Preparative Scale Achmatowicz and aza-Achmatowicz Rearrangements Catalyzed by Agrocybe aegerita Unspecific Peroxygenase

The unspecific peroxygenase (UPO) from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is an effective and practical biocatalyst for the oxidative expansion of furfuryl alcohols/amines on a preparative scale, using the Achmatowicz and aza-Achmatowicz reaction. The high activity and stability of the enzyme, which can be produced on large scale as an air-stable lyophilised powder, renders it a versatile and scalable biocatalyst for the preparation of synthetically valuable 6-hydroxypyranones and dihydropiperidinones. In several cases, the biotransformation out-performed the analogous chemo-catalysed process, and operates under milder and greener reaction conditions

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate

Imine Reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines are used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equivalents of three larger amines, in isolated yields of up to 93%

Catalytic Promiscuity of Unspecific Peroxygenases

This thesis describes multiple synthetic applications of the recombinantly expressed (in Pichia pastoris host) Agrocybe aegerita unspecific peroxygenase (rAaeUPO). Chapter One gives a description of the existing enzymatic and chemocatalytic methodologies, which allow the direct oxidation of non-activated sp3 C-H positions. In Chapter Two, the optimal operating conditions of rAaeUPO-catalysed reactions were studied, including the effect of substrate concentration, temperature, mode of H2O2 delivery and enzyme concentration. Building on these benchmarking studies, the oxidation of a variety of N�heterocycles were optimised for improved yields, mostly focusing on analogues of pyridine and tetrahydroquinolines. N-heterocyclic benzylic alcohols were generally yielded with high enantioselectivities. The optimised conditions also allowed the transformations to be performed on synthetically useful scales (few hundred milligrams). In Chapter Three, the potential carbene and nitrene insertion activities of rAaeUPO were explored. In our hands, this enzyme failed to support any of these transformations under the conditions employed. The possible reasons of this are described and discussed. Chapter Four describes the application of rAaeUPO for catalysing the oxidation of furan analogues. Direct oxidation of alkyl-substituted furans, followed by the Achmatowicz reaction of the product formed was not possible under the conditions used. On the other hand, the rAaeUPO-mediated oxidative ring expansion of a wide range of α-furylcarbinols and α�furylamine analogues was possible with high yields. The enzyme was also shown to tolerate high substrate concentrations, thereby making the approach easy to scale. In Chapter Five, the oxidation of ethylbenzenes, toluenes and the corresponding secondary and primary alcohols was studied first to understand the electronic, steric, and energetic factors influencing these oxidations. Whilst rAaeUPO is known to mediate benzylic oxidation with high enantioselectivity, the enzyme did not discriminate strongly between the secondary alcohol enantiomers. The Chapter also describes the oxidation of more complex benzylic and allylic aliphatic alcohols

Selective Oxidations of Toluenes and Benzyl Alcohols by an Unspecific Peroxygenase (UPO)

Unspecific Peroxygenases (UPOs) have emerged as robust biocatalysts for selective oxygenation reactions, as they are easily produced at scale and require only hydrogen peroxide as the external oxidant. UPOs can catalyze the oxygenation of the primary benzylic carbons of toluenes to give alcohol, aldehyde and carboxylic acid products. They can also catalyze hydroxylation at the benzylic position of ethylbenzenes, and the subsequent oxidation of the secondary alcohols to ketones. In this study, we have investigated factors that affect the balance of products in UPO-catalyzed benzylic oxygenations using a range of functionalised toluenes and ethyl benzenes, and a UPO from Agrocybe aegerita (rAaeUPO-PaDa-I-H variant). The product distribution is dependent upon a mixture of steric and electronic effects and, in selected cases, controlling the reaction conditions permits products from each product series to be generated chemoselectively. In this way, electron poor toluenes were converted directly into carboxylic acids in isolated yields of 36–99% on preparative scale

Preparative-Scale Biocatalytic Oxygenation of N-Heterocycles with a Lyophilized Peroxygenase Catalyst

A lyophilized preparation of an unspecific peroxygenase variant from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is a highly effective catalyst for the oxygenation of a diverse range of Nheterocyclic compounds. Scalable biocatalytic oxygenations (27 preparative examples, ≈100 mgs scale) have been developed across a wide range of substrates, including alkyl pyridines, bicyclic Nheterocycles and indoles. H2O2 is the only stoichiometric oxidant needed, without auxiliary electron transport proteins, which is key to the practicality of the method. Reaction outcomes can be altered depending on whether hydrogen peroxide was delivered by syringe pump or through in situ generation using an alcohol oxidase from Pichia pastoris (PpAOX) and methanol as a co-substrate. Good synthetic yields (up to 84%), regioselectivity and enantioselectivity (up to 99% ee) were observed in some cases, highlighting the promise of UPOs as practical, versatile and scalable oxygenation biocatalysts

Divergent Cascade Ring Expansion Reactions of Acryloyl Imides

Macrocyclic and medium-sized ring ketones, lactones and lactams can all be made from common acryloyl imide starting materials via divergent, one-pot cascade ring expansion reactions. Following either conjugate addition with an amine or nitromethane, or osmium(VIII) catalysed dihydoxylation, rearrangement via a 4-atom ring expansion takes place spontaneously to form the ring expanded products. A second ring expansion can also be performed following a second iteration of imide formation and alkene functionalisation/ring expansion. In the dihydroxylation series, 3- or 4-atom ring expansion can be performed selectively, depending on whether the reaction is under kinetic or thermodynamic control