Improvement of a stereoselective biocatalytic synthesis by substrate and enzyme engineering: 2-hydroxy-(4'-oxocyclohexyl)acetonitrile as the model.

Even if biocatalysis is finding increasing application, it still has to gain widespread use in synthetic chemistry. Reasons for this are limitations that enzymes have with regard to substrate range, reaction scope, and insufficient selectivity with unnatural compounds. These shortcomings can be challenged by enzyme and/or substrate engineering, which are employed to alter substrate specificity and enhance the enzyme selectivity toward unnatural substrates. Herein, these two approaches are coupled to improve the hydroxynitrile lyase catalyzed synthesis of 2-hydroxy-(4'-oxocyclohexyl)acetonitrile (4). The ketone functionality is masked as an enol ether, and the oxynitrilase of Hevea brasiliensis is engineered towards this masked substrate to give the product with a high optical purity and to drastically lower the amount of enzyme needed.

Ortho-lithiation of free ferrocenyl alcohols: a new method for the synthesis of planar chiral ferrocene derivatives.

An ortho-metalation method for free ferrocenyl alcohols has been developed, which allows preparation of planar chiral ferrocene derivatives with high yields and diastereoselectivities.

De-racemization of enantiomers versus de-epimerization of diastereomers--classification of dynamic kinetic asymmetric transformations (DYKAT).

The isolation of single stereoisomers from a racemic (or diastereomeric) mixture by enzymatic or chemical resolution techniques goes in hand with the disposal of 50% (racemate) or more (diastereomeric mixtures) of the "undesired" substrate isomer(s). In order to circumvent this drawback, dynamic systems have been developed for the de-racemization of enantiomers and the de-epimerizations of diastereomers. Key strategies within this area are discussed and are classified according to their underlying kinetics, that is, dynamic kinetic resolution (DKR), dynamic kinetic asymmetric transformations (DYKAT), and hybrids between both of them. Finally, two novel types of DYKAT are defined.

Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry.

The application of hydroxynitrile lyases (HNLs) as catalysts for the stereoselective condensation of HCN with carbonyl compounds has been reported as early as 1908. This enzymatic C-C bond coupling reaction furnishes enantiopure cyanohydrins which serve as versatile bifunctional building blocks for chemical synthesis. Screening of natural sources led to the discovery of both (R)- and (S)-selective HNLs, and several distinctly different classes of these enzymes with substantial differences concerning sequence, structure, and mechanism have been found. Especially during the last two centuries, HNLs have been developed into valuable biocatalysts, which can be produced in recombinant form by overexpression in microbial hosts, resulting in the implementation of industrial processes utilizing these enzymes. Recently, protein engineering in combination with in silico methods gave rise to the development of a tailor-made HNL for large-scale manufacturing of a specific target cyanohydrin.

Counteracting expression deficiencies by anticipating posttranslational modification of PaHNL5-L1Q-A111G by genetic engineering.

(R)-2-chloromandelic acid represents a key pharmaceutical intermediate. Its production on large scale was hampered by low turnover rates and moderate enantiomeric excess (ee) using enzyme as well as metal catalysts. The cloning and heterologous overexpression of an (R)-hydroxynitrile lyase from Prunus amygdalus opened a way to large-scale production of this compound. Especially the rationally designed mutation of alanine to glycine at amino acid position 111 of the mature protein tremendously raised the yield for enantioselective conversion of 2-chlorobenzaldehyde to (R)-2-chloromandelonitrile, which can be hydrolysed to the corresponding alpha hydroxy acid. However, expression of this mutein was less efficient than for the unmodified enzyme. Subsequent LC/MS/MS-analysis of the protein sequence revealed that mutation A111G triggered the posttranslational deamidation of the neighbouring residue asparagine (N110) to aspartic acid. This finding on the one hand could explain the decreased secretion efficiency of the mutant as compared to the wildtype enzyme, but on the other hand raised the question which of the two residues was truly accountable for the enhanced conversion. The muteins N110D, A111G and N110DA111G were constructed and compared in terms of protein productivity and performance in chemical syntheses. The expression level of the double mutein was augmented significantly and the enantioselectivity remained high. Reduced protein expression of mutein PaHNL5-L1Q-A111G was remedied by mutational anticipation of posttranslational deamidation.

Stereoselective biocatalytic synthesis of (S)-2-hydroxy-2-methylbutyric acid via substrate engineering by using "thio-disguised" precursors and oxynitrilase catalysis.

3-Tetrahydrothiophenone (4) and 4-phenylthiobutan-2-one (7) were used as masked 2-butanone equivalents to give the corresponding cyanohydrins 5 (79 % yield, 91 % ee) and 8 (95 % yield, 96 % ee) in an enzymatic cyanohydrin reaction applying the hydroxynitrile lyase (HNL) from Hevea brasiliensis. After hydrolysis and desulphurisation the desired intermediate (S)-2-hydroxy-2-methylbutyric acid (10) was obtained with 99 % ee. Interestingly, when applying (R)-selective HNL from Prunus amygdalus again the (S)-cyanohydrin 5 was formed (62 % ee). The absolute configuration of 5 was verified by crystal structure determination of the corresponding hydrolysis derived carboxylate. The fact that both enzymes yield the same enantiomer was analysed and interpreted by molecular modelling calculations.

Stereoselective hydroxylation of an achiral cyclopentanecarboxylic acid derivative using engineered P450s BM-3.

Substrate engineered, achiral carboxylic acid derivative was biohydroxylated with various mutants of cytochrome P450 BM-3 to give two out of the four possible diastereoisomers in high de and ee. The BM-3 mutants exhibit up to 9200 total turnovers for hydroxylation of the engineered substrate, which without the protecting group is not transformed by this enzyme.

The use of substrate engineering in biohydroxylation.

In the biohydroxylation of nonactivated carbon atoms, substrate engineering has been found to be a very useful and simple means to influence substrate acceptance and the regioselectivity and stereoselectivity of this transformation. Recently, this methodology has been applied to the hydroxylation of a large number of compounds including cycloalkane carboxylic acids, ketones, amines, amides and alcohols.

Oxidative biotransformations using oxygenases.

Considerable progress has been made in manipulating oxidative biotransformations using oxygenases. Substrate acceptance, catalytic activity, regioselectivity and stereoselectivity have been improved significantly by substrate engineering, enzyme engineering or biocatalyst screening. Preparative biotransformations have been carried out to synthesize useful pharmaceutical intermediates or chiral synthons on the gram to several-hundred-gram scale, by use of whole cells of wild type or recombinant strains. The synthetic application of oxygenases in vitro has been shown to be possible by enzymatic or electrochemical regeneration of NADH or NADPH.

In situ proton-NMR analyses of Escherichia coli HB101 fermentations in 1H2O and in D2O.

Experiments using one-dimensional Fourier-transform proton-NMR spectrometry for non-invasive analyses of microbial fermentations in situ, in vivo and in normal aqueous buffer are described. Analyses of the 'mixed acid' fermentation during growth of Escherichia coli on glucose and citrate were performed to identify and quantitatively estimate the concentrations of the two substrates provided and of the six products formed without sampling from the NMR tube. Identification of fermentation substrates and products was achieved by coincidence of selected diagnostic proton signals of individual compounds in the same solvent. The complete time course of growth of E. coli in the NMR tube correlated well with that of the same culture grown outside the magnet, with samples taken for proton-NMR analyses. The entire course of these in situ proton measurements during growth over 16-24 h was obtained automatically, usually unattended overnight. Thus, the utilization and formation of eight substances in the fermentation were monitored simultaneously, in normal 1H2O, without sampling and individual analysis. Several metabolic changes could be readily detected during the fermentations. Additionally, the pH changes were estimated from the chemical shifts of the acetate signal as growth progressed. The effect of varying D2O concentrations in the solvent on growth rates and product yields was examined, and the increase in the complexity of signals given by these fermentations is described. This versatile and rapid method for the simultaneous, direct and automatic analysis of mixtures of many compounds has the potential to be extended to routine on-line analyses of industrial fermentations.