Enzyme stereoselective hydrolysis

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). 

Esterases, proteases, and some lipases are used in stereoselective hydrolysis of esters bearing a chiral or a prochiral acyl moiety. The substrates are racemic esters and prochiral or meso-diesters. Pig liver esterase (PLE) is the most useful enzyme for this type of reaction, especially for the desymmetrization of prochiral or meso substrates. 

Enzyme-catalyzed stereoselective hydrolysis allows the preparation of enantio-merically enriched lactones. For instance. Pseudomonas sp. lipase (PSL) was found to be a suitable catalyst for the resolution of 5-undecalactone and 5-dodecalactone (Figure 6.20). Relactonization of the hydroxy acid represents an efficient method for the preparation of both enantiomers of a lactone [67]. 

The nitrilase from cyanobacterium Synechocystis sp. PCC6803 was found to effect the stereoselective hydrolysis of phenyl-substituted /3-hydroxy nitriles to (S)-enriched /3-hydroxy carboxylic acids. The enzyme also effected the conversion of y-hydroxynitrile, albeit with lesser enantioselectivity (Table 8.10). Interestingly, this enzyme was also was found to hydrolyze aliphatic dinitriles, such that for 1,2-dicyanoethane and 1,3-dicyanopropane the... 

This unnatural acid is used as a chiral intermediate for the synthesis of a number of products. Chemical asymmetric synthesis was very difficult and so the stereoselective synthetic properties of enzymes were exploited to carry out a selective reduction reaction. The stereoselective hydrolysis of protein amino acid esters had already been commercialised by Tanabe in Japan using immobilised aminoacylase, and selective reduction reactions using whole yeast cells are already used in a number of processes, such as the selective reduction of the anti-cancer drag Coriolin. 

Whole cells are used in stirred tanks with pH control, producing fS )-2-chloropropanoic acid in 50% yield from the racemate (0.3 M) with an enantiomeric excess of over 95%. This approach was selected in preference to other methods of resolution such as acylation of the racemate and then stereoselective hydrolysis. The dehalogenase enzyme is specific for substrates with a carboxyl group and a 2-chloro or bromo substituent. No cofactor or metal ion is required and reaction involves an inversion of configuration. 

Enzymic resolutions involve acceptance by the enzyme, which is a very finely honed chiral system, of one enantiomer of a racemic compound, but not the other. The selective acceptance arises because interactions between the enzyme and the enantiomers are diastereomeric. In its natural environment, the ability of an enzyme to discriminate between enantiomers is virtually absolute. In addition to their stereoselectivity, some enzymes can react at very high rates. Each round of catalysis by the enzyme carbonic anhydrase with its physiological substrate occurs in about 1.7 jus at room temperature, although for a small number of other enzymes, best exemplified by the more lethargic lysozyme, the corresponding figure is about a million times slower. Accordingly, the enzyme-catalysed hydrolysis of, say, one enantiomer of an ester proceeds at a finite rate and hydrolysis of the other not at all. Resolutions such as those of 39, 42 and 45 therefore have a kinetic basis and are also known as kinetic resolutions. 

A problem requiring special attention is the stereoselective in vivo activation of prodrugs derived from racemic mixtures, as exemplified by the stereoselective hydrolysis of 0-acetyl-propranolol (275), for which it was also found that the selectivity of plasma enzyme urase differs from that of liver and intestine en-... 

The racemic resolution of this molecule is very important because the 5 -enantiomer is 28-fold more active than the R-enantiomer. Sakaki and co-workers [4.69] realized the production of (5)-naproxen from the racemic naproxen methyl ester using lipase immobilized in hollow fibers. Their results showed that the MBR had good enzyme stability and enantiomeric excess of up to 0.92. The stereoselective hydrolysis of racemic 2-substituted propionates catalyzed by carboxyl esterase has been performed by Cretich and coworkers... 

For the enantioselective preparations of chiral synthons, the most interesting oxidations are the hydroxylations of unactivated saturated carbons or carbon-carbon double bonds in alkene and arene systems, together with the oxidative transformations of various chemical functions. Of special interest is the enzymatic generation of enantiopure epoxides. This can be achieved by epoxidation of double bonds with cytochrome P450 mono-oxygenases, w-hydroxylases, or biotransformation with whole micro-organisms. Alternative approaches include the microbial reduction of a-haloketones, or the use of haloperoxi-dases and halohydrine epoxidases [128]. The enantioselective hydrolysis of several types of epoxides can be achieved with epoxide hydrolases (a relatively new class of enzymes). These enzymes give access to enantiopure epoxides and chiral diols by enantioselective hydrolysis of racemic epoxides or by stereoselective hydrolysis of meso-epoxides [128,129]. 

Scheme 40 describes the second part of the Hoechst Marion Roussel process of 7-AC A (129) manufacture - the first enzymatic transformation has already been described in Scheme 6. Glutaric acid derivative 20 is now subjected to treatment with immobilized Pseudomonas sp, cu-amidodicarboxylate amido-hydrolase (recombinant in Escherichia coli). The enzyme catalyzes the chemo-and stereoselective hydrolysis of the amide and gives the free amine 129 in reasonable yield and optical purity. The whole process has also been established in several other companies, with minor modifications. Anbics, for example, is presently setting up a fermentation process with its subsidiary Bioferma Murcia in Spain for the production of 7-AC A. A typical isolated yield of 82% has been reported for 129, which can be further optimized to >85% by applying techniques such as reversed osmosis on the production scale [115].

Another example of the use of enzymes Is the resolution of racemic mixtures idilch are of value as chiral synthons. This Is Is Illustrated by the stereoselective hydrolysis of... 

The absorption and transport of the majority of drugs across biological membranes occurs by passive diffusion, a process dependent upon physicochemical properties, i.e., lipophilicity, ionization, and molecular size. Since enantiomers have identical physicochemical properties, stereoselectivity would not be expected even though membrane phospholipids are chiral, the significance of lipophilieity appears to outweigh that of compound chirality. In contrast, differences between diastereoisomers may occur as a result of their differential solubility. However, in the case of compounds transported via earrier-mediated meehanisms, e.g., facilitated diffusion or active transport, proeesses involving a direct interaction between a substrate and a carrier maeromoleeule, stereoselectivity is expected. Preferential absorption of the l- eompared to the D-enantiomers of dopa [96] and methotrexate [97,98] have been reported. In the case of the above examples, enantioseleetivity in absorption is observed, whereas in the case of eephalexin, a eephalosporin antibiotic, diastereoselectivity for the L-epimer oeeurs. The L-epimer has shown a greater affinity than, and acted as a competitive inhibitor of o-eephalexin transport [99]. The L-epimer is also more suseeptible to enzyme-mediated hydrolysis, with the result that it cannot be detected in plasma [99]. 

Enzyme-catalysed hydrolysis and esterification reactions are the most commonly exploited biotransformations. There are two main reasons for this state of affairs. Firstly, the reactions are very easy to perform, and no special apparatus is required. Secondly, there is a wide range of hydrolase enzymes available from commercial suppliers. Furthermore, the stereoselectivities and chemoselectivities of the enzymes are well known, and the likely stereochemical outcomes of such enzyme-catalysed reactions on previously unused substrates are now becoming predictable. 

Meso diacetates obtained from 1,4-diacetoxylation of conjugated dienes have been used for enzyme-catalyzed hydrolysis in enantioselective transformations [79-85]. In an application toward the carpenter bee pheromone (Scheme 11.18) [79], the meso-diacetate 54, obtained from stereoselective 1,4-diacetoxylation of (B,Z)-2,4-hexadiene, was enzymatically hydrolyzed to hydroxyacetate 55 with 92%... 

This multi-enzyme process (Scheme 9.8) starts with the enantioselective but often-reversible hydrolysis of one hydantoin (HYD, 28) enantiomer to the open-chain N-carbamoyl amino acid 29 by a hydantoinase, followed by stereoselective hydrolysis of the carbamate (CARB, 29), liberating ammonia, and carbon... 

Sinisterra et al. [85] reported the stereoselective hydrolysis of racemic A-benzoyl-phenylalanine methyl ester catalyzed by PEG-chymotrypsin in aqueous methanolic solution. The hydrolyzed products, A -benzoylphenylalanine, obtained by PEG-chymotrypsin as well as unmodified enzyme were extracted from the reaction mixture and analyzed by both polarimetry and proton nuclear magnetic resonance spectrophotometry. The enantiomeric ratios of the products were 98 2 and 50 50 (S R) for PEG-enzyme and unmodified enzyme, respectively. In this case, chymotrypsin acquired enantioselectivity by the chemical modification witii PEG. 

A very simple and elegant alternative to the use of ion-exchange columns or extraction to separate the mixture of D-amino add amide and the L-amino add has been elaborated. Addition of one equivalent of benzaldehyde (with respect to die D-amino add amide) to the enzymic hydrolysate results in the formation of a Schiff base with die D-amino add amide, which is insoluble in water and, therefore, can be easily separated. Add hydrolysis (H2SQ4, HX, HNO3, etc.) results in the formation of die D-amino add (without racemizadon). Alternatively the D-amino add amide can be hydrolysed by cell-preparations of Rhodococcus erythropolis. This biocatalyst lacks stereoselectivity. This option is very useful for amino adds which are highly soluble in die neutralised reaction mixture obtained after acid hydrolysis of the amide. 

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

The results presented in Tables 3 and 4 deserve some comments. First, a variety of enzymes, including whole-cell preparations, proved suitable for the resolution of different hydroxyalkanephosphorus compounds, giving both unreacted substrates and the products of the enzymatic transformation in good yields and, in some cases, even with full stereoselectivity. Application of both methodologies, acylation of hydroxy substrates rac-41 and rac-43 or the reverse (hydrolysis of the acylated substrates rac-42 and rac-44), enables one to obtain each desired enantiomer of the product. This turned out to be particularly important in those cases when a chemical transformation OH OAc or reverse was difficult to perform. As an example, our work is shown in Scheme 3. In this case, chemical hydrolysis of the acetyl derivative 46 proved difficult due to some side reactions and therefore an enzymatic hydrolysis, using the same enzyme as that in the acylation reaction, was applied. Not only did this provide access to the desired hydroxy derivative 45 but it also allowed to improve its enantiomeric excess. In this way. 

In this section, however, the stereoselective catalysis of hydrolysis will be described more fully in relation to the stereoselective nature of the enzyme reaction. According to Bunton et al. (1971b), (R)- and (S)-p-nitrophenyl O-methylmandelates [35] were hydrolyzed at an identical rate in borate buffers in the presence of CTAB, while a (R/S) mixture was hydrolyzed at a rate greater than either the (/ )- or (S)-enantiomer alone. They also reported that... 

G. Bellucci, G. Berti, R. Bianchini, P. Cetera, E. Mastrorilli, Stereoselectivity of the Epoxide Hydrolase Catalyzed Hydrolysis of the Stereoisomeric 3-ieri-Buty 1-1,2-epoxycyclohexane. Further Evidence for the Topology of the Enzyme Active Site ,. /. Org. Chem. 1982, 47, 3105 - 3112. 

---