Torsten Sehl

Efficient 2-step biocatalytic strategies for the synthesis of all nor(pseudo)ephedrine isomers

Chiral 1,2-amino alcohols are important building blocks for chemistry and pharmacy. Here, we developed two different biocatalytic 2-step cascades for the synthesis of all four nor(pseudo)ephedrine (N(P)E) stereoisomers. In the first one, the combination of an (R)-selective thiamine diphosphate (ThDP)-dependent carboligase with an (S)- or (R)-selective ω-transaminase resulted in the formation of (1R,2S)-NE or (1R,2R)-NPE in excellent optical purities (ee >99% and de >98%). For the synthesis of (1R,2R)-NPE, space–time yields up to ∼26 g L−1 d−1 have been achieved. Since a highly (S)-selective carboligase is currently not available for this reaction, another strategy was followed to complement the nor(pseudo)ephedrine platform. Here, the combination of an (S)-selective transaminase with an (S)-selective alcohol dehydrogenase yielded (1S,2S)-NPE with an ee >98% and a de >99%. Although lyophilized whole cells are cheap to prepare and were shown to be appropriate for use as biocatalysts, higher optical purities were observed with purified enzymes. These synthetic enzyme cascade reactions render the N(P)E-products accessible from inexpensive, achiral starting materials in only two reaction steps and without the isolation of the reaction intermediates.

TTC-based screening assay for ω-transaminases: a rapid method to detect reduction of 2-hydroxy ketones.

A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination of a wide range of various aliphatic, aliphatic-aromatic and aromatic-aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510 nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5-10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified.

Asymmetric synthesis of (S)-phenylacetylcarbinol – closing a gap in C–C bond formation

(S)-Phenylacetylcarbinol [(S)-PAC] and its derivatives are valuable intermediates for the synthesis of various active pharmaceutical ingredients (APIs), but their selective synthesis is challenging ...