Enantiomers preparative enantioseparation

In this chapter we will focus on the moleciilcir recognition niechanisms of the diverse chiral SOs and CSPs in combination with their spectra of applicability, but also aspects concerning the separation systems as well as on issues that are of interest for practical applications. This will include a discussion of structure resolution relationships as support for the selection of certain CSPs for a given separation problem, operation modes and mobile phase composition, stability, the ability to reverse the elution order to elute each of the enantiomers as the first peak, and loadability which is of primary importance for preparative enantioseparations. 

The early systematic preparative enantioseparations of drugs performed on cross-linked chiral polyacrylamides in low-pressure LC mode clearly showed that even this non-opti-mal technique, from the viewpoints of performance and costs, may be useful for comparative biomedical studies of enantiomers of chiral drugs. 

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. 

The pretzel-shaped molecule 96 (the first pretzelane ) was synthesized by intramolecular bridging of the two sulfonamide units of 79 with a bifunctionalized podand-like chain [54]. Again the enantioseparation of 96 was accomplished with a baseline quality separation and a large separation factor (a=5.20). Preparative separation of the enantiomers enabled the detection of the circular dichrogram of 96 (Figure 48). The optical rotation values of 79 and 96 were both determined to be [aD] = 168° (Troger base [aD] = 281°). 

The use of a convective macroporous polymer as an alternative support material instead of silica for the preparation of protein-based CSPs has successfully been demonstrated by Hofstetter et al. [221]. Enantioseparation was performed using a polymeric flow-through-type chromatographic support (POROS-EP, 20 pm polymer particles with epoxy functionalities) and covalently bound BSA as chiral SO. Using flow rates of up to 10 ml/min, rapid enantiomer separation of acidic compounds, including a variety of amino acid derivatives and drugs, could be achieved within a few minutes at medium efficiencies, typical for protein chiral stationary phases (Fig. 9.13). 

In another approach, reactive monodisperse porous poly(chloromethylstyrene-co-styrene-co-divinylbenzene) beads have been employed for the preparation of chiral HPLC packings. Thus, reactive chloromethyl groups were derivatized to yield amino functionalized beads onto which both rt-basic and rt-acidic type chiral. selectors, (/ )- -(l-naphthyl)ethylamine and (/ )-A -(3.5-dinitrobenzoyl)phenylglycine, respectively, were attached. The resulting chiral particles were chromatographically tested for the enantioseparation of model SAs. Despite the presence of strongly competitive it-TT-binding sites of the styrenic support these chirally modified beads afforded baseline separations for 2,2,2-trifluoro-l-(9-anthryl) ethanol and Af-(3.5-dinitro-benzoyl) leucine enantiomers, respectively [369. ... 

The chromatographic separation of enantiomers, often referred to as enantioseparation, has received a great deal of attention in recent years. Both liquid (LC) and gas (GC) chromatographic procedures are used. The former is extremely useful for enantioseparations because of the available variations in scale, mechanism, and technique. It has been used in enantioseparations from analytical to preparative in scale, taking advantage of various modes of diastereoisomeric interactions andusing elution and displacement techniques. All the chromatographic methods involve diastereoisomeric interactions between the enantiomers of interest and... 

Based on the general synthetic scope of this book, the subject of enantioseparations is treated in this chapter mainly from the viewpoint of micropreparative- and preparative-scale production of enantiomerically pure compounds. Analytical aspects are treated to a very limited extent and a quantification of enantiomers in biological fluids as well as stereoselec-... 

The resulting covalent diastereomeric compounds possessing different free energies may be resolved based on their different physico-chemical properties and then be transformed back to the optically pure starting material. The technique of covalent diastereomer formations is very laborious and time-consuming, and sometimes needs to be performed under conditions where a racemization may occur. Therefore, for preparative purposes this technique represents just a theoretical interest. However, it is still used in analytical-scale enantioseparations where the enantiomers must not necessarily be collected after the separation. 

The most important advantages of direct chromatographic enantioseparations for preparative purposes include the presence of resolved enantiomers in original and high optically pure form in different volume fractions of the mobile phase, no loss of chiral selectors, and amost no risk of contamination of desired enantiomers with the chiral selector. Parallel to enantioseparations other impurities may be removed from chiral compounds during this process. 

The indirect method is an efficient technique for the enantioseparation of amino acids. However, it is essential that the chiral derivatization reaction proceeds completely in both enantiomers, and that the racemization reaction does not occur. Furthermore, if the optical purity of the derivatization reagent is not known, and/or is not taken into consideration, the optical purity of the amino acid will not be determined precisely. Thus, the indirect method is unsuitable for the analysis of amino acid enantiomers in a standard sample and pharmaceutical preparations, where a low amount of antipode, at a level of 0.1 or 0.05%, should be determined. 

As a result, the baseline enantioseparation was obtained, as shown in Figure 19.1a. Monomeric Pro is one of these rare amino acids, which develop yellow (and not bluish) color when visualized with ninhydrin. l-Pto used as an external standard (Figure 19.1b) confirmed the identity of the lower yellow spot number 2 as enantiomer L and the upper yellow spot number 3 as enantiomer d, as shown in Figure 19.1a. The respective Rp values were 0.57 0.02 and 0.74 0.02. Brownish-purple spot number 1 (Rp = 0.32 0.02 Figure 19.1) apparently originates from the Pro-derived peptides and it is also fully separated from the monomeric l-Pto spot number 2. The presence of peptides in the two freshly prepared Pro samples wimesses to rapid peptidization of this amino acid (although contamination of the commercial monomeric DL-Pro and L-Pro samples with peptides cannot be excluded). 

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