Hans Iding

Benzoylformate Decarboxylase from Pseudomonas putida as Stable Catalyst for the Synthesis of Chiral 2-Hydroxy Ketones

The thiamin diphosphate- and Mg2+-dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2-hydroxy ketones in a benzoin-condensation type reaction. Carboligation constitutes a side reaction of BFD, whereas the predominant physiological task of the enzyme is the non-oxidative decarboxylation of benzoylformate. For this purpose the enzyme was obtained in sufficient purity from Pseudomonas putida cells in a one-step purification using anion-exchange chromatography. To facilitate the access to pure BFD for kinetical studies, stability investigations, and synthetical applications, the coding gene was cloned into a vector allowing the expression of a hexahistidine fusion protein. The recombinant enzyme shows distinct activity maxima for the decarboxylation and the carboligation beside a pronounced stability in a broad pH and temperature range. The enzyme accepts a wide range of donor aldehyde substrates which are ligated to acetaldehyde as an acceptor in mostly high optical purities. The enantioselectivity of the carboligation was found to be a function of the reaction temperature, the substitution pattern of the donor aldehyde and, most significantly, of the concentration of the donor aldehyde substrate. Our data are consistent with a mechanistical model based on the X-ray crystallographic data of BFD. Furthermore we present a simple way to increase the enantiomeric excess of (S)-2-hydroxy-1-phenyl-propanone from 90% to 95% by skillful choice of the reaction parameters. Enzymatic synthesis with BFD are performed best in a continuously operated enzyme membrane reactor. Thus, we have established a new enzyme tool comprising a vast applicability for stereoselective synthesis.

The synthetic development of the anti-influenza neuraminidase inhibitor oseltamivir phosphate (Tamiflu®): A challenge for synthesis & process research

The evolution of the synthesis of oseltamivir phosphate (Tamiflu®), used for the oral treatment and prevention of influenza virus infections (viral flu) is described. Oseltamivir phosphate is the ethyl ester prodrug of the corresponding acid, a potent and selective inhibitor of influenza neuraminidase. The discovery chemistry route and scalable routes used for kilo laboratory production as well as the technical access to oseltamivir phosphate from (-)-shikimic acid proceeding via a synthetically well-developed epoxide building block followed by azide transformations are reviewed. Synthesis and process research investigations towards azide-free conversions of the key epoxide building block to oseltamivir phosphate are discussed. The search for new routes to oseltamivir phosphate independent of shikimic acid including Diels-Alder approaches and transformations of aromatic rings employing a desymmetrization concept are presented in view of large-scale production requirements.

Application of α-keto acid decarboxylases in biotransformations

The advantages of using enzymes in the synthesis of organic compounds relate to their versatility, high reaction rates, and regio- and stereospecificity and the relatively mild reaction conditions involved. Stereospecificity is especially important in the synthesis of bioactive molecules, as only one of the enantiomeric forms usually manifests bioactivity, whereas the other is often toxic. Although enzymes which catalyze asymmetric carbon-carbon bond formation are of great importance in bioorganic chemistry, only a few examples are known for thiamin diphosphate (ThDP)-dependent enzymes, whereas transformations using e.g. aldolases, lipases and lyases are well documented already. The present review surveys recent work on the application of pyruvate decarboxylase and benzoylformate decarboxylase in organic synthesis. These enzymes catalyze the synthesis of chiral alpha-hydroxy ketones which are versatile building blocks for organic and pharmaceutical chemistry. Besides the substrate spectra of both enzymes amino acid residues relevant for substrate specificity and enantioselectivity of pyruvate decarboxylase have been investigated by site-directed mutagenesis.

New, Efficient Synthesis of Oseltamivir Phosphate (Tamiflu) via Enzymatic Desymmetrization of a meso-1,3-Cyclohexanedicarboxylic Acid Diester

A new, enantioselective synthesis of the influenza neuraminidase inhibitor prodrug oseltamivir phosphate 1 (Tamiflu) and its enantiomer ent-1 starting from cheap, commercially available 2,6-dimethoxyphenol 10 is described. The main features of this approach comprise the cis-hydrogenation of 5-(1-ethyl-propoxy)-4,6-dimethoxy-isophthalic acid diethyl ester (6a) and the desymmetrization of the resultant all-cis meso-diesters 7a and 7b, respectively. Enzymatic hydrolysis of the meso-diester 7b with pig liver esterase afforded the (S)-monoacid 8b, which was converted into cyclohexenol 17 via a Curtius degradation and a base-catalyzed decarboxylative elimination of the Boc-protected oxazolidinone 14. Introduction of the second amino function via S(N)2 substitution of the corresponding triflate 18 with NaN3 followed by azide reduction, N-acetylation, and Boc-deprotection gave oseltamivir phosphate 1 in a total of 10 steps and an overall yield of approximately 30%. The enantiomer ent-1 was similarly obtained via an enzymatic desymmetrization of meso-diester 7a with Aspergillus oryzae lipase, providing the (R)-monoacid ent-8a.

Asymmetric benzoin reaction catalyzed by benzoylformate decarboxylase

Abstract Aromatic aldehydes are converted into benzoins by benzoylformate decarboxylase catalyzed C–C bond formation. The reaction affords ( R )-benzoins with high enantiomeric excess and in good chemical yields. A broad range of aromatic aldehydes can be used as substrates in aqueous buffer or buffer/DMSO-solutions.

Identification of (S)-selective transaminases for the asymmetric synthesis of bulky chiral amines

The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.

Expanding the Imine Reductase Toolbox by Exploring the Bacterial Protein-Sequence Space.

Recent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C‐terminal domain clustering of the bacterial protein‐sequence space was performed to identify the novel IRED candidates. Each of the identified enzymes was characterized against a set of nine cyclic imine model substrates. A refined clustering towards putative active‐site residues was performed and was consistent both with our screening and previously reported results. Finally, preparative scale experiments on a 100 mg scale with two purified IREDs, IR_20 from Streptomyces tsukubaensis and IR_23 from Streptomyces vidiochromogenes, were carried out to provide (R)‐2‐methylpiperidine in 98 % ee (71 % yield) and (R)‐1‐methyl‐1,2,3,4‐tetrahydroisoquinoline in >98 % ee (82 % yield).

Opportunities and challenges for combining chemo- and biocatalysis

AbstractThe past decade has seen a substantial increase in successful examples of the combination of chemo- and biocatalysis for multistep syntheses. This is driven by obvious advantages such as higher yields, decreased costs, environmental benefits and high selectivity. On the downside, efforts must be undertaken to combine the divergent reaction conditions, reagent tolerance and solvent systems of these ‘different worlds of catalysis’. Owing to progress in enzyme discovery and engineering, as well as in the development of milder and more compatible conditions for operating with various chemocatalysts, many historical limitations can already be overcome. This Review highlights the opportunities available in the chemical space of combined syntheses using prominent examples, but also discusses the current challenges and emerging solutions, keeping in mind the fast progress in transition metal-, organo-, photo-, electro-, hetero- and biocatalysis.Chemical and biological catalysts provide distinct advantages and disadvantages to the synthetic chemist. This Review focuses on efforts to combine chemo- and biocatalysts, outlining the opportunities achievable by this approach and also efforts to overcome any incompatibilities between these different systems.

Asymmetric Reductive Amination of Ketones Catalyzed by Imine Reductases

Biocatalysis employing imine reductases is a promising approach for the one‐step generation of chiral amines from ketones. The enzymes reported for this process suffer from low activity and moderate stereoselectivity. We identified a set of enzymes that facilitate this reaction with high to quantitative conversions from a library of 28 imine reductases. This enabled the conversion of ketones with ammonia, methylamine, or butylamine into the corresponding amines. Most importantly, we performed preparative (>100 mg) scale syntheses of amines such as (1S,3R)‐N,3‐dimethylcyclohexylamine and (R)‐N‐methyl‐2‐aminohexane with excellent stereochemical purities (98 % de, 96 % ee) in good yields.

Protein-engineering of an amine transaminase for the stereoselective synthesis of a pharmaceutically relevant bicyclic amine

Application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones represents an environmentally benign and economically attractive alternative to transition metal catalyzed asymmetric synthesis. However, the restrictive substrate scope has limited the conversion typically to non-sterically demanding scaffolds. Recently, we reported on the identification and design of fold class I ATAs that effect a highly selective asymmetric synthesis of a set of chiral aromatic bulky amines from the corresponding ketone precursors in high yield. However, for the specific amine synthetic approach extension targeted here, the selective formation of an exo- vs. endo-isomer, these biocatalysts required additional refinement. The chosen substrate (exo-3-amino-8-aza-bicyclo[3.2.1]oct-8-yl-phenyl-methanone), apart from its pharmacological relevance, is a demanding target for ATAs as the bridged bicyclic ring provides substantial steric challenges. Protein engineering combining rational design and directed evolution enabled the identification of an ATA variant which catalyzes the specific synthesis of the target exo-amine with >99.5% selectivity.