomeric purity of isolated L-isomer of 196 was 99%, the radiochemical purity was 99%, and the r.y. was 24%, with a specific radioactivity of 55 mCi mmol-1. 

It seems tc, also be worth mentioning that the described procedure has been used for micro-preparative separations of mephenytoin and hexobarbital enantiomers (26) p -CD solutions were also successfully used for resolution of 1-[2-(3-hydroxyphenyl)-l-phenylethylJ-4-(3-me-thyl-2-buteny1) piperazine enantiomers in RP systems (.20). An especially interesting example of the application of -CD is the separation of optical isomers of D,L - norgestrel (27). 

Stereoselective addition of a chiral cyclic phosphite to a cyclic imine was applied to the synthesis of phosphonic analogs of d- and L-penicillamine41. Enantiomerically pure dioxaphospholane oxide 6 underwent addition to 2,5-dihydro-2,2,5,5-tetramethylthiazole in the presence of boron trifluoride as a catalyst. The diastereomeric adducts 7, obtained in a 2 1 ratio, were easily separated on silica gel and their configuration assigned by X-ray analysis the (4/ )-isomer was shown to be the major diastereomer. Hydrolysis of each diastereomer gave pure enantiomers of phosphopenicillamine 841. 

The template la can be split off by water or methanol to an extent of up to 95% (see Scheme 4.II). The accuracy of the steric arrangement of the binding sites in the resulting imprinted cavity can be tested by the ability of the polymer to resolve the racemate of the template, namely phenyl-a-D,L-mannopyranoside. The polymer was equilibrated in a batch procedure with a solution of the racemate under conditions that allowed a thermodynamically controlled partition of the enantiomers between polymer and solution. The enrichment of the antipodes in the polymer and in solution was determined and the separation factor a, i.e. the ratio of the distribution coefficients of the d- and L-enantiomer between polymer and solution, was calculated. After extensive optimisation of the procedure, a values between 3.5 and 6.0 were obtained [4]. This is an extremely high selectivity for racemic resolution that cannot be reached by most other methods. 

Even in the best case, some racemic product is produced and must be separated out. This separation is easy or hard, depending on the nature of the racemate. If the racemic modification has a different crystalline form to that of the pure d or l, then separation of the pure excess enantiomer will be inefficient. If one achieves a 90% ee value, then it is quite possible to get out only 75-80% pure enantiomer. With lower ee values, the losses become prohibitive. For such a system, a catalyst of very high efficiency must be used. Unfortunately, most compounds are of this type their racemic modifications do not crystallize as pure d- or l-forms. If, on the other hand, the racemic modification is a conglomerate or an equal mix of d- and L-crystals, then recovery of the excess the L-form can be achieved with no losses. Since the l- and D,L-forms are not independently soluble, a 90% ee value easily gives a 90% recovery of pure isomer. In our L-dopa process, the intermediate is just such a conglomerate and separations are efficient. This lucky break was most welcome. If one thinks back, ours was the same luck that Pasteur encountered in his classical tartaric acid separations, 150 years ago. 

Figure 1. Typical HPLC chromatograms of the OPA-NAC (II) derivitized amino acids detected from the spark discharge reactions. Chromatograms labeled with roman numerals I.) Amino acid standard, II.) CO2/N2 not sparked, III.) CO2/N2 + CaCOs, sparked, hydrolyzed- ascorbate. IV.) CO2/N2, sparked, hydrolyzed - ascorbate V.) CO2/N2, sparked + CaCOs, hydrolyzed + ascorbate. Amino acids I.) DL aspartic acid 2.) DL glutamic acid 3.) DL serine 4.) glycine 5.) P-alanine 6.) DL alanine 7.) a-amino isobutyric acid 8.) DL norleucine (internal standard). The D and L enantiomers of glutamic acid and serine are not separated under these chromatographic conditions.

The conventional C-18 and the CD columns do interact differently with solutes of certain classes of isomers. For example, C-18 columns cannot separate enantiomers unless special additives are introduced into the mobile phase. The cyclodextrin bonded phases, however, can easily separate enantiomeric species as illustrated in Figure 9. The D and L enantiomers of dansyl-DL-leucine and of dansyl-DL-norleucine are resolved using a beta-CD column, but attempts to separate these isomers were unsuccessful using a C-18 column. The nature of the interactions between enantiomers and the cyclodextrin cavity has been described elsewhere (20. 21). 

The preparative separation of mixtures of D- and L-amino acids into the D-and L-enantiomers, already a problem in E. Fischer s laboratory, has been perfected nowadays but it still takes much time. In order to obtain analytical results as fast as possible, chromatographic methods were attempted early, and gas chromatography turned out to be the method of choice. In 1965, Emanuel... 

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