Chymotrypsin chirality

The structural basis of one of the classic examples of such stereospecificity, that of chymotrypsin for L-amino acid derivatives, is immediately obvious on examination of the crystal structure of the enzyme. D-Amino acid derivatives differ from those of L-amino acids by having the H atom and the side chain attached to the chiral carbon interchanged (structure 8.10). The d derivatives cannot bind because of steric hindrance between the side chain and the walls of enzyme around the position normally occupied by the H atom of L derivatives (Chapter 1). 

The separation of enantiomers can be effected either by transforming them into diastereoisomers using a chiral reagent and separating them on conventional phases or by separating the enantiomers on chiral phases. The utilization of chiral phases has not yet become routine, but studies of enantiomeric dipeptides have been carried out (115,116). Pirkle et al. (117) and Hyun et al. (118) separated enantiomeric di- and tripeptides (methyl esters of /V-3-5-dinitrobenzoyl derivatives) on chiral stationary phases (CSPs) derived from (R)-a-arylalkylamines, (S)-N-(2-naphthyl) valine, or (S)-1 -(6,7-dimethyl-1 -naphthyl) isobutylamine. These workers were able to separate four peaks for each dipeptide derivative, corresponding to the two enantiomeric pairs (R,R)/(S,S) and (R,S)/(S,R). Cyclodextrin-bonded stationary phases and chiral stationary immobilized a-chymotrypsin phases were used to separate enantiomeric peptides (118a,b). 

One of the most investigated type of reaction in the field of catalytic imprinted polymers, as indicated by the large number of publications available, is certainly ester hydrolysis. In particular, a great deal of work has been carried out on systems inspired by hydrolytic enzymes since 1987. In 2000, Shea et al. [37] reported the preparation of enantioselective imprinted polymers for the hydrolysis of N-tert-butoxycarbonyl phenylalanine-p-nitrophenyl ester (55), using a system already developed by the same group in 1994 [19]. The system was inspired by the natural hydrolytic enzyme chymotrypsin and polymerisable imidazole units (27) were used as functional monomers coupled via ester linkages to a chiral phosphonate (56), analogue of (d)- or (L)-phenyl-alanine. After template removal, the imprinted polymers showed selectivity towards the hydrolysis of the enantiomer with which they were imprinted. The ratio of the rate constants, k /k, was 1.9 for the polymer imprinted with the D-enantiomer and kjku was 1.2 for that imprinted with the L-enantiomer. Moreover, the imprinted polymer showed a 2.5-fold increase in the rate of the reaction when compared with the control polymer, imprinted with a... 

Wainer et al. [77] presented a CSP based on a-chymotrypsin protein, and, initially, the chiral resolution of certain amino acids and amino ester was achieved on this protein CSP [14,77]. Later, this CSP was used for the chiral resolution of dipeptides and profens [78,79]. Recently, Felix and Descorps [80,81] used immobilized a-chymotrypsin for the chiral resolution of a variety of racemic compounds. Cellobiohydrolase-I (CBH-I) immobilized to silica gel was found to... 

Most of the enzymes show extremely strict chiral recognitions, and only one of the enantiomers can be the substrate of the enzyme. For example, chymotrypsin incorporates L-peptides only to the enzyme-substrate binding site to form enzyme-substrate complex, so it shows very high enantioselectivity (Figure 3 (a)). Oxidoreductases also form the enzyme-substrate complex of only one enantiomer, so enantioselectivities are high when isolated enzymes are used for reactions instead of whole cells containing both (R)- and (.S )-specific enzymes, which leads to overall low enantioselectivities. 

I. Marie, A. Karlsson, and C. Pettersson, Separation of enantiomers using a-chymotrypsin-silica as a chiral stationary phase, J. Chromatogr., 604 185 (1992). 

Asymmetrization of a prochiral dicarboxylic acid diester catalyzed by lipases, where the stereo center of the product is located on the acyl side, becomes a single-step process because the polar carboxylic acid and/or amide formed are not well accepted as substrates by the Upase. One example is the enantioselective hydrolysis or ammonolysis of diethyl 3-hydroxyglutarate, as shown in Scheme 7.4, a reaction which leads to the formation of a precursor for the important chiral side chain of atorvastatin, lipitor [40, 41]. The S-enantiomer was formed with high e.e. (98%), but unfortunately this is the undesired enantiomer for the production of the pharmaceutically important product. Only a-chymotrypsin gave a predominance of the... 

A CSP consists of a chiral selector, which either alone constitutes the stationary phase or which has been immobilised to a solid phase. The chiral selector is a low molecular weight compound or a polymer, either synthetic or natural. A broad range of CSPs has been developed. Examples of CSPs that have been used successfully include polysaccharides, such as cellulose and its derivatives [6] and cyclodex-trins [7], and proteins, e.g. bovine serum albumin, aj-acid glycoprotein, cellulase, trypsin and a-chymotrypsin [8]. Several different synthetic polymers have also proven to be useful CSPs, for example the Blaschke-type CSPs (polyacrylamides and polymethacrylamides) [9] and the Pirkle-type CSPs [10]. 

Of particular interest is the study of the biological mechanisms associated with enzyme stereoselectivity and enantioselectivity. For example, MD simulations have been successful in explaining the different affinities of trypsin and acetylcholinesterase to the diastereomers of soman inhibitors [154] and the ability of subtilisin Carlsberg and a-chymotrypsin to discriminate between R-and S- configurations of chiral aldehyde inhibitors [155, 156]. 

---