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.