In the Classroom
Differentiations of Enantiomers via Their Diastereomeric Association Complexes—There Are Two Ways of Shaking Hands Albrecht Mannschreck* and Roland Kiesswetter Department of Organic Chemistry, University of Regensburg, D-93 040 Regensburg, Germany; *
[email protected] The intermolecular interaction of one enantiomer with a chiral auxiliary molecule may differ from the interaction of the other enantiomer with the same auxiliary molecule (1). This observation has been used since Louis Pasteur’s time in several ways, for example the separation and analysis of enantiomers, the study of their influence on biosystems, and their resolution by kinetic means.
Figure 1. Unequal handshakes, related to unequal associations of enantiomers with chiral auxiliary molecules. Top: Right hand with black sleeve (one enantiomer) and right hand with gray sleeve (a chiral auxiliary molecule) perform a usual handshake (an association). Bottom: Left hand with black sleeve (the other enantiomer) and right hand with gray sleeve (an auxiliary molecule with the same sense of chirality as before) perform a rarely experienced, unequal handshake (an unequal association).
Table 1. Situations Where Diastereomeric Association Complexes Are Important Situation
Diastereomeric Complexes
(R )
+
aux
(R ) ..... aux
Preparative separation of enantiomers via crystallization
Substrate–auxiliary salts
(S )
+
aux
(S ) ..... aux
Analysis of enantiomers by NMR spectroscopy
Substrate–additive complexes
Separation of enantiomers by chromatography
Substrate–sorbent complexes
Biological activities of enantiomers
Substrate–receptor complexes
Reactivities of enantiomers in enzymatic resolution
Reactants–enzyme complexes
Enantiomers of the substrate
Chiral auxiliary molecule
Diastereomeric association complexes
Scheme I. Simplified scheme for the associations of enantiomers with optically pure chiral auxiliary molecules.
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These topics relating to enantiomers can be found in organic chemistry textbooks and are included in the university curriculum for chemistry students. In textbooks and in teaching, the aspects to be treated in the present article are not commonly discussed together, in connection with a concept of interaction-based differentiation of enantiomers. This aim might be achieved by a textbook writer or a teacher by referring other aspects of enantiomers that are presented earlier or later in the curriculum. Such an approach provides an opportunity to reiterate and integrate the concept. However, in our opinion a single lecture, possibly resembling this article, is more effective to presenting an integrated view of enantiomers. To benefit most from the lecture, a student should have been exposed to the following topics: basic stereochemistry, in particular the notions of chiral molecules, enantiomers, diastereomers, and racemic and chiral nonracemic samples (2a); the NMR chemical shift; the principle of chromatography including the retention time; the basis of protein structure; and elements of chemical kinetics, particularly the notions of ground and transition states, catalysis, and free energy. When presenting this lecture we started by shaking hands with one of the students, then unexpectedly taking her or his other hand for the same purpose (Figure 1). After this experiment, it was not difficult to introduce chiral nonracemic auxiliaries of all types. We used this presentation at three universities in the second or third year of chemistry and our teaching experience was positive. Two enantiomers are represented (2b), in Scheme I and later in this article, by the abbreviations (R ) and (S ). Similarly, the abbreviation “aux” represents the auxiliary molecules. The interactions between the latter and an enantiomer are symbolized by a dotted line. This line represents the sum of the intermolecular forces between the substrate and auxiliary molecules. It has been shown that a single intermolecular
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force, for example, one hydrogen bond, is not sufficient, but at least three intermolecular forces are required for a differentiation of enantiomers (1, 3). The two combinations (R )....aux and (S )....aux are called diastereomeric association complexes. The physical and chemical properties of these complexes may be unequal. There are several occasions in which the diastereomeric association complexes are important in a practical sense. In the present article, five such occasions (Table 1) will be briefly characterized and a single example of each will be described. Emphasis will be placed upon their common basis—the differentiation of enantiomers via their diastereomeric complexes. Preparative Separation of Enantiomers via Crystallization of Diastereomeric Salts Enantiomers are related to one another like an object and its mirror image. Therefore, they can neither be easily distinguished nor easily separated. Louis Pasteur achieved separation in 1853 by adding optically pure auxiliary molecules to these enantiomers. To demonstrate this type of separation, we choose (+)-10-camphorsulfonic acid, (+)-HO3SR (Scheme II and Figure 2), as the auxiliary compound and (RS )-2-amino-2-phenylacetic acid as the substrate. Cation– anion attraction (after reprotonation) and cation–anion hydrogen bonding provide interactions to form the diastereomeric ion pairs (R )....aux and (S )....aux or corresponding aggregates. Contrary to the free (R ) and (S ) molecules, those two pairs or aggregates are not related to one another like an object and its mirror image, but the enantiomeric relationship between (R ) and (S ) has been transformed into a diastereomeric relationship (Scheme I). Experience shows that physical and chemical properties of such species differ, including their solubilities. Therefore, one of the diastereomeric substrate–auxiliary salts (Scheme II) preferentially crystallizes, whereas the other one preferentially remains in solution. After removing the auxiliary, enantiomerically enriched free (R )- and (S )-amino acids are isolated and may be obtained pure after repetition of crystallization (4a, 5). Noncovalently-bound auxiliaries can be added and removed under mild conditions, which is an advantage (6) over covalently-bound diastereomeric derivatives such as carboxylic amides and esters. The molecular structures (7) of the two salts (Figure 2) show conformational differences in the cations and also in the methylenesulfonate segments of the anions. These differences are connected with unequal networks of hydrogen bonds in the lattices of the two salts, which, in turn, are related to the lower solubility of (R)....aux in water (7). (R)-2-Amino-2-phenylacetic acid does not occur in nature but is required for the synthesis of important antibiotics. The separation described is therefore carried out on a scale of more than 1000 t per year (4a, 8a). For this purpose, the resulting less useful (S ) product is racemized and recycled in the crystallization process. The separation of enantiomers via crystallization of ionic or nonionic diastereomeric complexes with a variety of auxiliaries is used for the small- and largescale preparation of many chemicals and pharmaceuticals (2c). Such separations work best if the auxiliary is enantiomerically pure; on the other hand, they can only work if the latter is nonracemic (2c). www.JCE.DivCHED.org
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Analysis of Enantiomers by NMR Spectroscopy In the present respect, analysis primarily consists of the quantitative determination of the relative quantities of enantiomers in their mixtures, a task occurring frequently, for example, after enantioselective syntheses, upon isolations of natural products, and after preparative separations like the one in Scheme II. As it would be difficult to use such a crystallization procedure for analysis, other methods, for example, NMR spectroscopy, are used for this purpose. The NMR spectra of enantiomers are identical. However, the spectrum of a racemate may show two separate superimposed spectra for its constituents if an enantiomerically pure (or highly enriched) auxiliary is added to the solution (2d).
Ph
CH NH2 CO2H
(RS ) precipitate
ⴙ
Ph
C
NH3 ⴚ
ⴙ HO3SR aux
filtrate
O3SR
H CO2H
(R )
ⴙ
Ph
C
NH3 ⴚ
O3SR
HO2C H
aux
(S )
aux
work up Ph
work up
NH2
C
Ph
⫹
HO3SR
HO3SR
H CO2 H
NH2
C
⫹
HO2C H
(R )
aux
aux
(S )
Scheme II. Separation of the enantiomers of 2-amino-2-phenylacetic acid via preferential crystallization of one of its diastereomeric salts with the auxiliary (+)-10-camphorsulfonic acid, (+)-HO3SR, in H2O.
Figure 2. Diastereomeric salts prepared from (RS)-2amino-2-phenylacetic acid and (+)-10-camphorsulfonic acid.
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As an additive, we choose (+)-1-(9-anthryl)-2,2,2trifluoroethanol (Figure 3). The substrate consists of a racemic chromene (9). Hydrogen bonding between the acidic OH group and an acetal oxygen atom as well as π–π association (10) provide intermolecular interactions for the formation of the diastereomeric substrate–additive complexes (R)....aux and (S )....aux that may differ with respect to their chemical shifts. Instead of the single peak for the CH3 protons of the (RS )chromene before the addition of the auxiliary, two signals with equal areas are seen after the addition (Figure 3, right). Another sample of the chromene shows two peaks with areas of 90:10, which are the relative quantities of the enantiomers (Brandl, F., Mannschreck, A., unpublished results). Some insight into the observations in the NMR is obtained by examining the dynamics of the processes going on. The two equilibria in Scheme I are brought about by formations and cleavages of noncovalent bonds, all of which are fast on the so-called NMR time scale (2e). This means that neither the CH3 protons of the free (R) enantiomers nor the CH3 protons of (R)....aux are observed separately, but one averaged CH3 peak is seen for the equilibrium of the (R) molecule (2d). The same is true for the (S ) molecule. Therefore, the two observed CH3 signals stem from the two complexes and the two noncomplexed enantiomers. This description of the dynamics means that the interactions should not be too strong and the associations should be reversible. Otherwise no averaging would occur and the two
spectra of the complexes might be obscured by the spectrum of the free substrate. The NMR analysis of enantiomers continues to be successful for many cases in which substrate–additive complexes are favorable (11, 12). No calibration by a sample with known enantiomeric composition is required. Separation of Enantiomers by Chromatography The time required for the elution of a substrate from a column is called the retention time tR. The time needed to elute a standard sample that does not interact with the sorbent (stationary phase) is t0. Therefore, (tR − t0), the net retention time, represents a measure for the interaction between the compound and the sorbent (13a). The net retention times of enantiomers are identical in chromatography on a stationary phase containing no chiral molecules, for example, on silica gel. The (tR − t0) values of enantiomers may be, however, unequal if the latter interact with a chiral nonracemic sorbent. As an example, the enantiomers of carvone are separated using microcrystalline tribenzoylcellulose (Figure 4), which means that they are temporarily included (or partially included) in the holes of a cellulose derivative. For the racemate, two peaks with equal areas are found (Figure 4). Another sample shows one peak for (S )-carvone in the chromatogram (14); that is, it is enantiomerically pure.
Me
(R )
(S )
A
O
H H3CO
CH2
H Me
17.6
3.50 3.48
O
δ
(R )
(R ) HO C
20.9
t R / min
O CH2OCPh O
CF3 H
O
PhCO PhCO
O
O
n
aux
aux
(S ) A
Me O
H H3CO
O
3.51 3.49
(S )
δ
Figure 3. Structures of the chromene enantiomers and the auxiliary compound, (+)-1-(9-anthryl)-2,2,2-trifluoroethanol. NMR spectra (400 MHz) of the methyl protons of the enantiomers of chromene in CDCl3 with 5.0 equiv of the auxiliary compound. The peaks cannot yet be assigned to the enantiomers. Top NMR: An (RS)-chromene (3.0 mg/mL). Bottom NMR: Another sample of the chromene (2.1 mg/mL) containing 90% of the enantiomer with the higher δ value and 10% of the other enantiomer.
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H Me
CH2
20.9
t R / min
(S ) Figure 4. Structures of the carvone enantiomers and the chiral nonracemic sorbent, tribenzoylcellulose, as an auxiliary. Chromatograms of carvone enantiomers with MeOH as a mobile phase (column: 250-mm long, 8-mm i.d., 60 bar pressure, 1.0 mL/min flow rate). Absorbance measured by a UV detector at 330 nm. Right, Top: ( RS )-carvone (0.40 mg). Bottom: Another sample (0.20 mg) of carvone consists of the pure (S ) enantiomer.
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In analogy to NMR, the interactions should not be too strong, so that the transient substrate–sorbent complexes (Table 1) (2f, 13b) are subsequently cleaved upon elution; that is, the desorbed substrate molecules leave the column for detection. Many different chiral nonracemic stationary phases (2f, 13b) have been commercialized and it appears that the majority of enantiomers can now be separated. Again, no calibration sample is necessary for the determination of enantiomeric composition. Essentially, two types of this method are useful in the present context: (i) gas chromatography (13) in which a gas is flowing through a capillary containing the dissolved sorbent and (ii) liquid chromatography (9, 13) in which a liquid flows through a column containing the solid sorbent. For preparative work, this type of method can be applied on a kilogram scale (15), such as, for the production of enantiomerically pure pharmaceuticals, if the alternative approaches fail. In the case of a sufficiently large peak separation of the enantiomers upon their direct chromatography, this procedure should be preferred (6) to the use of diastereomeric derivatives that requires the formation and cleavage of covalent bonds before and after the chromatographic run.
HO
HO
HO
HO
C H
C
OH MeNH CH2
CH2NHMe
(R )
OH
H
(S )
Figure 5. The enantiomers of adrenaline.
(R ) + receptor
(R ) ..... receptor
(S ) + receptor
(S ) ..... receptor
activity of (R )-enantiomer activity of
(S )-enantiomer
Scheme III. Simplified scheme of biological activities of enantiomers. Single full-line arrow indicates the formation of one of the diastereomeric complexes, for example, the (R )....receptor species, which generates a biological effect. The dotted-line arrow indicates the behavior of the other enantiomer, which differs widely.
+ achiral reagent
For the biological activity of substrates, the so-called physiological receptors are essential. These are primarily proteins embedded in a membrane. They are chiral and can bind, for instance, the molecules of a drug by noncovalent interactions to form drug–receptor complexes, thereby eventually producing some biological effect in the cell, indicating that the drug is active (4b, 16). The enantiomers of adrenaline (epinephrine) were chosen as substrates (Figure 5). As a hormone and a drug, (R)adrenaline causes an increase in blood pressure within the human body. It has been shown that this increase is generated by the formation of a (R)....receptor complex (Scheme III) with the α-adrenergic receptor. A favorable hydrogen bond of the adrenaline aliphatic hydroxy group inside this complex has been suggested. On the other hand, the (S) enantiomer as a drug gives rise to a weaker increase in blood pressure. A corresponding (S )....receptor species is conceivable but it lacks a favorable inside hydrogen bond (3, 17). For examples like adrenaline, the use of one of the enantiomers as a drug is preferred to the application of the racemate. Widely differing situations were encountered for many other pharmaceutical products (2g, 18). These include the case of activities of the same type and the case of qualitatively different effects of the enantiomers, which means that one effect may be useful, the other one harmful to the body. Therefore, the decision, whether a particular drug should be applied in one enantiomerically pure form or as a racemate, must be made on the basis of detailed stereochemical and pharmacological studies. In such studies, the possibility of interconversion of the enantiomers under physiological conditions, for example, by removal and addition of a labile proton, must be taken into account (19). As a whole, the number of therapeutic applications in the form of one of the enantiomers is growing. In addition to drugs, a variety of further substrates, consisting of chiral molecules, may exert influences on a biosystem via binding to a receptor (Scheme III). Such substrates are exemplified by compounds for plant protection (herbicides, fungicides, insecticides), pheromones, taste substances, and odorants. Again, unequal activities of the enantiomers are proven in many cases (2g, 8b). (R )- and (S )-carvones (Figure 4) with odors of spearmint and caraway, respectively, represent an example (20–22). These different flavors are presumably brought about by the odor receptors in the nose. A few synthetic materials for plant protection are even applied in agriculture in the form of one of the enantiomers (23). Reactivities of Enantiomers in Enzymatic Resolution
(+)-catalyst
(R)-substrate
Biological Activities of Enantiomers
(R)-product slow (+)-catalyst
(S)-substrate + achiral reagent
(S)-product fast
Scheme IV. Reactivities of enantiomers in kinetic resolution in the presence of a chiral nonracemic catalyst. If the (R ) substrate reacts more slowly than the (S ) substrate, the compounds in the boxes prevail after some time.
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Before discussing the use of enzymes, the application of chiral nonracemic catalysts in general (Scheme IV) will be mentioned (2h). (R ) and (S ) substrates show the same reactivities towards an achiral reagent, which means that the same quantities of the (R) and (S ) products are formed. This must not be true if this reaction is carried out in the presence of a chiral nonracemic catalyst. The two rates of reaction now differ, which means that, after a certain period of time, the quantities of the (R ) and the (S ) products formed are unequal. As
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Me2CH H C O HO2C
+ H 2O
NH CMe
acylase
(R )
slow
Me2CH
H C
HO 2C
HO2C
H C
O
+
HO CMe
NH 2
(R ) O
+ H 2O
Me2CH NH CMe
(S )
acylase
HO2C
fast
H C
O
+
Me2CH NH2
HO CMe
(S ) Scheme V. Hydrolysis of (RS)-N-acetylvaline, catalyzed by an acylase. The compounds in the boxes prevail after some time. Conditions are 10 h at 37 °C in H2O at pH 7. O
O
R⬘NH CR + H2O + E
R⬘NH2 + HO CR + E
amide
amine
(R)-amide
acid
‡
(R)-amide
(R)-amine + acid + E
E
+ H2O + E
H2O
‡
(S)-amide
(S)-amide + H2O + E
(S)-amine
E
+ acid + E
H2O ‡
(R)-amide E H2O
‡
(S)-amide E
G
G
H 2O
(R)-amide + H2O + E
(R)-amine + acid + E
Reaction Coordinate
Conclusion (S)-amine
(S)-amide + H2O + E
+ acid + E
Reaction Coordinate
Scheme VI. Simplified scheme for enzymatic resolutions via hydrolysis of a carboxylic (RS)-amide forming mainly (S)-amine (in the box) and carboxylic acid. R´: A chiral substituent; compare, for instance, Scheme V. E: Enzyme. [(R)-amide ···H2O··· E]‡ and [(S)-amide ···H2O··· E]‡: Conceivable diastereomeric transition states of the hydrolyses, comprising reactants-enzyme complexes; diastereomeric ground-state complexes are also involved, but not shown.
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a consequence, the quantities of the remaining (R ) and (S ) substrates are unequal, too. These results are brought about by a kinetic resolution of a racemate via catalysis. The method is called an enzymatic resolution if the catalyst is an enzyme. As an example, we consider the hydrolysis of synthetic (RS)-N-acetylvaline (Scheme V) (24, 25). In the presence of an acylase, the rates of the two reactions differ considerably. After a suitable time, the (R) substrate and the (S ) product prevail. Their polarities and solubilities are unequal, which means that these materials can be separated easily. Enantiomerically pure (S )-valine is useful for several medicinal purposes; the less useful (R ) substrate is therefore racemized and recycled. The present type of hydrolysis has developed, beyond the production of (S )-valine, to a general procedure for obtaining the enantiomers of amino acids (24, 26a), some of which are required as food supplements for cattle. Such processes play a role for the preparation of many nonracemic organic compounds (2h, 27, 28), several examples even being adapted for use in the teaching laboratory (29– 33). The course of an enzymatic hydrolysis can be visualized by the free energy of the reacting system in a simplified way (Scheme VI) (1, 26b, 34). Carboxylic amide molecules form ground-state amide–enzyme complexes (2b, 35, 36) in a way similar to the substrate–auxiliary species (Scheme I) discussed in the earlier sections of this article. These ground-state complexes are not shown; essentially, the experimental facts are brought about kinetically and, therefore, the transition-state complexes (Scheme VI) are more important in the present context. Such species probably contain the amide, water, and the enzyme (36); they are certainly formed in more than one step. It remains open whether, besides noncovalent interactions (36), covalent bonding is involved in some cases. When starting from the (R )-amide or the (S )-amide, we have to pass diastereomeric transition states with unequal free energies. The corresponding observations are slow reaction (or none at all) of the (R )-amide and fast reaction of the (S )amide to form the (S )-amine and the carboxylic acid. The transition states, comprising reactants–enzyme complexes, are included here because they are the result of the procedure performed throughout the present article: transformation of an enantiomeric relationship into a diastereomeric one by means of association, as shown, for instance, in Scheme VI.
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We have briefly characterized a few aspects of organic stereochemistry that stem from the intermolecular interaction of the enantiomer (R) with a chiral auxiliary molecule and the interaction of (S) with a chiral auxiliary (Scheme I). These complexations are shown to be relevant upon the following occasions: • Separation of enantiomers via crystallization of diastereomeric salts • Analysis of enantiomers by NMR spectroscopy • Separation of enantiomers by chromatography • Biological activities of enantiomers • Reactivities of enantiomers in enzymatic resolution
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In all cases, an interaction-based recognition is accomplished by mechanisms such as hydrogen bonding, cation–anion attraction or π–π interaction as well as by inclusion (or partial inclusion). The reason is that the physical and chemical properties of the two possible diastereomeric complexes may be unequal. The following practical applications of this phenomenon are mentioned: the small- and large-scale preparation of the enantiomers of many organic compounds; the direct analysis of chiral nonracemic mixtures by chromatography and by NMR spectroscopy; and the growing therapeutic use of drugs in the form of one of the enantiomers. We propose to present a lecture to the students that emphasizes the basic similarity of the above association-based differentiations. This similarity helps to make useful connections between some seemingly unrelated subjects of the curricula. Acknowledgments We thank the “Stiftungsinitiative Johann Gottfried Herder” for funding one-semester visiting professorships at Comenius University, Bratislava, Slovak Republic, and at Masaryk University, Brno, Czech Republic, where teaching along the above lines was tried and the present article was written. We are grateful to E. von Angerer, G. Hauska, J. Sauer, Regensburg, and P. Kois, Bratislava for useful discussions. Literature Cited 1. Greer, J.; Wainer, I. W. In Chirality in Natural and Applied Science; Lough, W. J., Wainer, I. W., Eds.; Blackwell: Oxford, 2002; pp 87–108. 2. Eliel, Ernest L.; Wilen, Samuel H.; Doyle, Michael P. Basic Organic Stereochemistry; Wiley-Interscience: New York, 2001; (a) pp 4, 108, 142; (b) p 268; (c) pp 209–236; (d) pp 153– 160; (e) pp 398–403; (f ) pp 160–175; (g) pp 132–138; (h) pp 257–273. 3. Wermuth, Camille G. In The Practice of Medicinal Chemistry, 2nd ed.; Wermuth, Camille G., Ed.; Academic Press: Amsterdam, 2003; pp 275–288. 4. Buxton, S. R.; Roberts, S. M. Guide to Organic Stereochemistry; Longman: Harlow, NY, 1996; (a) pp 194–199; (b) pp 172–179. 5. Clark, George L.; Yohe, G. Robert J. Am. Chem. Soc. 1929, 51, 2796–2807. 6. Lough, W. J. In Chirality in Natural and Applied Science; Lough, W. J., Wainer, I. W., Eds.; Blackwell: Oxford, 2002; pp 179–202. 7. Yoshioka, Ryuzo; Hiramatsu, Hajime; Okamura, Kimio; Tsujioka, Ikuko; Yamada, Shinichi. J. Chem. Soc., Perkin Trans. 2000, 2, 2121–2128. 8. Sheldon, Roger A. Chirotechnology. Industrial Synthesis of Optically Active Compounds; Marcel Dekker: New York, 1993; (a) pp 78–80, 188–191; (b) pp 61–69. 9. Mannschreck, Albrecht; Lorenz, Klaus; Schinabeck, Michael. In Organic Photochromic and Thermochromic Compounds; Crano, John C., Guglielmetti, Robert, Eds.; Kluwer Academic/
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Plenum Publishers: New York, 1999; Vol. 2, pp 261–295. 10. Cox, James R. J. Chem. Educ. 2000, 77, 1424–1428. 11. Parker, David. Chem. Rev. 1991, 91, 1441–1457. 12. Wenzel, Thomas J.; Wilcox, James D. Chirality 2003, 15, 256– 270. 13. Poole, Colin F. The Essence of Chromatography; Elsevier: Amsterdam, 2003; (a) pp 8–9; (b) pp 793–834. 14. Brandl, Friedrich; Pustet, Nikola; Mannschreck, Albrecht. J. Chromatogr. A 2001, 909, 147–154. 15. Francotte, Eric R. J. Chromatogr. A 2001, 906, 379–397. 16. Patrick, Graham L. An Introduction to Medicinal Chemistry; Oxford University Press: Oxford, 1995; pp 45–50. 17. Patil, Popat N.; Miller, Duane D. In Chirality in Natural and Applied Science; Lough, W. J., Wainer, I. W., Eds.; Blackwell: Oxford, 2002; pp 139–178. 18. Triggle, David J. In Chirality in Natural and Applied Science; Lough, W. J., Wainer, I. W., Eds.; Blackwell: Oxford, 2002; pp 109–138. 19. Testa, Bernard; Carrupt, Pierre-Alain; Gal, Joseph. Chirality 1993, 5, 105–111. 20. Murov, Steven L.; Pickering, Miles J. Chem. Educ. 1973, 50, 74–75. 21. Koenig, Wilfried A. In Chirality in Natural and Applied Science; Lough, W. J., Wainer, I. W., Eds.; Blackwell: Oxford, 2002; pp 261–284. 22. Uffelman, Erich S.; Cox, Elizabeth H.; Goehring, J. Brown; Lorig, Tyler S.; Davis, C. Michele. J. Chem. Educ. 2003, 80, 1368–1372. 23. Tombo, Gerardo M. Ramos; Bellus, Daniel. Angew. Chem. 1991, 103, 1219–1241. Tombo, Gerardo M. Ramos; Bellus, Daniel. Angew. Chem., Int. Ed. Engl. 1991, 30, 1193–1215. 24. Chenault, H. Keith; Dahmer, Juergen; Whitesides, George M. J. Am. Chem. Soc. 1989, 111, 6354–6364. 25. Baker, Carl G.; Sober, Herbert. J. Am. Chem. Soc. 1953, 75, 4058–4060. 26. Faber, Kurt. Biotransformations in Organic Chemistry. A Textbook, 4th ed.; Springer: Berlin, 2000; (a) pp 58–60; (b) pp 18–21. 27. Sime, John T. J. Chem. Educ. 1999, 76, 1658–1661. 28. Roberts, Stanley M. J. Chem. Educ. 2000, 77, 344–348. 29. Clement, G. E.; Potter, R. J. Chem. Educ. 1971, 48, 695–696. 30. Mohrig, Jerry R.; Shapiro, Samuel M. J. Chem. Educ. 1976, 53, 586–589. 31. Sheardy, Richard; Liotta, L.; Steinhart, E.; Champion, R.; Rinker, J.; Planutis, M.; Salinkas, J.; Boyer, T.; Carcanague, D. J. Chem. Educ. 1986, 63, 646–647. 32. Poire, C.; Rabiller, C.; Chon, C. J. Chem. Educ. 1996, 73, 93–95. 33. Stetca, Delia; Arends, Isabel W. C. E.; Hanefeld, Ulf. J. Chem. Educ. 2002, 79, 1351–1352. 34. Robinson, Michael J. T. Organic Stereochemistry; Oxford University Press: Oxford, 2000; pp 78–81. 35. Sih, Charles J.; Wu, Shih-Hsuing. Top. Stereochem. 1989, 19, 63–125. 36. Nelson, David L.; Cox, Michael M. Lehninger Principles of Biochemistry, 3rd ed.; Worth: New York, 2000; pp 250–265
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