Resolution with Enzymes

Adam, W., Lazarus, M., Boss, B. et al. (1997) Enzymic resolution of chiral 2-hydroxy carboxylic acids by enantioselective oxidation with molecular oxygen catalyzed by the glycolate oxidase from spinach (Spinacia oleracea). The Journal of Organic Chemistry, 62 (22), 7841-7843. 

Larsen, T.M., Wedeking, J.E., Rayment, I. and Reed, G.H. (1996) A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 A resolution, Biochemistry, 30, 4349-4358. 

Using the principles outlined in this article, the crystal structures of the following complexes of RNase A have been determined the free enzyme, both with and without a sulfate ion in the active site, the enzyme-dinucleotide complex, the enzyme-cyclic phosphate intermediate complex, the enzyme-transition state complex, and the enzyme-product complex, all at or near atomic resolution. This structural informa-... 

DKR of secondary alcohol is achieved by coupling enzyme-catalyzed resolution with metal-catalyzed racemization. For efficient DKR, these catalyhc reactions must be compatible with each other. In the case of DKR of secondary alcohol with the lipase-ruthenium combinahon, the use of a proper acyl donor (required for enzymatic reaction) is parhcularly crucial because metal catalyst can react with the acyl donor or its deacylated form. Popular vinyl acetate is incompatible with all the ruthenium complexes, while isopropenyl acetate can be used with most monomeric ruthenium complexes. p-Chlorophenyl acetate (PCPA) is the best acyl donor for use with dimeric ruthenium complex 1. On the other hand, reaction temperature is another crucial factor. Many enzymes lose their activities at elevated temperatures. Thus, the racemizahon catalyst should show good catalytic efficiency at room temperature to be combined with these enzymes. One representative example is subtilisin. This enzyme rapidly loses catalytic activities at elevated temperatures and gradually even at ambient temperature. It therefore is compatible with the racemization catalysts 6-9, showing good activities at ambient temperature. In case the racemization catalyst requires an elevated temperature, CALB is the best counterpart. 

It is emphasized that in the case of kinetic resolution, the MS measurements must be performed in the appropriate time window (near 50% conversion). If this is difficult to achieve due to different amounts or activities of the mutants being screened in the wells of microtiter plates, the system needs to be adapted in terms of time resolution. This means that samples for MS evaluation need to be taken as a function of time. Finally, it is useful to delineate the possibility of multi-substrate ee screening using the MS-based assay, which allows for enzyme fingerprinting with respect to the enantioselectivity of several substrates simultaneously. 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, enzymes that catalyze the hydrolysis of hydantoins 7-11,147). Because synthetic hydantoins are accessible by a variety of chemical syntheses, including Strecker reactions, enan-tioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, because hydantoins are easily racemized chemically or enzymatically by appropriate racemases, dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases have been reported 147). However, if asymmetric induction is poor or if inversion of enantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of L-methionine as part of a whole cell system E. coll) (Figure 22) 148). 

Usually a biocatalyst-based process is in competition with other approaches, for instance enzyme resolution processes to produce single isomer products are in competition with their isolation from natural sources and chemical asymmetric synthesis processes. 

The enzyme-catalyzed regio- and enantioselective reduction of a- and/or y-alkyl-substituted p,5-diketo ester derivatives would enable the simultaneous introduction of up to four stereogenic centers into the molecule by two consecutive reduction steps through dynamic kinetic resolution with a theoretical maximum yield of 100%. Although the dynamic kinetic resolution of a-substituted P-keto esters by chemical [14] or biocatalytic [15] reduction has proven broad applicability in stereoselective synthesis, the corresponding dynamic kinetic resolution of 2-substituted 1,3-diketones is rarely found in the literature [16]. 

Solvent-accessible surface representation of the GlnRS enzyme complexed with tRNA and ATP. The region of contact between tRNA and protein extends across one side of the entire enzyme surface and includes interactions from all four protein domains. The acceptor end of the tRNA and the ATP are seen in the bottom of the deep cleft. Protein is inserted between the 5 and 3 ends of the tRNA and disrupts the expected base pair between Ul and A72. (From M. G. Rould, J. J. Persona, D. Soil, and T. Steitz, Structure of E. coli glutamyl-tRNA synthetics complexed with tRNA ln and ATP at 2.8-A resolution, implications for tRNA discrimination, Science 246 1135-1142, 1989, 1989 by the AAAS.)... 

When solutions of individual pesticides were used, concentrations as low as 0.5 pgl-1 (10 9 M) could be determined. When binary mixtures with pesticide levels from 0 to 5 pgl-1 were measured, the concentration of each pesticide could be determined within the range with errors of 0.4 pgl-1 for paraoxon and 0.5 pgl-1 for carbofuran. Similar levels were obtained when river water samples spiked with pesticide were used but with a higher degree of inaccuracy. When different mutant AChEs were utilised, binary mixtures of the very similar pesticides paraoxon and malaoxon could be analysed in the range 0-5 pgl-1, with resolution of the two components with accuracies of the order of 1 pgl-1. The use of more sensitive and selective mutant enzymes together with the addition of extraction and concentration steps to the assay could greatly enhance the methods range and accuracy. 

M. J. Kim, Y. Ahn, and J. Park, Dynamic kinetic resolutions and asymmetric transformations by enzymes couples with metal catalysis, Curr. Opin. Biotechnol. 2002, 13, 578-587. 

Chiral alcohols are valuable products mainly as building blocks for pharmaceuticals or agro chemicals or as part of chiral catalysts. Cheap biotransformation methods for the selective reduction of particular ketone compounds are known for many years rather catalyzed by fermentation than with isolated enzymes. Products prepared with whole cells such as baker s yeast often lack high enantioselectivity and there were several attemps to use isolated enzymes. Resolution of racemates with hydrolases are known in some cases but very often the reduction of the prochiral ketone using alcohol dehydrogenases are much more attractive. 

The tertiary structure of HLADH, the representing enzyme of this group, was refined to 2.4 A resolution with X-ray diffraction [102 105], and the three-dimensional modeling of other medium-chain ADHs is based upon this structure [106, 107], The tertiary structure of yeast ADH was found to be similar to that of HLADH although the primary structure of YADH is quite different. 

The alternative approach to the determination of the stereochemistry of hydroxylation )3 to the nitrogen occurring in the biosynthesis of haemanthamine involved the synthesis of stereospecifically labeled tyrosine. Catalytic hydrogenation of suitable acylaminocinnamic acids labeled with isotopic hydrogen in fi position proceeds stereoselectively to furnish an equimolecular mixture of L-(/3i -3H]-and D-[j8 -3H]tyrosine (419) and (420). Enzymic resolution of the racemic amino acid yielded the L and d isomers. The two optically active forms of tyrosine were... 

Biological catalysts for asymmetric transformations have been used in specific cases for a considerable period of time, excluding the chiral pool materials. However, until recently, the emphasis has been on resolutions with enzymes rather than asymmetric transformations (Chapter 19 see also Chapters 20 and 21). With our increasing ability to produce mutant enzymes that have different or broad-spectrum activities compared to the wild types, the development of biological catalysts is poised for major growth. In addition to high stereospecificities, an organism can be persuaded to perform more than one step in the overall reaction sequence and may even make the substrate (Chapter 3). 

High resolution crystallographic studies have demonstrated that AspAT has open and closed conformations. Upon the binding of a substrate which favors the closed conformation, the small domain rotates so that the active site residues are brought closer to the substrate moiety. This movement of the small domain is important for initiation of the catalytic reaction. Therefore, the closed conformation is believed to represent the functional states of the enzyme.291 Three distinct crystal forms of mAspAT were analyzed, namely triclinic crystal for open conformation, monoclinic and orthorhombic crystals for closed conformation. The enzymes cocrystallized with substrate analogs seemed to have a tendency to assume the closed conformation.28 291 The order of this tendency was shown to be apoenzyme1 > PLP form of the enzyme > PMP form of the enzyme. 

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