Amide bond formation is one of the most fundamental chemical transformations utilised in the generation of bioactive molecules, with amide bond forming reactions accounting for an appreciable proportion of all reactions carried out in medicinal chemistry laboratories. Despite widespread usage, traditional synthetic approaches towards amide bond synthesis present with a number of drawbacks in terms of selectivity, efficiency and environmental outlook. Enzymatic approaches towards amide bond synthesis have the potential to address many of these issues, and as such investigations into the use of biocatalysts for amide bond formation are beginning to receive significant attention. An amide bond forming enzyme known as CfaL is involved in amide bond formation during the production of the phytotoxic secondary metabolite coronatine in several pathovars of Psuedomonas syringae. Herein, we have biochemically characterised the activity of CfaL and have identified a number of recombinant CfaL homologues from bioinformatics searches. We have engineered CfaL variants with improved biocatalytic properties based on a crystal structure solved to a resolution of 2.10 Ã
, and have carried out a thorough investigation into the substrate scope tolerance and biocatalytic potential of a number of different CfaL enzymes. We have found the wild type CfaL enzymes to be accepting of a remarkably wide range of acyl donor and nucleophile substrates, and have found CfaL catalysed amide bond formation to be both selective and scalable. Beyond rational structure guided mutagenesis, we have attempted to broaden the substrate scope of the CfaL enzymes through the exchange of structural units of the CfaL enzymes with other structurally analogous enzymes such as McbA from Marinactinospora thermotolerans. We have successfully managed to generate a chimeric CfaL enzyme with an altered substrate preference through such structural unit exchange methodologies. We have also attempted to expand the biocatalytic potential of the CfaL enzymes through their incorporation into tandem catalytic processes. In doing so, we have established a biocatlytic cascade capable of furnishing amide products from an un-functionalised styrene starting material. This cascade utilises the Cfal enzymes in tandem with a decarboxylase enzyme known as AnFDC, and proceeds through the generation of a trans-cinnamic acid intermediate, which is subsequently reacted with L-isoleucine in a CfaL catalysed amide bond formation. Due to the serious global impact of the SARS-CoV-2 pandemic, a part of my PhD studies have also been focused on the development of bisubstrate inhibitors aimed at the SARSCoV-2 NSP10/NSP16 MTase complex. The work conducted throughout this largely distinct project is discussed in chapter 4, and concerns synthesis efforts aimed at the generation of aziridine-SAM analogues. It is intended that these aziridine-SAM analogues will eventually be tethered to RNA molecules using the SARS-CoV-2 NSP10/NSP16 MTase complex, in an effort to generate potent and selective viral MTAse inhibitors for eventual clinical development.
Enzymatic Approaches to Amide Bond Synthesis
Rowlinson, M. (Author). 1 Aug 2023
Student thesis: Phd