Abstract
The Diels-Alder reaction is an important synthetic transformation involving the [4 + 2]-cycloaddition of a diene and dienophile to construct cyclohexene rings with up to four new
stereocentres. This reaction has been extensively applied in the total synthesis of complex natural
product scaffolds but achieving high selectivity under mild conditions is often challenging. This
thesis describes investigations on Diels-Alderase enzymes from tetronate biosynthetic pathways
to probe enzyme selectivity and explore their potential use as biocatalysts.
Chapter 2 focuses on TedJ, a decalin
synthase from the biosynthetic pathway
of tetrodecamycin. Previous work
demonstrated TedJ catalyses a
stereoselective [4 + 2]-cycloaddition and
this transformation was exploited in the
chemoenzymatic synthesis of antibiotic
(–)-13-deoxytetrodecamycin. Herein, we
explore TedJ substrate tolerance by
modifying substituent pattern and
carbon backbone chain length. Four Omethylated tetronate analogues were
synthesised (92, 93, 96 and 97) and a combination of TedJ in vitro assays, computational
studies and scale-ups using immobilised enzyme showed that TedJ can accept unnatural
substrates but in some cases cannot override inherent unfavourable interactions. This work
culminated in the production of three novel cycloadducts (125, 132 and 142) which will be used
in future chemoenzymatic syntheses of tetrodecamycin analogues.
Chapter 3 focuses on putative Diels-Alderase QmnH, proposed to catalyse an unusual
intermolecular Diels-Alder reaction cascade in antiviral complex quartromicin biosynthesis.
Bioinformatic analysis and AlphaFold modelling indicate QmnH is a heterodimeric protein with
two β-barrel domains, each suggested to catalyse one set of intermolecular Diels-Alder reactions.
We synthesised proposed natural substrate analogues (216 and 217) and cloned constructs of
variant QmnH* and separated N- and C- terminal domains to use in biochemical assays.
However, the QmnH constructs were recalcitrant to all expression conditions tested including
various E. coli cell lines and different induction techniques, possibly because of incorrect disulfide
bond formation. Future directions including alternative heterogeneous hosts or gene cluster
knock-out studies are discussed.
| Date of Award | 9 Dec 2025 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Chris L Willis (Supervisor) & Marc W Van der Kamp (Supervisor) |
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