Resolution, of enantiomers

The separation of a racemic mixture into its enantiomeric components is termed resolution. The first resolution, that of tartaric acid, was carried out by Louis Pasteur in 1848. Tartaric acid is a byproduct of wine making and is almost always found as its dextrorotatory 2R,3R stereoisomer, shown here in a perspective drawing and in a Fischer projection. 

A description of Pasteur s work, as part of a broader discussion concerning crystal structure, can be found in the article Molecules, Crystals, and Chirality in the July 1997 issue of the Journal of Chemical Education, 

PROBLEM 7.19 There are two other stereoisomeric tartaric acids. Write their Fischer projections, and specify the configuration at their stereogenic centers. 

Occasionally, an optically inactive sample of tartaric acid was obtained. Pasteur noticed that the sodium ammonium salt of optically inactive tartaric acid was a mixture of two mirror-image crystal forms. With microscope and tweezers, Pasteur carefully separated the two. He found that one kind of crystal (in aqueous solution) was dextrorotatory, whereas the mirror-image crystals rotated the plane of polarized light an equal amount but were levorotatory. 

TUthough Pasteur was unable to provide a structural explanation— that had to wait for van t Hoff and Le Bel a quarter of a century later—he correctly deduced that the enantiomeric quality of the crystals was the result of enantiomeric molecules. The rare form of tartaric acid was optically inactive because it contained equal amounts of (+)-tartaric acid and ( )-tartaric acid. It had earlier been called racemic acid (from Latin racemus, a bunch of grapes ), a name that subsequently gave rise to our present term for an equal mixture of enantiomers. 

Could the unusual, optically inactive form of tartaric acid studied by Pasteur have been meso-tartaric acid  

When a chiral compound is synthesized from achiral reagents, however, a racemic mixture of enantiomers is obtained. For example, we saw that the reduction of 2-butanone (achiral) to 2-butanol (chiral) gives a racemic mixture  

If we need one pure enantiomer of 2-butanol, we must find a way of separating it from the other enantiomer. The separation of enantiomers is called resolution, and it is a different 

In 1848, Louis Pasteur noticed that a salt of racemic ( )-tartaric acid crystallizes into mirror-image crystals. Using a microscope and a pair of tweezers, he physically separated the enantiomeric crystals. He found that solutions made from the left-handed crystals rotate polarized light in one direction and solutions made from the right-handed crystals rotate polarized light in the opposite direction. Pasteur had accomplished the first artificial resolution of enantiomers. Unfortunately, few racemic compounds crystallize as separate enantiomers, and other methods of separation are required. 

The traditional method for resolving a racemic mixture into its enantiomers is to use an enantiomerically pure natural product that bonds with the compound to be resolved. When the enantiomers of the racemic compound bond to the pure resolving agent, a pair of diastereomers results. The diastereomers are separated, then the resolving agent is cleaved from the separated enantiomers. 

Let s consider how we might resolve a racemic mixture of (/ )- and (5)-2-bu-tanol. We need a resolving agent that reacts with an alcohol and that is readily available in an enantiomerically pure state. A carboxylic acid combines with an alcohol to form an ester. Although we have not yet studied the chemistry of esters (Chapter 21), the following equation shows how an acid and an alcohol can combine with the loss of water  

Hydrolytic enzymes are often able to catalyze the esterification of one enantiomer of a racemic alcohol, thus providing one enantiomer of the alcohol and the ester of the other enantiomer, readily separable by extraction or other means. Candida rugosa lipase, immobilized on DEAE-Sephadex-A-25, has been used in such a resolution of ( )-menthol (Eq. 3.2) [20]. 

The resolutions described above are based on the formation of diaste-reomeric complexes with a column stationary phase or an enzyme. The more common alternative is to bond the enantiomers covalently to a chiral resolving agent to make stable diastereomeric molecules, separate those diastereomers by chromatography or recrystallization, and then disassemble each purified diastereomer to obtain the resolved enantiomers. 

Racemic A,A -dimethyl-l,2-diphenylethylenediamine was treated with L-(+)-tartaric acid to afford a pair of diastereomeric salts which were separated overnight by selective precipitation from ethanol. The (R,R)-diamine was separated by filtration, followed by extraction with base to remove the tartartic acid. Removal of the solvent produced the crude diamine in 90% yield and greater than 99% ee. The (5,5)-diamine was isolated by concentration of the mother liquor and the same extraction procedure to 

Both enantiomers of mandelic acid are commercially available and these are suitable resolving agents for a variety of functional groups, and often used to isolate chiral alcohols. A sequential use of (R)- and (5)-mandelic acid allowed resolution of racemic amino alcohols (Eq. 3.3) [22]. Subsequent extraction gave 90% recovery of the amino alcohols with 99% ee or better. Mandelic acid was recovered in 93% yield. 

The diastereomeric differentiations in the preceding cases involve physical interactions in chromatography or crystallization. Another possibility is to use a chemical reaction that is fast with one stereoisomer and slow with the other (kinetic resolution). A strain of baker s yeast engineered to express cyclohexanone monooxygenase was used to effect a Baeyer Villiger reaction on a variety of 2-alkylcyclohexanones, an example of which is shown in Equation 3.4 [23]. The ketone and lactone are readily separated by silica gel chromatography. 

We have seen that individual enantiomers have identical physical properties and only can be distinguished in a chiral environment. Plane-polarized light is such a chiral environment, and one enantiomer is dextrorotatory and one is levorotatory. Another way to distinguish enantiomers is to allow them to react (or interact) with other chiral molecules. The interaction of a mixture of enantiomers with a single enantiomer of a chiral molecule produces a mixture of diastereomers as illustrated. 

Since diastereomers have different physical properties, diey can be separated on the basis of those physical properties. After separation of the diastereomers, 

The use of enzymes to resolve enantiomers has become an extremely popular method only recently. Enzymes are chiral catalysts which often exhibit very high selectivity for one enantiomer of a racemic mixture. Since enzymes are soluble in aqueous solution, it was often impossible to get sufficiently high concentrations of 

Thus it is very easy to acetylate a racemic alcohol and treat the racemic mixture of acetates with a lipase. One enantiomer is hydrolyzed to the alcohol and the other remains as die ester. These are separated chromatographically and each component is obtained widi high optical purity. This technique is becoming more important and could be die most general technique for resolution in the future. 

The use of kinetic resolution to obtain a single enantiomer from a mixture of enantiomers is often useful for particular functional groups. Since individual 

As mentioned earlier, enantiomers have the same physical properties (boiling point, melting point, solubility, etc.). Since traditional separation techniques generally rely on differences in physical properties, they cannot be used to separate enantiomers from each other. The resolution (separation) of enantiomers can be achieved in a variety of other ways. 

The first resolution of enantiomers occurred in 1847, when Pasteur successfully separated enantiomeric tartrate salts from each other. Tartaric acid is a naturally occurring, optically active compound found in grapes and easily obtained during the wine-making process  

Only this stereoisomer is found in nature, yet Pasteur was able to obtain a racemic mixture of tartrate salts from the owner of a chemical plant  

The tartrate salts were then allowed to crystallize, and Pasteur noticed that the crystals had two distinct shapes that were nonsuperimposable mirror images of each other. Using only a pair of tweezers, he then physically separated the crystals into two piles. He dissolved each pile in water and placed each solution in a polarimeter to discover that their specific rotations were equal in amount but opposite in sign. Pasteur correctly concluded that the molecules themselves must be nonsuperimposable mirror images of each other. He was the first to describe molecules as having this property and is therefore credited with discovering the relationship between enantiomers. 

Most racemic mixtures are not easily resolved into mirror-image crystals when allowed to crystallize, so other methods of resolution are required. Two common ways will now be discussed. 

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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). 

Quantitative Analysis of Selectivity. One of the principal synthetic values of enzymes stems from their unique enantioselectivity, ie, abihty to discriminate between enantiomers of a racemic pair. Detailed quantitative analysis of kinetic resolutions of enantiomers relating the extent of conversion of racemic substrate (c), enantiomeric excess (ee), and the enantiomeric ratio (E) has been described in an excellent series of articles (7,15,16). 

The variety of enzyme-catalyzed kinetic resolutions of enantiomers reported ia recent years is enormous. Similar to asymmetric synthesis, enantioselective resolutions are carried out ia either hydrolytic or esterification—transesterification modes. Both modes have advantages and disadvantages. Hydrolytic resolutions that are carried out ia a predominantiy aqueous medium are usually faster and, as a consequence, require smaller quantities of enzymes. On the other hand, esterifications ia organic solvents are experimentally simpler procedures, aHowiag easy product isolation and reuse of the enzyme without immobilization. 

As was the case for kinetic resolution of enantiomers, enzymes typically exhibit a high degree of selectivity toward enantiotopic reaction sites. Selective reactions of enaiitiotopic groups provide enantiomerically enriched products. Thus, the treatment of an achiral material containing two enantiotopic functional groups is a means of obtaining enantiomerically enriched material. Most successful examples reported to date have involved hydrolysis. Several examples are outlined in Scheme 2.11. 

Crystallization methods are widely used for the separation, or resolution, of enantiomer pairs. Enantiomer mixtures may essentially crystallize in two different ways. In around 8 per cent of cases, each enantiomer crystallizes separately, giving rise to a mechanical mixture of crystals of the two forms, known as a conglomerate. Conglomerates may usually be separated by physical methods... 

Asymmetric epoxidation of racemic unsaturated fluoro alcohols by the chiral Sharpless reagent can be exploited for kmetic resolution of enantiomers The recovered stereoisomer has 14-98% enantiomeric excess [55] (equation 50)... 

In this chapter a number of the preparative techniques used in the resolution of enantiomers is presented. Some of these techniques will be developed more fully in following chapters. 

HPLC separations are one of the most important fields in the preparative resolution of enantiomers. The instrumentation improvements and the increasing choice of commercially available chiral stationary phases (CSPs) are some of the main reasons for the present significance of chromatographic resolutions at large-scale by HPLC. Proof of this interest can be seen in several reviews, and many chapters have in the past few years dealt with preparative applications of HPLC in the resolution of chiral compounds [19-23]. However, liquid chromatography has the attribute of being a batch technique and therefore is not totally convenient for production-scale, where continuous techniques are preferred by far. 

With regard to the resolution of enantiomers, some applications can be found with modified silica gel supports. Thus, a Pirkle-type CSP was used for the separation of 200 mg of a racemic benzodiazepinone [75]. Also tris-(3,5-dimethylphenyl)carba-mate of cellulose coated on silica [91, 92] was applied successfully to the resolution of the enantiomers of 2-phenoxypropionic acid and to oxprenolol, alprenolol, propranolol among other basic drugs. However, the low efficiency of this technique and the relative high price of the CSPs limits its use to the resolution of milligram range of sample. 

From the pioneering studies of Ito et al. [117], CCC has been mainly used for the separation and purification of natural products, where it has found a large number of applications [114, 116, 118, 119]. Moreover, the potential of this technique for preparative purposes can be also applied to chiral separations. The resolution of enantiomers can be simply envisaged by addition of a chiral selector to the stationary liquid phase. The mixture of enantiomers would come into contact with this liquid CSP, and enantiodiscrimination might be achieved. However, as yet few examples have been described in the literature. 

Although some applications for preparative-scale separations have already been reported [132] and the first commercial systems are being developed [137, 138], examples in the field of the resolution of enantiomers are still rare. The first preparative chiral separation published was performed with a CSP derived from (S -N-(3,5-dinitrobenzoyl)tyrosine covalently bonded to y-mercaptopropyl silica gel [21]. A productivity of 510 mg/h with an enantiomeric excess higher than 95% was achieved for 6 (Fig. 1-3). 

Gas chromatography (GC) has also been used for preparative purposes, but is restricted to relatively volatile racemates such as anesthetics, pheromones or monoterpenes and, therefore, very few applications are reported. Nevertheless, in the cases to which GC may be applied, it could be considered as an economical alternative to HPLC. Most of the resolutions of enantiomers were performed on cyclodex-trin-derived CSPs [109, 144-153], and only on very few occasions were other chiral selectors used [153]. 

Proteins (BSA or ovomucoid, OVM) have also been successful in the preparative resolution of enantiomers by liquid-liquid extraction, either between aqueous and lipophilic phases [181] or in aqueous two-phase systems (ATPS) [123, 180]. The resolution of d,l-kynurenine [180] and ofloxacin and carvediol [123] were performed using a countercurrent extraction process with eight separatory funnels. The significant number of stages needed for these complete resolutions in the mentioned references and others [123, 180, 189], can be overcome with more efficient techniques. Thus, the resolution of d,l-kynurenine performed by Sellergren et al. in 1988 by extraction experiments was improved with CCC technologies 10 years later [128]. 

W. H. Pirkle and B. C. Hamper, The direct preparative resolution of enantiomers by liquid chromatography on chiral stationary phases in Preparative Liquid Chromatography, B. A. Bidling-meyer (Ed.), Journal Chromatography Library Vol. 38, 3 Edition, Elsevier Science Publishers B. V, Amsterdam (1991) Chapter 7. 

The past two decades have seen remarkable advances in chiral chromatography, as only 20 years ago, the direct resolution of enantiomers by chromatography was still considered to be an impressive technical achievement. 

Most methods for the resolution of enantiomers contained in a reaction mixture consist in the conversion of the compounds into stable or transient diastereoisomers and separation of the latter on the basis of their different physico-chemical properties. 

Numerous endogenous substances and commercially available pharmaceuticals are racemic mixtures. Therefore, it is an important problem of clinical chemistry to develop methods for resolution of enantiomers and for establishing enantiomeric purity, because these substances exhibit different biological and physiological... 

The lipases demonstrated very high stability in media partially or totally composed of organic solvent. In such media, the lipases catalyze esterification, transesterification, and resolution of enantiomers [19,45,75,97-100]. Nevertheless, several biphasic systems (organic-aqueous) are used for hydrolysis of lipid and fats [7,34,101]. Kinetic studies in biphase media or in inverted micelles demonstrate that the lipase behavior is different... 

The resolution of enantiomers by liquid chromatography using chiral stationary phases is based on the formation of reversible diastereomeric complexes of different stability between the sample and stationary phase. Since the formation of the complexes is strongly dependent on the structure of the sample, there are no universal chiral stationary phases. The specific advantages of TLC for enantiomeric separations result from its low cost, convenience and speed (10,97,98). The main limitation, particularly with respect to column liquid chromatography, is the small number of phases currently available. 

Miller, L., Orihuela, C., Fronek, R., and Murphy, J., Preparative chromatographic resolution of enantiomers using polar organic solvents with polysaccharide chiral stationary phases,. Chromatogr. A, 865, 211, 1999. 

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