Enzyme-Catalyzed Kinetic Resolution

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. 

Various racemic secondary alcohols with different substituents, eg, a-hydroxyester (60), are resolved by PFL neatly quantitatively (75). The effect of adjacent unsatuiation on enzyme-catalyzed kinetic resolutions was thoroughly studied for a series of aHyUc (61), propargyUc (62), and phenyl-substituted 2-aIkanols (76,77). Excellent selectivity was observed for (E)-aHyhc alcohols whereas (Z)-isomers showed poor selectivity (76). 

Enzymes may be used either directly for chiral synthesis of the desired enantiomer of the amino acid itself or of a derivative from which it can readily be prepared, or for kinetic resolution. Resolution of a racemate may remove the unwanted enantiomer, leaving the intended product untouched, or else the reaction may release the desired enantiomer from a racemic precursor. In either case the apparent disadvantage is that the process on its own can only yield up to 50% of the target compound. However, in a number of processes the enzyme-catalyzed kinetic resolution is combined with a second process that re-racemizes the unwanted enantiomer. This may be chemical or enzymatic, and in the latter case, the combination of two simultaneous enzymatic reactions can produce a smooth dynamic kinetic resolution leading to 100% yield. 

As shown in Table 12,H202 and fBuOOH have been used frequently as oxygen donors in peroxidase-catalyzed sulfoxidations. Other achiral oxidants, e.g. iodo-sobenzene and peracids, are not accepted by enzymes and, therefore, only racemic sulfoxides were found (c.f. entries 34-36). Interestingly, racemic hydroperoxides oxidize sulfides to sulfoxides enantioselectively under CPO catalysis [68]. In this reaction, not only the sulfoxides but also the hydroperoxide and the corresponding alcohol were produced in optically active form by enzyme-catalyzed kinetic resolution (cf. Eq. 3 and Table 3 in Sect. 3.1). 

Determination of Enantiomeric Excess Without Separation of Products. To determine the enantioselectivity in a given enzyme-catalyzed kinetic resolution, the reaction product very often must be isolated before the determination. This is very time-consuming when one... 

Preparation of Enantiomerically Pure (2R)- and (2S)-Ethyl-2-Fluorohexanoate by Enzyme-Catalyzed Kinetic Resolution... 

With the increased use of enzymes in polymer chemistry, the enzymology terminology to describe the reaction kinetics and the enantioselectivity of a reaction has become more and more common in polymer literature. The parameter of choice to describe the enantioselectivity of an enzyme-catalyzed kinetic resolution is the enantiomeric ratio E. The enantiomeric ratio is defined as the ratio of the specificity constants for the two enantiomers, R) and S) (1) ... 

Scheme 7.14 Enzyme-catalyzed kinetic resolution of racemic trans-2-aminocyclopentanol and its benzyl ether.

Yeast-mediated reductions predominantly form a single enantiomer and it is often difficult to find conditions which produce the opposite stereoisomer selectively. It has, however, been possible to obtain both enantiomers in 50% yield in 100% via enzymatic optical resolution. Chiral fluorinated secondary alcohols possessing the mono-, di- and/or trifluoromethyl group have been prepared by enzyme-catalyzed kinetic resolutions [27]. 

Optically pure trans-2-phenylcyclohexanol can also be prepared by resolution of the phthalate esters using brucine to obtain the (-t-)-alcohol and strychnine to obtain the (-)-alcohol (after basic hydrolysis of their respective salts).11 Enzyme-catalyzed kinetic resolution of the acetate esters using pig liver esterase4 and pig liver acetone powder12 has been used to prepare both enantiomers of this chiral auxiliary. The hydroboration of 1-phenylcyclohexene with isopinocampheylborane has been reported to give the chiral auxiliary in 97% enantiomeric excess.13... 

ENANTIOMERICALLY PURE ETHYL (R)- AND (S)-2-FLUOROHEXANOATE BY ENZYME-CATALYZED KINETIC RESOLUTION (Hexanolc acid, 2-1luoro-, ethyl ester, (R)- and (S)-)... 

The development of accurate methods for the determination of enantiomeric purity, which began in the late 1960 s, has been critical for the assessment of enantioselective synthesis. Thus a prerequisite in the enzyme-catalyzed kinetic resolution of racemates is a precise and reliable assessment of the degree of enantioselectivity (E), enantiomeric excess (ee) and conversion (c). Among these methods are 1) polarimetric methods, 2) gas chromatographic methods, 3) liquid chromatographic methods and 4) NMR spectroscopy. The most convenient and sensitive methods used are chiral GC and HPLC. 

An attractive method for the determination of the enantiomeric excess of substrates and products resulting from the enzyme-catalyzed kinetic resolution of secondary alcohols is chiral gas chromatography (GC).48,49 This sensitive method is quick, simple to carry out and unaffected by the presence of impurities in the analyzed sample, therefore, isolation and purification of the analyzed sample is not required. Very small sample size is required for the analysis hence, reactions can be done on small scale. 

Kurokawa et al81 reported the enzyme-catalyzed kinetic resolution of racemic N-carbamoyl, A-Boc, N-Cbz proline esters and prolinols using protease and Candida antarctica lipase B. The latter was efficient in the enantioselctive hydrolysis of both N-Boc and N-Cbz proline derivatives with E > 100. 

ENZYME-CATALYZED KINETIC RESOLUTION 29.4.1 Synthesis of Leukotriene B4 (LTB4)... 

One way in which a small company can keep abreast of new developments is to collaborate with universities. Moreover, such collaboration helps to motivate staff (and publication of results provides advertising). One fruit of such a collaboration was a synthesis of LTB4, in which chiral moieties of the molecule are derived from the enantiomers of a common intermediate (ll).11 Several routes have been devised for enzyme-catalyzed kinetic resolution of bicyclo[3.2.0]heptenones.12 An efficient one that was used for the synthesis of LTB4 (12) is shown in Scheme 29.4. 

ENANTIOMERICALLY PURE ETHYL (R)- AND (S)-2-FLUORO-HEXANOATE BY ENZYME-CATALYZED KINETIC RESOLUTION... 

Hoft E, Hamann H-J, Kunath A, Adam W, Hoch U, Saha-Moller CR, Schreier P (1995) Enzyme-catalyzed kinetic resolution of racemic secondary hydroperoxides. Tetrahedron Asymmetry 6 603-608... 

More than 60% of all biotransformations using isolated enzymes are hydrolase-dependent processes. Several reviews have summarized the achievements in this field [33-37]. From the numerous examples published, two will be discussed in order to highlight the potential of enzyme-catalyzed kinetic resolution for the production of chiral pharmaceuticals. 

The extension of this method to the enzyme-catalyzed kinetic resolution of 1-phenylethanol by transesterification [Eq. (7)] was published by two groups nearly simultaneously (145,146). 

The dynamic kinetic resolution (DKR) of secondary alcohols and amines (Scheme 11.11) is a prominent, industrially relevant, example of chemo-enzymatic chemistry in which a racemic mixture is converted into one enantiomer in essentially 100% yield and in high ee. This is in sharp contrast to enzyme-catalyzed kinetic resolutions that afford the desired end-product in a yield of at most 50%, while 50% of the starting material remains unreacted. In DKR processes, hydrolases are typically employed as the enantioselective acylation catalyst (which can be either R or S selective) while a concurrent racemization process racemizes the remaining substrate via an optically inactive intermediate. This ensures that all starting material is converted into the desired end-product. The importance of optically pure secondary alcohols and amines for the pharmaceutical industry triggered the development of a number of approaches that enable the racemization of sec-alcohols and amines via their corresponding ketones and imines, respectively [42],... 

Most substrates for enzyme-catalyzed kinetic resolution reactions do not undergo spontaneous racemization under conditions that are suitable for enzyme activity. One solution to this problem has been to design mild transition metal-catalyzed methods for in situ racemization 17. In order to achieve this goal, the racemisation method must be able to function without an adverse effect on the enzyme. Additionally, the enzyme must not inhibit the racemization method. 

The success of an enzyme-catalyzed kinetic resolution is limited by the maximum chemical yield of 50% for each enantiomer. However, this drawback can be overcome by a process called dynamic kinetic resolution. The key idea of this principle is to racemize the slow reacting enantiomer continuously reproducing the faster one. In an ideal case at the end of the conversion one enantiomer is formed in 100% yield with 100% of enantiomeric excess[13S 1371. The kinetic requirements for a dynamic kinetic resolution are shown in Scheme 11.1-16[8bl. 

Other limitations of enzyme-catalyzed kinetic resolutions of enantiomers are that the enzymes are easily denatured, they often require stoichiometric amounts of co-factors, and they are expensive and substrate-specific. 

In an enzyme-catalyzed kinetic resolution process of a racemic substrate, one enantiomer is converted preferentially. For an irreversible conversion of a substrate S into a product P starting from a racemic substrate with = C, the enantiomeric ratio depends on conversion x according to ... 

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