Steven Paul Hanlon

The use of enzymes in organic synthesis and the life sciences: perspectives from the Swiss Industrial Biocatalysis Consortium (SIBC)

The potential of biotechnology by means of biocatalysis or biosynthesis in organic synthesis is far from being fully exploited. For this reason a group of life science companies active in pharmaceuticals, flavour and fragrance, vitamin and fine chemicals businesses describe some examples of the use of enzymes in industrial organic synthesis and discuss why enzymes are still the exception rather than the rule in organic synthesis.

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).

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.

Metabolite Identification via LC-SPE-NMR-MS of the In vitro Biooxidation Products of a Lead mGlu5 Allosteric Antagonist and Impact on the Improvement of Metabolic Stability in the Series

Detailed information on the metabolic fate of lead compounds can be a powerful tool for an informed approach to the stabilization of metabolically labile compounds in the lead optimization phase. The combination of high performance liquid chromatography (HPLC) with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) has been used to give comprehensive structural data on metabolites of novel drugs in development. Recently, increased automation and the embedding of on‐line solid‐phase extraction (SPE) into a integrated LC‐SPE‐NMR‐MS system have improved enormously the detection limits of this approach. The new technology platform allows the analysis of complex mixtures from microsome incubations, combining low material requirements with relatively high throughput. Such characteristics make it possible to thoroughly characterize metabolites of selected compounds at earlier phases along the path to lead identification and clinical candidate selection, thus providing outstanding guidance in the process of eliminating undesired metabolism and detecting active or potentially toxic metabolites. Such an approach was applied at the lead identification stage of a backup program on metabotropic glutamate receptor 5 (mGlu5) allosteric inhibition. The major metabolites of a lead 5‐aminothiazole‐4‐carboxylic acid amide 1 were synthesized and screened, revealing significant in vitro activity and possible involvement in the overall pharmacodynamic behavior of 1. The information collected on the metabolism of the highly active compound 1 was pivotal to the synthesis of related compounds with improved microsomal stability.

The Organic–Synthetic Potential of Recombinant Ene Reductases: Substrate‐Scope Evaluation and Process Optimization

In this study an evaluation of the synthetic potential of a broad range of recombinant ene reductases was performed. In detail, a library of 23 ene reductases was used to screen the CC reduction of 21 activated alkenes from different compound classes as substrates. The chosen set of substrates comprises nitroalkenes with an aryl substituent at the β‐position and a methyl substituent at the α‐ or β‐position, α,β‐unsaturated carboxylic acids and their esters with and without substituents at the β‐position, a range of cyclic α,β‐unsaturated ketones with different ring sizes and substitution patterns and one α,β‐unsaturated boronic acid. After we obtained insight into the substrate scope, several biotransformations were prioritised and further investigated in a screening of 41 reaction parameters (which included chaotropic and kosmotropic salts, polyols, buffer solutions, amino acids and organic solvents) towards their impact on the activity and enantioselectivity of the applied ene reductases. Under the optimised conditions, selected reduction processes were performed on an increased lab scale (up to 30 mL) with up to 10 % substrate concentration, which led in general to both high conversion and (if chiral products were formed) enantioselectivity.

Amine Transaminase Engineering for Spatially Bulky Substrate Acceptance

Amine transaminase (ATA) catalyzing stereoselective amination of prochiral ketones is an attractive alternative to transition metal catalysis. As wild‐type ATAs do not accept sterically hindered ketones, efforts to widen the substrate scope to more challenging targets are of general interest. We recently designed ATAs to accept aromatic and thus planar bulky amines, with a sequence‐based motif that supports the identification of novel enzymes. However, these variants were not active against 2,2‐dimethyl‐1‐phenyl‐propan‐1‐one, which carries a bulky tert‐butyl substituent adjacent to the carbonyl function. Here, we report a solution for this type of substrate. The evolved ATAs perform asymmetric synthesis of the respective R amine with high conversions by using either alanine or isopropylamine as amine donor.

Human xanthine oxidase recombinant in E. coli: A whole cell catalyst for preparative drug metabolite synthesis.

Human xanthine oxidoreductase (XOR), which is responsible for the final steps of the purine metabolism pathway and involved in oxidative drug metabolism, was successfully expressed in Escherichia coli BL21(DE3) Gold. Recombinant human (rh) XOR yielded higher productivity with the gene sequence optimized for expression in E.coli than with the native gene sequence. Induction of XOR expression with lactose or IPTG resulted in complete loss of activity whereas shake flasks cultures using media rather poor in nutrients resulted in functional XOR expression in the stationary phase. LB medium was used for a 25L fermentation in fed-batch mode, which led to a 5 fold increase of the enzyme productivity when compared to cultivation in shake flasks. Quinazoline was used as a substrate on the semi-preparative scale using an optimized whole cell biotransformation protocol, yielding 73mg of the isolated product, 4-quinazolinone, from 104mg of starting material.

Human Enzymes for Organic Synthesis

Abstract Human enzymes have been widely studied in various disciplines. The number of reactions taking place in the human body is vast, and so is the number of potential catalysts for synthesis. Herein, we focus on the application of human enzymes that catalyze chemical reactions in course of the metabolism of drugs and xenobiotics. Some of these reactions have been explored on the preparative scale. The major field of application of human enzymes is currently drug development, where they are applied for the synthesis of drug metabolites.