Optical purity determination enantiomers

Methods for determining the optical purity (= minority enantiomer / L of the two enantiomers) have considerably evolved over the years. 

A NMR study on the formation of diastereoisomeric inclusion complexes between fluorinated amino acid derivatives and a-CD in 10% D2O solution shows that the chemical shifts of the D-amino acid derivatives included by a-CD are upheld from those of their L analogues [77]. The shift difference between the diastereoisomers formed with D and L enantiomers can be used for chiral analysis and optical purity determinations. For example, the interaction of -CD with propanolol hydrochloride produces diastereomeric pairs. The protons of the antipode give NMR signals which differ in chemical shifts in D2O solution at 400 MHz. The intensity of the resonance signals for each diastereoisomer has been used for optical purity determination. By adding racemate to pure (—) isomer, this technique is able to measure optical purity of propanolol hydrochloride in water down to the level of 1%. 

The ee is determined by liberution of (3S)-3-hydroxy-5-nicthylhcxanoic acid by acidic hydrolysis, which is then derivalized by heating with isopropylisocyanatc and analyzed 011 a chiral capillary GC column (Chirasil-L-Val, 50 m, 190 C, carrier 90 kPa) (,V)-cnantiomcr / 9.35 min 97% (fJ)-enantiomer 9.58 min 3% 94% ee. Material of 99% optical purity is obtained by rccrystallization. 

In 1960, Montanari and Balenovic and their coworkers described independently the first asymmetric oxidation of sulfides with optically active peracids. However, the sulphoxides were formed in this asymmetric reaction (equation 130) with low optical purities, generally not higher than 10%. The extensive studies of Montanari and his group on peracid oxidation indicated that the chirality of the predominantly formed sulphoxide enantiomer depends on the absolute configuration of the peracid used. According to Montanari the stereoselectivity of the sulphide oxidation is determined by the balance between one transition state (a) and a more hindered transition state (b) in which the groups and at sulphur face the moderately and least hindered regions of the peracid,... 

Our approach for chiral resolution is quite systematic. Instead of randomly screening different chiral acids with racemic 7, optically pure N-pMB 19 was prepared from 2, provided to us from Medicinal Chemistry. With 19, several salts with both enantiomers of chiral acids were prepared for evaluation of their crystallinity and solubility in various solvent systems. This is a more systematic way to discover an efficient classical resolution. First, a (+)-camphorsulfonic acid salt of 19 crystallized from EtOAc. One month later, a diastereomeric (-)-camphorsulfonic acid salt of 19 also crystallized. After several investigations on the two diastereomeric crystalline salts, it was determined that racemic 7 could be resolved nicely with (+)-camphorsulfonic acid from n-BuOAc kinetically. In practice, by heating racemic 7 with 1.3equiv (+)-camphorsulfonic acid in n-BuOAc under reflux for 30 min then slowly cooling to room temperature, a cmde diastereomeric mixture of the salt (59% ee) was obtained as a first crop. The first crop was recrystallized from n-BuOAc providing 95% ee salt 20 in 43% isolated yield. (The optical purity was further improved to -100% ee by additional recrystallization from n-BuOAc and the overall crystallization yield was 41%). This chiral resolution method was more efficient and economical than the original bis-camphanyl amide method. 

Specification in Table 9. b Yield of a mixture of 75 and 76 which was separated from the crude reaction product by a silica gel chromatography. All [a]D values were measured in CHC13 at c 1.0.d Optical purity was determined by HPLC on Chiralcel.e Since optically active 75 a and 76 a could not be separated, it is not clear whether both enantiomers are (—)-ones or not. Therefore, both are tentatively shown as (—)-enantiomers. Since [a]D value of each enantiomer is also not clear, [otJD value of the mixture is shown.f Compound is inert to irradiation. Optical purity was not determined. h When an acetone solution of the mixture of (+)— 75c and 76c was kept, racemic 76c crystallized out, mp 135-137 °C. 

BA trans-3.4-dihvdrodiol cannot be separated from BA trans-8.9-dihydrodiol in several HPLC conditions (27-29). Quantification of BA trana-3,4-dihydrodiol by HPLC can only be accomplished after converting the 3,4-dihydrodiol to its diacetate (25.26). The BA trans-3.4-dihydrodiol formed in BA metabolism by liver microsomes from pheno-barbital-treated rats was determined to have a 3R,4R/3S,4S enantiomer ratio of 69 31 (30). Recently we have determined the optical purity of the BA trans-3.4-dihvdrodiol formed in the metabolism of BA by three liver microsomes prepared from untreated rats and rats that had been pretreated with an enzyme inducer. As shown in Table II, cytochrome P-450 isozymes contained in liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats had similar stereoselectivity toward the 3,4-double bond of BA. BA trans-3.4-dihydrodiol is formed via the 3,4-epoxide intermediate (31). 

One of the terms for describing enantiomer composition is optical purity. It refers to the ratio of observed specific rotation to the maximum or absolute specific rotation of a pure enantiomer sample. For any compound for which the optical rotation of its pure enantiomer is known, the ee value may be determined directly from the observed optical rotation. 

Recent advances in gas chromatographic separations of enantiomers allow precise determination of the enantiomeric purity of the algal pheromones. The czs-disubstituted cyclopentenes, such as multifidene, viridiene, and caudoxirene, are of high optical purity [ 95% enantiomeric excess (e.e.)] whenever they have been found (32,33). The situation is different with the cyclopropanes and the cycloheptadienes, as shown in Table 2 and Figure 1. Hormosirene from female gametes or thalli of... 

For a nonracemic mixture of enantiomers prepared by resolution or asymmetric synthesis, the composition of the mixture was given earlier as percent optical purity (equation 1), an operational term, which is determined by dividing the observed specific rotation (Mobs) of a particular sample of enantiomer with that of the pure enantiomer ( max), both of which were measured under identical conditions. Since at the present, the amount of enantiomers in a mixture is often measured by nonpolarimetric methods, use of the term percent optical purity is obsolete, and in general has been replaced by the term percent enantiomeric excess (ee) (equation 2) introduced in 197163, usually equal to the percent optical purity, [/ ] and [5] representing the relative amounts of the respective enantiomers in the sample. 

NMR spectroscopy was found to be a valuable technique for differentiation between the enantiomers of optically active compounds. The principles of the methods used to distinguish between enantiomers by means of NMR have been developed and reviewed by Mis-low and Raban (217). The best results from the point of view of the determination of optical purity and absolute configuration of chiral sulfur compounds, especially of sulfinyl compounds, have been obtained with the help of chiral solvents (218). Pirkle (86) was the first to demonstrate that enantiomeric sulfoxides have nonidentical NMR spectra when dissolved in chiral alcohols having the following general formula ... 

As in the case of other chiral compounds, the optical and enantiomeric purity of chiral organosulfur compounds can be determined by various methods (241). The simplest and most common method for the determination of optical purity of a mixture of enantiomers is based on polarimetric measurements. However, this method requires a knowledge of the specific rotation of the pure enantiomer. In the... 

An interesting method for the estimation of optical purity of sulfoxides, which consists of the combination of chemical methods with NMR spectroscopy, was elaborated by Mislow and Raban (241). The optical purity is usually determined by the conversion of a mixture of enantiomers into a mixture of diastereomers, the ratio of which may be easily determined by NMR spectroscopy. In contrast to this, Mislow and Raban used as starting material for the synthesis of enantiomeric sulfoxides a diastereomeric mixture of pinacolyl p-toluenesulfinates 210. The ratio of the starting sulfinates 210 was 60.5 39.5, as evidenced by the H NMR spectrum. Since the Grignard reaction occurs with full stereospecificity, the ratio of enantiomers of the sulfoxide formed is expected to be almost identical to that of 210. This corresponds to a calculated optical purity of the sulfoxide of 20%. In this way the specific rotations of other alkyl or aryl p-tolyl sulfoxides can conveniently be determined. 

As was already mentioned, the phenomenon of nonequivalence of NMR spectra of enantiomers in chiral solvents is a basis for the determination of enantiomeric purity of a variety of chiral sulfur compounds. This method, developed by Pirkle, has the advantage over other methods of being absolute that is, the chemical shift difference between enantiotopic nuclei induced by the chiral solvent increases with increasing optical purity of the solvent, whereas the relative intensities of the signals that are used to measure the enantiomeric composition of the solute are not affected. 

An extremely important aspect in pharmaceutical research is the determination of drug optical purity. The most frequently applied technique for chiral separations in CZE remains the so-called dynamic mode where resolution of enantiomers is carried out by adding a chiral selector directly into the BGE for in situ formation of diastereomeric derivatives. Various additives, such as cyclodextrins (CD), chiral crown ethers, proteins, antibiotics, bile salts, chiral micelles, and ergot alkaloids, are reported as chiral selectors in the literature, but CDs are by far the selectors most widely used in chiral CE. 

A new and detailed tetralin study deals with the resolution and the determination of the optical purity of the enantiomers of 5-OH-DPAT (21). Of particular interest is that an analytical HPLC method that measures very small amounts of optical impurity was used [72]. It could be shown that the i -enantiomer really possesses antagonistic properties, as measured biochemically, in non-pretreated animals [73]. These findings support the modelling results of Froimowitz and co-workers [74,75]. Other studies have not revealed such properties of (/ -21) [64,76]. These new and interesting findings have implications for the atypical D2 antagonsits with preferential action on D2 autoreceptors, developed from D2 agonists, as discussed below. 

A racemic mixture contains equal amounts of the two enantiomeric forms of the compound and has an optical rotation of zero the optical rotations arising from each of the two types of molecule are cancelled out. It follows that a mixture of enantiomers in unequal proportions will have a rotation that is numerically less than that of an enantiomer. Here, we see how to use the measured optical activity to determine the proportions of each enantiomer in the mixture, and therefore its optical purity. Optical purity is a measure of the excess of one enantiomer over the other in a sample of a compound. 

NIRA provides a non-destructive alternative to differential scanning calorimetry for the determination of polymorphic forms of drugs, e.g. the polymorphic forms of caffeine. NIRA has also been used to determine optical purity. While the pure opposite enantiomers of a substance have identical NIR spectra, mixing two... 

Enantiomeric Purity (ep) - General measure of the composition of a mixture of enantiomers, to be applied except when the determination is based on optical rotation measurements see Optical Purity. 

Optical Purity (op) - This measure of the composition of a mixture of enantiomers must not be applied, unless the composition was determined via measurement of optical rotation (see Section 1.2.2.2.) see Enantiomeric Purity. 

A requirement for determination of optical purity (P) is that the specific rotation of the pure enantiomer, [a]max, is known with certainty. This maximum rotation can be established by calculation, e.g., via competitive reaction methods41,42, or by direct determination employing an enantiomerically pure sample. Enantiomerically pure samples are generally believed to arise from enzymatic reactions performed on biogenic substrates, an assumption which in some instances is incorrect31, or are obtained in most cases by crystallization2. As many direct, nonchiroptical methods are available for determining enantiomeric purities, maximum rotations can also be extrapolated from the specific rotation of a sample of known ee. 

The precision of enantiomeric purity determinations by gas chromatography is high123 124-1 >s. This statement holds not only for small enantiomeric purities ( 0% ee), e.g., in the differentiation of a true racemate from enantiomerically slightly enriched mixtures (in reactions devoted to the amplification of optical activity under prebiotic conditions), but also for very high enantiomeric purities (— 100% ee), with detection of 1.0 to 0.1% (and less) of enantiomeric impurities (see Section 3.1.5.8). It is always advantageous if the enantiomer present as an impurity is eluted as the first peak from the gas chromatographic column (Section 3.1.5.3.). This is achieved by the proper selection of the chirality of the nonracemic stationary phase147-188 which, unfortunately, is not possible for the cyclodextrin phases. 

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