Ulrike T. Strauss

Biocatalytic transformation of racemates into chiral building blocks in 100% chemical yield and 100% enantiomeric excess

Biocatalytic techniques, which lead to the highly efficient transformation of a racemate into a single stereoisomeric product in (theoretically) 100% chemical yield and 100% enantiomeric excess are reviewed and their specific merits and limitations are discussed. The processes known so far can be classified into the following categories: (i) A range of methods are based on the improvement of classic kinetic resolution processes, for instance reracemization followed by repeated resolution, dynamic resolution and follow-up reactions, such as stereoinversion reactions. (ii) On the contrary, more elegant solutions are derived by employing enantioconvergent processes, which are based on the transformation of each enantiomer through stereochemically different pathways, which can be achieved by using combined chemo- and/or biocatalysis. (iii) Finally, a novel type of process for the deracemization of compounds possessing a sec-alcohol and -amino group makes use of a cyclic oxidation–reduction sequence, which is combined in a cyclic mode.

Deracemization of (±)-mandelic acid using a lipase–mandelate racemase two-enzyme system

Abstract Deracemization of (±)-mandelic acid was achieved by using a novel two-enzyme process consisting of: (i) Pseudomonas sp. lipase catalyzed O-acylation of (±)-mandelic acid in diisopropyl ether; followed by (ii) mandelate racemase catalyzed racemization of the remaining unreacted (R)-mandelic acid in aqueous buffer. When this one-pot sequence was repeated four times, (S)-O-acetylmandelic acid was obtained in 80% isolated yield and >98% ee as the sole product.

Selectivity-enhancement in enantioselective hydrolysis of sec-alkyl sulfates by an alkylsulfatase from Rhodococcus ruber DSM 44541

Abstract The Enantioselectivity of the biohydrolysis of sec -alkyl sulfate esters using a bacterial alkylsulfatase from Rhodococcus ruber DSM 44541 was dramatically enhanced in presence of additives (‘enhancers’) such as carbohydrates, polyethylene glycol, detergents, metal ions and through enzyme immobilization. In presence of iron, the E value for the kinetic resolution of (±)-3- and (±)-4-octyl sulfate was improved from E =3.9 to ≥200 and E =1.1 to 10, respectively.

Substrate spectrum of mandelate racemase: Part 2. (Hetero)-aryl-substituted mandelate derivatives and modulation of activity

Efficient enzymatic racemization of 2-hydroxy-2-heteroaryl-acetic acid derivatives by mandelate racemase under mild conditions is reported for the first time. (i) Steric limitations for aryl-substituted mandelate derivatives were elucidated to be particularly striking for o-substituents, whereas m- and p-analogues were freely accepted, as well as heteroaryl- and naphthyl-analogs. (ii) The electronic character of substituents was found to play an important role: whereas electron-withdrawing substituents dramatically enhanced the racemization rates, electron-donating analogs caused a depletion. This effect could be ascribed to an α-carbanion-stabilization in accordance with the known enzyme mechanism. The latter was modeled by comparison of gas phase deprotonation energies as a useful parameter to describe resonance stabilization. The calculated data nicely correlate with the experimentally observed activities for a specific substrate as long as other parameters, such as steric restrictions, are absent or play a minor role.

Large-scale preparation of a nitrile-hydrolysing biocatalyst: Rhodococcus R 312 (CBS 717.73)

Abstract Lyophilized cells of Rhodococcus R 312 (CBS 717.73) can be employed as an easy-to-use biocatalyst for the biocatalytic hydrolysis of nitriles to furnish the corresponding carboxamides or acids on a preparative scale. The following practical aspects are advantageous: (i) Fermentation yields a high biomass (∼10 g dry cell weight l−1), (ii) enzyme induction is not required, (iii) a maximum of activity is obtained at the late exponential growth phase (25.7 μmol min−1 mg−1), which can be monitored by a simple photometic assay and (iv) the cells can be stored at +4°C for several months without significant loss of activity.

Substrate spectrum of mandelate racemase. Part 1 : Variation of the α-hydroxy acid moiety

Enzymatic racemization of mandelic acid derivatives modified at the α-hydroxy acid moiety was achieved using mandelate racemase [EC 5.1.2.2]. Whereas α-amino acid derivatives, such as phenyl glycine and mandelic acid hydrazide were not accepted, the mandelic acid amide was racemized at an acceptable rate. The latter was significantly enhanced by an electron-withdrawing substituent in the phenyl moiety. Based on the catalytic mechanism of the enzyme, the relative activities of non-natural substrates could be explained by steric and electronic reasons.

Stabilization and activity-enhancement of mandelate racemase from Pseudomonas putida ATCC 12336 by immobilization

Mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12336 was efficiently immobilized through ionic binding onto DEAE- and TEAE 23-cellulose. The activity of the immobilized enzyme was significantly enhanced as compared to the native protein, i.e., 2.7- and 2.5-fold, respectively. DEAE-cellulose-immobilized mandelate racemase could be efficiently used in repeated batch reactions for the racemization of (R)-mandelic acid under mild conditions.

Novel Carboxyl Esterase Preparations for the Resolution of Linalyl Acetate

Summary. Biocatalytic resolution of the tertiary terpene alcohol (±)-linalool was accomplished via hydrolysis of its corresponding acetate ester using two highly enantiospecific enzymes (E > 100). The latter were identified in a crude cell-free extract of Rhodococcus ruber DSM 43338 and could be separated by (partial) protein purification. Since they showed opposite enantiopreference, they were termed (R)- and (S)-linalyl acetate hydrolase (LAH). The activity and selectivity of the enzyme preparations was markedly dependent on the fermentation conditions.

Simultaneous direct high-performance liquid chromatographic enantioseparation of 2-methylglycidol-1-benzyl ether and 2- methylglycerol-1-benzyl ether using a solvent-switching technique

Simultaneous HPLC separation of the enantiomers of 3-benzyloxy-2-methyl-1,2-propanediol and the corresponding 3-benzyloxy-2-methyl-1,2-propene oxide could be accomplished on amylose derived Chiralpak AD switching between 10% 2-propanol and 3% 1,2-dimethoxyethane as polar modifier in n-heptane.