Expanding the GenoChemetic toolbox : radical halogenases and aqueous alkyl halide cross-coupling

Abstract

Natural products are a very important class of bioactive and often medicinally relevant molecules, however strategies for their chemical diversification are often hindered by their complexity and high number of unactivated aliphatic carbons. The GenoChemetic strategy represents a powerful tool for natural product diversification, merging the biosynthetic production of reactive metabolites with mild aqueous chemistries to enable modification in biological conditions. The merger of biosynthetic production of aliphatically halogenated compounds by radical halogenases with aqueous alkyl halide coupling chemistries remains an unexploited but promising strategy for enabling functionalisation at C(sp³)-H centres in this way.

This PhD thesis focuses on the stepwise process to expand the GenoChemetic method of natural product diversification through biocatalytic halogenation from the sp² realm to the sp³ realm. To this end in silico techniques have been used to mine novel radical halogenase from genomic libraries to discover five novel radical halogenases from cyanobacteria, myxobacteria, and bacteroidetes. Furthermore, one vanadium dependent halogenase was discovered from algae which produce a distinct halo-metabolite signature when virally infected. These novel enzymes have been examined in a phylogenetic, structural, and genomic context, and recombinantly produced in Escherichia coli. Halogenation assays were designed, and a wide range of substrates used to probe these novel proteins for halogenation activity.

Methods for the aqueous coupling of sp³ alkyl halides have also been explored. By screening catalysts and ligands, it has been shown that that the water-soluble palladium source Na₂PdCl₄ can be used in conjunction with a tris(2,4-dimethyl-5-sulfophenyl)phosphine trisodium salt (TXPTS) ligand to enable the first fully aqueous, air tolerant, and mild Suzuki-Miyaura coupling. This aqueous coupling was able to derivatise a wide range of functionalised hydrophilic and hydrophobic primary alkyl halides. It was also used to tag two halogenated natural products with fluorescent and ionisable groups in a method for natural product analysis and analogue generation.