Natural product generation and diversification through blending synthetic biology and synthetic chemistry

Abstract

Antimicrobial resistance is a modern threat to our society, with more than 33000 cases of deaths reported in Europe in 2015 relating to human infection by antibiotic resistant bacteria. Since 1981, 70% of approved antibacterial agents have been obtained, derived, or inspired from natural products. The generation of natural product analogues can be very challenging, but can lead to improved bioactivity, bioavailability, and also help understanding the mechanism and interaction between the natural product antibiotic and the bacterial target.

By blending synthetic chemistry with synthetic biology, we can use the best of both worlds and access more efficiently the desired analogues. Synthetic biology approaches can be used to engineer a chemically reactive and orthogonal handle such as an epoxide or halogen into a molecule, providing a site for selective synthetic diversification. Using synthesis, precursor-directed biosynthesis and GenoChemetics, a new approach pioneered by the Goss group, I worked toward accessing analogues of 4 natural products.

Chapter 2 describes GenoChemetic studies on biosynthetic generation of new-to-nature brominated violacein analogues using a precursor-directed biosynthesis approach. This was achieved by heterologous expression of violacein biosynthetic gene cluster in E. coli; then feeding these cultures with brominated tryptophan analogues, leading to the production of 3 new brominated violacein analogues. Although all efforts for purification of the complex mixtures could not afford any pure bromo-violacein for NMR characterisation, a detailed LC-MS and MS-MS analysis allowed confirmation of the identity of these new natural products.

The mixture was used in chapter 3 to generate a further 29 analogues via Suzuki- Miyaura cross-coupling reactions. These products could be characterised by LC- MS. To complement the work presented, a total synthesis of one of the brominated analogues was achieved, NMR and LC-MS characterised, to compare with the biosynthetically generated bromo-violaceins. The synthetic material was used to generate 4 of the previously mentioned cross-coupling analogues and characterised to confirm the assignments made in chapter 3. These methods can be useful to generate libraries of violacein analogues using this GenoChemetic approach.

Chapter 4 details identification of cyclo-(Phe-Pro) diketopiperazine (DKP) natural product (isolated from microbial cultures in our labs) and chemical synthesis of various diastereomers to elucidate the stereochemistry of this natural product. Cyclo-(Phe-Pro) was investigated via a fully synthetic approach to generate analogues, comparing these analogues with naturally obtained samples. All 4 stereoisomers were generated, and 2 brominated analogues were also synthesised and characterised to test the robustness of the method. The antibiotic bioassays carried out were inconclusive. Relative stability and interconversion of diastereoisomers were also studied. This study provides a foundation for synthetic access to derivatives of DKP natural products.

In chapter 5, other natural non-ribosomal peptides, telomycin and kutznerides, were selected for analogue generation. Precursor-directed biosynthesis was employed to generate brominated analogues of both natural products.