Module No. 20: Methods of Resolution Subject

Subject Chemistry

Paper No and Title 1; Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No and 21: Methods of Resolution Title Module Tag CHE_P1_M21

CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

TABLE OF CONTENTS

1. Learning Outcomes

2. Introduction

3. Methods of Resolution

3.1 Resolution by mechanical Separation of Crystals

3.2 Resolution by formation of Diastereoisomers

3.3 Resolution by Formation of molecular Complexes

3.4 Resolution by Chromatography

3.5 Resolution Through Equilibrium Asymmetric Transformation

3.6 Resolution Through Asymmetric Transformation

3.7 Resolution by Biochemical Transformation

3.8 Resolution Through inclusion Compounds

3.9 Methods Based on NMR Spectroscopy

3.9.1 Use of Diastereomers

3.9.2 Use of Shift Reagents

3.9.3 Use of Chiral Solvating Agents

4. Summary

CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

1. Learning Outcomes

After studying this module, you shall be able to

 Know that pure form of isomers can be obtained from a racemic mixture.

 Understand the process of resolution.

 Learn about the different methods of resolution.

 Analyse the efficacy of various methods of resolution.

2. Introduction

In the previous modules we have discussed in detail various types of isomers, viz., enantiomers, diastereomers, erythro and threo isomers etc. When an enantiomer is converted into a racemic mixture or a racemic modification (usually through chemical reactions), this process is known as racemisation. Conversely, when a racemic modification is separated into its constituent enantiomers, the process is known as resolution. In this module, we shall focus our attention to various methods of resolution and their efficacy.

Preparation of pure stereoisomeric forms or resolution is an indispensable part of many stereochemical investigations. Only after this is achieved, many further steps can be undertaken- the study of the physical properties of stereoisomers and of their chemical reactions. Another route to the preparation of pure stereoisomers is furnished by stereospecific reactions, which involve the formation of the desired stereoisomer free from impurities of the other forms.

3. Methods of resolution

Most of the natural products, foodstuffs, drugs, flavouring agents, perfumes and other biologically active materials usually show their desirable or beneficial effects in one enantiomeric form only, e.g., (+)-morphine is a powerful analgesic, on the other hand its (-)- isomer is not. Thus, resolution is a method of considerable practical importance. CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

Nearly all modern methods of resolution of racemates rely on the classical experiments carried out by Louis Pasteur about a hundred and fifty years ago in 1848. The basic resolution methods are developed in and around the experiments carried out by Pasteur.

1. Mechanical separation, a method also known as spontaneous resolution. It depends on the crystallization of the two forms separately, which are then separated by hand. 2. Chemical resolution based on the formation of diastereomers. 3. Resolution by the biochemical method which makes use of the ability of the microorganisms, their enzymatic systems, to destroy one enantiomer more rapidly than the other. 4. Adsorption methods, in particular chromatographic techniques.

A considerable improvement has been made in this area since then. In this module we shall discuss the various methods of resolution in depth.

Efficiency of Resolution

The efficiency of a resolution method is estimated by optical purity of the pure product obtained and is expressed by p (in per cent).

[] of product p 100 [] of pure enantiomer

Numerically the optical purity corresponds to the excess of one enantiomer over the other in per cent: it does not coincide with the fraction of an optical antipode in the mixture.

The various methods of resolution and its details are discussed at length below:

3.1 Resolution by Mechanical Separation of Crystals

The first resolution ever to be brought about was achieved in this way as stated earlier by Louis Pasteur in 1848. Pasteur prepared the sodium ammonium salt of racemic tartaric acid and allowed it to crystallize in large crystals by slow evaporation of the aqueous solution. He

CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

then picked apart the two kinds of crystals, making use of the fact that they showed dissymmetry in the crystal state.

A more useful variation of mechanical separation is the method of inoculation, originally discovered by Gernez. If a saturated solution of a racemic mixture is carefully inoculated with a pure crystal of one of the enantiomers, the crystal will grow and an appreciable amount of the active isomer shall be separated from the racemic mixture. For example, (+)- sodium ammonium tartarate can be crystallized from a solution of the racemic modification not only by inoculation with a crystal of the (+) salt but also by inoculation with (-)-

asparagine, H2NCOCH2CH(NH2)CO2H Crystallization methods by themselves are rarely practical methods of resolution, but there are often used in a practical methods of resolution, but they are often used in practical way in conjunction with other methods. For example, phenylmethylcarbinyl hydrogen phthalate,

After bring resolved to the extent of about 95% or so by means of brucine is dissolved in carbon disulphide and seeded with a small crystal of the racemic compound. In this particular case, the racemic compound is less soluble than enantiomers and so most of the excess of it left the resolved phthalate crystallizes out. The mother liquor is decanted and problem ether added to it, whereupon the enantiomeric phthalate crystallizes in turn, in other cases, where a racemic mixture is formed or where the racemic compound is more soluble than the enantiomers, the active from may be purifies by crystallization, any residual racemic material remaining in the mother liquor.

3.2 Resolution by formation of diastereoisomers When a racemic modification is allowed to interact with an optically active material to give a derivative (such as a salt), in actual fact two diastereoisomeric derivatives result. For example, in a reaction of a racemic acid (+)-A with an active base, (-)-B, the individual molecules of the acid are either (+) or (-), and therefore, the individual molecules of the salt formed are either (+) or (-), and, therefore, the individual molecules of the salt formed are either (+)-A•(-)-B or (-)-A•(-)-B. These two types of salt molecules are evidently no longer enantiomers, but diasteroisomers. Therefore, they have different properties and may in general be separated on the basis of difference in properties.

CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

Several conditions should be fulfilled by a good resolving agent. First, the compound between the resolving agent and the substance to be resolved should be easily formed and should also be easily broken up, for once one of the diastereoisomers, e.g.,(-)-A•(-)B, is obtained in the pure state, it must be decomposed chemically so that pure (-)-A may be recovered. This condition is generally met by salts, which are usually formed readily by mixing the organic acid base in a solvents and may be decomposed, following resolution, by treatment with mineral acid (if the organic acid is to be recovered) or mineral base (if the organic base is desired).

3.3 Resolution Through the Formation of Molecular Complexes Instead of forming stable salts or covalent compounds with the substrates and the resolving reagents, it is possible, in a few cases to have molecular which form easily and thus are ideally suited for resolution. The first observation was made by Pasteur in the formation of molecular compounds between amides of (-)-malic acid and of tartaric acid. Digitonin (XXI) as shown in the figure below, which is steroidal saponin forms addition complexes with various alcohols, e.g., α-terpineol, isocarvomenthol, and phenolic compounds which can be preferentially crystallized from appropriate solvents and then decomposed to give enantiomerically pure alcohols and phenols.

Figure 1: Some chiral complexing agents

CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

Compound Resolution: complex formation (+)-2-Naphthylcamphylamine (XXII) Used to resolve N-s-butylpicramide Α-(2,4,5,7-tetranitro-9- Forms charge transfer complexes (π-complexes) fluorenylideneaminooxy)propionic acid with many aromatic hydrocarbons (TAPA) (XXIII) Pt (IV) reagent containing R-1- Through complexation phenylethylamino moiety (XXIV) Chiral complexing agent (XXV) Through enantioselective portioning between an aqueous phase and a solvent containing a chiral complexing agent (like chiral crown ether)

Table 1: Complex formation by some resolution agents

3.4 Resolution by chromatography The chromatographic method of resolution of racemic mixtures is carried out generally under four different conditions: (i) Formation of diastereomeric mixture by derivatisation with optically active reagents and separation by classical chiral using achiral adsorbents based on the different adsorption coefficients of the diastereomer; (ii) Direct resolution of the racemic mixture using chiral of the adsorbent materials either as solid or as liquid stationary phase; (iii) Direct resolution on an achiral solid phase using a mobile chiral liquid phase; and finally, (iv) Direct resolution using an achiral solid stationary phase modified by a chiral reagents. (First method is an alternative to separation by crystallisation of diastereomeric salts. The method is of general use and may illustrated the resolution of 2-butanol by forming esters with (-) mandelic acid followed by chromatography on Dowex-50W-X2 to give both the diastereomeric esters.

The second method, namely, use of chiral stationary solid phase is more interesting. The naturally occurring polysaccharides and their derivatives provide useful chiral stationary phase materials. One, namely, microcrystalline triacetlyl cellulose (MCTC) forms a versatile

CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

column on which a numbers of compounds, e.g., racemic olefins, biphenyls, ketones, alcohols, esters, acids, and salts have been resolved (Hesse and Hagel 1976). The problem is its relative unavailability, An alternative method of preparation of a chiral stationary phase is the adsorption of chemical binding of a resolving agents to an achiral stationary phase, thus (-)-TAPA (XXIII) adsorbed or covalently including helicenes. Chiral crown ethers bounded to silica or organic polymers have been used by cram and cram (1958). In the third variation, many cationic chelated metal complexes, e.g., tris(diamino)metal complex have been solved of chromatography on a column of cation-exchange sephadex (dextran cross-linked with epichlorohydrin) by elution with an aqueous solution of (+)- tartrate. In the fourth variation, an ordinary adsorbent like silica gel is made stereoselective by special treatment with optically active substances, e.g., (+)-tartaric acid and (+) camphorsulphonic acids which are later removed by elution with solvent leaving behind a ‘memory’. Gas chromatography has also been extensively used for analysis as well as separation of enantiomers working on the above principles. The trifluoroacetyl derivatives of optically active amino acids have been used for gas chromatographic resolution of racemic alcohols via the corresponding esters. Paper chromatography can also effect partial resolution; in the paper itself (cellulose material) can act as chiral adsorbents or it may be impregnated with solution of optically active compound such as camporsulphonic acids.

3.5 Resolution Through Equilibrium Asymmetric Transformation: The resolution technique based on the principle of equilibrium asymmetric transformation involves two steps: epimerisation of a diastereomeric species, be it covalent compound, a salt or a complex, in solution and precipitation of the predominant epimer. If the asymmetric centre undergoing the configurational change can be separated from the rest of the diastereomeric species, only one enantiomer is obtained. In ordinary resolution, the yield of the enantiomer can never exceed 50% but in asymmetric transformation, the racemic form can, in principle be completely converted into one of the enantiomers. When the racemic form of 2-(p-caroxybenzyl)-1-hydrinanone (Fig. 2a, XXVI) is treated with brucine in acetone solution, one of the diastereomers precipitates in over 90% yield. On acidification, the (+)-

CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

enantiomer of the ketone (Fig. 2b, XXVII) is obtained which, however, racemises spontaneously.

Fig. 2: Equilibrium asymmetric transformation

3.6 Resolution Through Kinetic Asymmetric Transformation The kinetic asymmetric transformation is based on the principle that one of the diastereomer is formed, destroyed or transformed selectively by a chemical reaction. This is possible because the activation energy of the diastereomeric transition states in the reaction of the racemate with a chiral reagent are different and so the two enantiomers react at different rates.

3.7 Resolution by Biochemical Transformation In the kinetic method of resolution as discussed above, the chiral reagents may be replaced by microorganisms or enzymes which are often highly stereoselective in their reactions. Pasteur first observed that when a solution of the ammonium salt of the (±)-tartaric acid is fermented by yeast or a mould (Pencillium glaucum), the natural (+)-form is completely consumed leaving behind the ammonium salt of the (-)-tartaric acid. The biochemical method has found important application in the resolution of (±)-amino acids. The shortcomings of the biochemical transformation are as follows: i. Very dilute, usually aqueous solutions have to be used for fermentation, thus the product may be hard to isolate; ii. The enantiomer which is biologically more important is often selectively consumed; iii. It is very difficult to find an appropriate microorganism for a particular substrate.

3.8 Resolution Through Inclusion Compounds Some compounds crystallise in such a way that a hole is formed inside the crystal which can accommodate another guest molecule without forming any chemical bond. These complexes CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

are known as clathrate or inclusion compounds. The inclusion of the guest molecule depends on the steric fit inside the crystal lattice and is often very selective. Desoxycholic acid, a steroidal compound forms such crystals in which a particular enantiomer of selected molecules can be included. It can used for the resolution of camphor.

3.9 Methods Based on NMR Spectroscopy NMR spectroscopy is extensively used for the determination of enantiomeric purity.

3.9.1 Use of Diastereomers Since any two corresponding ligands (or nuclei) in two enantiomers are enantiotopic by external comparison, they are isochronous and cannot be distinguished by NMR working under achiral condition. But if the enantiomers can be first derivatized with an optically pure reagent, two such ligands will be diastereotopic by external comparison and will have different chemical shifts.

3.9.2 Use of Shift Reagents Chiral shift reagents as shown in figure 3 are also extensively used to determine the enantiomeric excess by NMR. They form diastereomeric complexes with chiral substrates having a variety of functional groups and at the same time, induce increased anisochrony in two diastereotopic groups of protons.

Figure 3: Chiral shift reagents and chiral solvating agents

3.9.3 Use of Chiral Solvating Agents: In yet another variation of NMR method, a ‘chiral solvating agent’ (CSA) is used which forms diastereomeric solvates with the substrate through weak solvent-solute interactions. CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution

The CSA may be either a solvent, or a cosolvent or even a solid auxiliary. Two most common CSA’s are 2,2,2-trifluoro-1-phenylethanol and the corresponding anthracyl derivative (Figure 4).

Figure 4: Diastereomeric associates with CSA through H-bonding

4. Summary

In this module, we have taught you that:  Preparation of pure stereoisomeric forms or resolution is an indispensable part of many stereochemical investigations. Only after this is achieved, many further steps can be undertaken- the study of the physical properties of stereoisomers and of their chemical reactions.  Nearly all modern methods of resolution of racemates rely on the classical experiments carried out by Louis Pasteur about a hundred and fifty years ago in 1848.  The basic resolution methods are developed in and around the experiments carried out by Pasteur.

 The efficiency of a resolution method is estimated by optical purity of the pure product obtained and is expressed by p (in per cent). CHEMISTRY Paper No. 1: Organic Chemistry-I (Nature of bonding and Stereochemistry) Module No. 20: Methods of Resolution