Diastereomeric derivatization

Three general methods exist for the resolution of enantiomers by Hquid chromatography (qv) (47,48). Conversion of the enantiomers to diastereomers and subsequent column chromatography on an achiral stationary phase with an achiral eluant represents a classical method of resolution (49). Diastereomeric derivatization is problematic in that conversion back to the desired enantiomers can result in partial racemization. For example, (lR,23, 5R)-menthol (R)-mandelate (31) is readily separated from its diastereomer but ester hydrolysis under numerous reaction conditions produces (R)-(-)-mandehc acid (32) which is contaminated with (3)-(+)-mandehc acid (33). 

Diastereomeric derivatization 129 Reaction with OPA and chiral thiols, Ai-isobutyryl-L,D-cysteine separation on C,8 column application to many food types... 

Barksdale, J.M. Clark, C.R. Liquid chromatographic determination of the enantiomeric composition of amphetamine and related drugs by diastereomeric derivatization. J. Chromatogr. Sci. 1985, 23 (4), 176-180. 

GC) (HPLC] 1 FC) (Capillary) gas, (high-performance) liquid, or supercritical fluid chromatography. Where used for ee determination, either chiral columns or diastereomeric derivatization is implied. 

Several alternative routes can be used in order to derivatize the carboxy function (Fig. 7-7). Ketones can be transferred by hydrazines and diols to the corresponding hydrazines or acetals. 2,2,2-Trifluoro-l-phenylethylhydrazine [19] is an example of the first group, while 2,3-butanediol or l,4-dimethoxy-2,3-butanediol can be used to form diastereomeric acetals. 

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). 

The second approach for the synthesis of 2-amino-3-hydroxycarboxylic acids starts with a chiral isothiocyanate which is added, via the tin enolate, to aldehydes. The initially formed adducts are immediately derivatized to the heterocycles, from which. yj 7-2-amino-3-hy-droxycarboxylic acids result after a three-step procedure. The diastereomeric ratios of the intermediate bis-heterocyclic products range from 93 7 to 99 1 (desired isomer/sum of all others)104. 

A variety of methods are also available when the compound under investigation can be converted with a chiral reagent to diastereomeric products, which have readily detectable differences in physical properties. If a derivatizing agent is employed, it must be ensured that the reaction with the subject molecule is quantitative and that the derivatization reaction is carried out to completion. This will ensure that unintentional kinetic resolution does not occur before the analysis. The derivatizing agent itself must be enantiomerically pure, and epi-merization should not occur during the entire process of analysis. 

Various chiral derivatizing agents have been reported for the determination of enantiomer compositions. One example is determining the enantiomeric purity of alcohols using 31P NMR.28 As shown in Scheme 1-8, reagent 20 can be readily prepared and conveniently stored in tetrahydrofuran (THF) for long periods. This compound shows excellent activity toward primary, secondary, and tertiary alcohols. To evaluate the utility of compound 20 for determining enantiomer composition, some racemic alcohols were chosen and allowed to react with 20. The diastereomeric pairs of derivative 21 exhibit clear differences in their 31P NMR spectra, and the enantiomer composition of a compound can then be easily measured (Scheme 1-8). 

One of the most powerful methods for determining enantiomer composition is gas or liquid chromatography, as it allows direct separation of the enantiomers of a chiral substance. Early chromatographic methods required the conversion of an enantiomeric mixture to a diastereomeric mixture, followed by analysis of the mixture by either GC or HPLC. A more convenient chromatographic approach for determining enantiomer compositions involves the application of a chiral environment without derivatization of the enantiomer mixture. Such a separation may be achieved using a chiral solvent as the mobile phase, but applications are limited because the method consumes large quantities of costly chiral solvents. The direct separation of enantiomers on a chiral stationary phase has been used extensively for the determination of enantiomer composition. Materials for the chiral stationary phase are commercially available for both GC and HPLC. 

The use of diazaphospholidines as chiral derivatizing agents for the determination of the enantiomeric composition of choro- or bromohydrins has been reported. Thus, 31P NMR spectra of a range of diastereomeric derivatives have been described to show a systematic deshielding from 0.2 to 12.9 ppm of isomers 48 compared to isomers 49 <2000TA1273>. 

CE has been applied extensively for the separation of chiral compounds in chemical and pharmaceutical analysis.First chiral separations were reported by Gozel et al. who separated the enantiomers of some dansylated amino acids by using diastereomeric complex formation with Cu " -aspartame. Later, Tran et al. demonstrated that such a separation was also possible by derivatization of amino acids with L-Marfey s reagent. Nishi et al. were able to separate some chiral pharmaceutical compounds by using bile salts as chiral selectors and as micellar surfactants. However, it was not until Fanali first showed the utilization of cyclodextrins as chiral selectors that a boom in the number of applications was noted. Cyclodextrins are added to the buffer electrolyte and a chiral recognition may... 

Since dorzolamide possesses two chiral centers, an indirect chiral separation has been developed [18]. The procedure employs the chemical derivatization of the secondary amino group of the inhibitor, formation of diastereomeric urea derivatives (each with three chiral centers in the molecule), and their separation under non-ehiral HPLC conditions. Using... 

The direct gas-chromatographic method is especially suited as an analytical tool for enantiomer analysis when no sample derivatization is required. In the absence of diastereomeric effects between enantiomers ( EE-effect )25, the chiral compound may be investigated in situ, i.e., without isolation and purification using a minute amount of sample, e.g., by head-space analysis with 10 9 g using flame ionization detector (FID) or 1 (F 11 g using selected ion monitoring (GLC-MSSIM). 

As discussed earlier, the concepts of chiral chromatography can be divided into two groups, the indirect and the direct mode. The indirect technique is based on the formation of covalently bonded diastereomers using an optically pure chiral derivatizing agent (CDA) and reacting it with the pair of enantiomers of the chiral analyte. The method of direct enantioseparation relies on the formation of reversible quasi diastereomeric transient molecule associates between the chiral selector, e.g., i /t)-SO, and the enantiomers of the chiral selectands, [R,S)-SAs [(Ry SA + (S)-SA] (Scheme 1). 

Derivatization of chiral arylalkylamines and NMR measurement of the diastereomeric ratios can be conveniently performed using d-camphor-10-sulfonic acid, having the (l/ ,4/ ) configuration49. 

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