Enzyme racemization

A /3-hydroxybutyric acid dehydrogenase that uses DPN has been known for many years. It is specific for i/-/3-hydroxybutyrate, and does not act with the CoA derivative. The original description of the CoA-requiring system included a specific requirement for D-hydroxy acids. Later a racemase that converts ii-/3-hydroxy CoA compounds to d-was reported, but it appears that the racemase is the resultant of an D-dehydrogenase together with the previously described D-enzyme. Racemization requires DPN. ... 

One approach called enzymatic resolution, involves treating a racemic mixture with an enzyme that catalyzes the reaction of only one of the enantiomers Some of the most commonly used ones are lipases and esterases enzymes that catalyze the hydrol ysis of esters In a typical procedure one enantiomer of the acetate ester of a racemic alcohol undergoes hydrolysis and the other is left unchanged when hydrolyzed m the presence of an esterase from hog liver... 

The enzyme is a single enantiomer of a chiral molecule and binds the coenzyme and substrate m such a way that hydride is transferred exclusively to the face of the carbonyl group that leads to (5) (+) lactic acid Reduction of pyruvic acid m the absence of an enzyme however say with sodium borohydride also gives lactic acid but as a racemic mixture containing equal quantities of the R and S enantiomers... 

Microorganisms and their enzymes have been used to functionalize nonactivated carbon atoms, to introduce centers of chirahty into optically inactive substrates, and to carry out optical resolutions of racemic mixtures (1,2,37—42). Their utifity results from the abiUty of the microbes to elaborate both constitutive and inducible enzymes that possess broad substrate specificities and also remarkable regio- and stereospecificities. 

One of the homochiral starting materials (45) for the acetylcholinesterase (ACE) inhibitor captopril [62571 -86-2] (47) is produced by a Hpase enzyme-catalyzed resolution of racemic 3-methyl-4-acetylthiobutyric acid (44) and L-proline (46) (65). 

Both pure L- and D-amino acids can be made using hydantoinase enzymes. These enzymes catalyze the stereoselective hydrolysis of racemic hydantoins such as (50) which is used for the production of D-alanine (15) (58). 

Although most anesthetics are achiral or are adininistered as racemic mixture, the anesthetic actions are stereoselective. This property can define a specific, rather than a nonspecific, site of action. Stereoselectivity is observed for such barbiturates as thiopental, pentobarbital, and secobarbital. The (3)-enantiomer is modestly more potent (56,57). Additionally, the volatile anesthetic isoflurane also shows stereoselectivity. The (3)-enantiomer is the more active (58). Further evidence that proteins might serve as appropriate targets for general anesthetics come from observations that anesthetics inhibit the activity of the enzyme luciferase. The potencies parallel the anesthetic activities closely (59,60). 

In many cases only the racemic mixtures of a-amino acids can be obtained through chemical synthesis. Therefore, optical resolution (42) is indispensable to get the optically active L- or D-forms in the production of expensive or uncommon amino acids. The optical resolution of amino acids can be done in two general ways physical or chemical methods which apply the stereospecific properties of amino acids, and biological or enzymatic methods which are based on the characteristic behavior of amino acids in living cells in the presence of enzymes. 

Enzymatic Method. L-Amino acids can be produced by the enzymatic hydrolysis of chemically synthesized DL-amino acids or derivatives such as esters, hydantoins, carbamates, amides, and acylates (24). The enzyme which hydrolyzes the L-isomer specifically has been found in microbial sources. The resulting L-amino acid is isolated through routine chemical or physical processes. The D-isomer which remains unchanged is racemized chemically or enzymatically and the process is recycled. Conversely, enzymes which act specifically on D-isomers have been found. Thus various D-amino acids have been... 

The primary disadvantage of the conjugate addition approach is the necessity of performing two chiral operations (resolution or asymmetric synthesis) ia order to obtain exclusively the stereochemicaHy desired end product. However, the advent of enzymatic resolutions and stereoselective reduciag agents has resulted ia new methods to efficiently produce chiral enones and CO-chain synthons, respectively (see Enzymes, industrial Enzymes in ORGANIC synthesis). Eor example, treatment of the racemic hydroxy enone (70) with commercially available porciae pancreatic Hpase (PPL) ia vinyl acetate gave a separable mixture of (5)-hydroxyenone (71) and (R)-acetate (72) with enantiomeric excess (ee) of 90% or better (204). 

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). 

Enzyme-Catalyzed Asymmetric Synthesis. The extent of kinetic resolution of racemates is determined by differences in the reaction rates for the two enantiomers. At the end of the reaction the faster reacting enantiomer is transformed, leaving the slower reacting enantiomer unchanged. It is apparent that the maximum product yield of any kinetic resolution caimot exceed 50%. 

It is generally beheved that selectivity of hydrolytic enzymes strongly depends on the proximity of the chiral center to the reacting carbonyl group, and only a few examples of successful resolutions exist for compounds that have the chiral center removed by more than three bonds. A noticeable exception to this rule is the enantioselective hydrolysis by Pseudomonasfluorescens Hpase (PEL) of racemic dithioacetal (5) that has a prochiral center four bonds away from the reactive carboxylate (24). The monoester (6) is obtained in 89% yield and 98% ee. 

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). 

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

Unprotected racemic amines can be resolved by enantioselective acylations with activated esters (110,111). This approach is based on the discovery that enantioselectivity of some enzymes strongly depends on the nature of the reaction medium. For example, the enantioselectivity factor (defined as the ratio of the initial rates for (3)- and (R)-isomers) of subtiHsin in the acylation of CX-methyl-ben zyl amine with tritiuoroethyl butyrate varies from 0.95 in toluene to 7.7 in 3-methyl-3-pentanol (110). The latter solvent has been used for enantioselective resolutions of a number of racemic amines (110). 

Alcohol dehydrogenase-catalyzed reduction of ketones is a convenient method for the production of chiral alcohols. HLAD, the most thoroughly studied enzyme, has a broad substrate specificity and accommodates a variety of substrates (Table 11). It efficiendy reduces all simple four- to nine-membered cycHc ketones and also symmetrical and racemic cis- and trans-decalindiones (167). Asymmetric reduction of aUphatic acycHc ketones (C-4—C-10) (103,104) can be efficiendy achieved by alcohol dehydrogenase isolated from Thermoanaerohium hrockii (TBADH) (168). The enzyme is remarkably stable at temperatures up to 85°C and exhibits high tolerance toward organic solvents. Alcohol dehydrogenases from horse Hver and T. hrockii... 

Deprotection using enzymes can be quite useful. An added benefit is that a racemic or meso substrate can often by resolved with excellent enantiose-lectivity. 

NH2). The acid crystallises readily when 4g in 50mL H2O is treated with abs EtOH at 4°/ 3hrs, and is collected washed with cold abs EtOH and Et20 and dried in vac. Also recrystallises from aqueous Me2CO, Rp on Si02 TLC plates with n-BuOH-H20-AcOH (4 1 1) is 0.26. The racemate has m 238-240°. [Leukart et al. Helv Chim Acta 59 2181 1976 Eberle and Zeller Helv Chim Acta 68 1880 1985 Jansen et al. Reel Trav Chim Pays-Bas 88 819 7969.] It is a suicide inhibitor of y-cystathionase and other enzymes [Washtier and Abeles Biochemistry 16 2485 7977 Shinozuka et al. Eur J Biochem 124 377 7982]. 

Forskolin is an activator of the enzyme adenylate cyclase which has therapeutic utility. Outlined below are stereocontrolled routes to racemic and natural chiral forms of forskolin derived by multistrategic retrosynthedc analysis. 

The use of enzymes for the hydrolysis of acylals is effective, and in the case of racemic derivatives some enantioenrichment of the aldehyde is possible. ... 

In a catalytic asymmetric reaction, a small amount of an enantio-merically pure catalyst, either an enzyme or a synthetic, soluble transition metal complex, is used to produce large quantities of an optically active compound from a precursor that may be chiral or achiral. In recent years, synthetic chemists have developed numerous catalytic asymmetric reaction processes that transform prochiral substrates into chiral products with impressive margins of enantio-selectivity, feats that were once the exclusive domain of enzymes.56 These developments have had an enormous impact on academic and industrial organic synthesis. In the pharmaceutical industry, where there is a great emphasis on the production of enantiomeri-cally pure compounds, effective catalytic asymmetric reactions are particularly valuable because one molecule of an enantiomerically pure catalyst can, in principle, direct the stereoselective formation of millions of chiral product molecules. Such reactions are thus highly productive and economical, and, when applicable, they make the wasteful practice of racemate resolution obsolete. 

The reversibility of halohydrin dehalogenase-catalyzed reactions has been used for the regioselective epoxide-opening with nonnatural nucleophiles (an example is given in Scheme 10.34) [133]. The stereoselectivity of the enzyme results in the resolution of the racemic substrate. At the same time, the regioselectivity imposed by the active site geometry yields the anti-Markovnikov product. [128]... 

Before we leave the enzymatic modification of terpenoids, we should point out that enzymes are also employed to resolve racemic mixtures of terpenoids. The principles of Bus are similar to those employed in the resolution of racemic mixtures of amino acids (see Chapter 8). 

Explain how this enzyme may be used to resolve a racemic mixture of DL-menthol. Assume you want to prepare both D-menthol and L-menthol. 

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