The team has developed a novel computer-aided synthesis software called retrobiocat powered by a comprehensive biocatalyst database (Nature Catalysis, 2021, 4, 98-104; ACS Catal. 2023, 13, 11771-11780 ). The software and database has been developed as part of this project with the retrobiocat tool freely available online (retrobiocat.com).
This database has been used to develop enzyme cascades towards amino polyols and iminosugars, which are highly sought-after pharmaceutical targets, but their chemical synthesis is lengthy and can suffer from poor scalability and purification. Using retrobiocat for design of the cascades, we have developed efficient routes to amino diols and amino polyols (Chem. Commun., 2020, 56, 7949-7952;JACS Au 2022, 2, 10, 2251–2258). We have developed cell-free protocols for isolation of highly polar iminosugar products from biotransformations in a single step through development of a gradient-elution cation exchange purification. The two-step pathway provides a short synthetic route that can be used as a cell-free platform for broader iminosugar synthesis (ACS Cent. Sci. 2023, 9, 1, 103–108).
A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof-of-principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross-reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O-methylnorbelladine (Angew. Chem. Int. Ed. 2021, 60, 18660-18665).
The retrobiocat database has also highlighted gaps in our toolbox of biocatalysts. During the project we have addressed two of these. Firstly, the activation of C-H bonds in synthetic chemistry is not trivial and requires harsh conditions and often heavy metal catalysis. Here, the team employed a mild pathway to formal C-H bind activation by employing a P450 enzyme. Subsequent lactonization yielded lactone derivatives in high selectivity which have a range of application in flavours, fragrance and in pharmaceuticals (Angew. Chem. 2019, 131, 5724 –5727). This work was expanded to develop a family of biocatalysts for the regioselective mid- and end-chain C−H activation of fatty acids to generate hydroxy acids . Ancestral sequence reconstruction was successfully applied to the multidomain enzyme family of CYP116B cytochrome P450 monooxygenases to generate ancestor variants with distinct product and thermostability profiles.(Angew.Chem. IE, 2024, 63, e202314869). Secondly, biocatalytic amide bond formation was used effectively to selectively form commercially relevant surfactants (Angew.Chem. IE, 2022, 61, e202205054) in single steps with very high selectivity.
