The cis¬-dihydrodiol compounds, produced by the highly regio- and stereo-selective Rieske non-heme iron oxygenases, such as toluene dioxygenase (TDO), are versatile synthons for chiral, asymmetric synthesis. The cis-dihydroxylation of ortho- and meta-substituted phenols by TDO produce cyclohexenone cis-diols. The keto group of cyclohexenone cis¬-diol tautomers increases the functionalisation and stability compared to other arene cis-dihydrodiols. (4S,5S)-dihydroxy-3-iodocyclohexe-2-en-1-one, formed from 3-iodophenol by the TDO-expressing bacteria Pseudomonas putida UV4 and Escherichia coli LK, has been identified as a highly versatile synthon for chemoenzymatic synthesis. Earlier attempts at the production of significant quantities of this compound had been unsuccessful, due to its biodegradation by P. putida UV4. E. coli LK accumulated the target compound but struggled with substrate toxicity and formation of toxic catechol metabolites, which inhibit toluene dioxygenase.
The project aimed to optimise the biotransformation of 3-iodophenol by P. putida UV4 for the production of preparative amounts of (4S,5S)-dihydroxy-3-iodocyclohex-2-en-1-one, eventually producing the compound in multigram quantities (~15 g). Additionally, catechol-2,3-dioxygenase (todE) of P. putida UV4 was introduced to E. coli LK, creating a strain able to degrade catechols. This new strain carries the todE gene on a Lac operon regulated pMCL210 plasmid and was designated E. coli 1/210LK. After optimisation of the biotransformation, it showed an increase in the concentration of (4S,5S)-dihydroxy-3-iodocyclohex-2-en-1-one of ~40% compared to E. coli LK, thus competing with P. putida UV4 but at a lower reaction rate.
Using AutoDock (Vina), an analysis of the active site of TDO and its substrate interactions provided a rationale for earlier results and predicted new synthetically useful tricyclic cis-dihydrodiol metabolites. Molecular docking showed His311 and Gln215 as primary interaction partners for the orientation of phenols within the active site. Hydrogen bonding between the phenolic OH group and the side chain of Gln215 was highlighted as a possible reason for the formation of catechols via 1,2-dihydroxylation of phenols.
Several tricyclic arenespreviously found to be acceptable substrates for naphthalene dioxygenase were also successfully docked to TDO. Dibenzofuran, dibenzothiophene and carbazole were biotransformed by P. putida UV4, fluorene was not. The dibenzofuran cis-dihydrodiol was produced in high yields (>60%).
This project produced an improved biocatalyst, E. coli 1/210LK, for the production of cyclohexenone cis-diols, serving as basis for future research. Molecular docking has given insight into the active site and its substrate interactions, paving the way for further analysis. Molecular docking also predicted the formation of dibenzofuran ¬cis-dihydrodiol, which was applied in the total synthesis of ribisins A-C by Dr. C. McGivern.
|Date of Award||2019|
- Queen's University Belfast
|Supervisor||Leonid Kulakov (Supervisor) & Christopher Allen (Supervisor)|
- toluene dioxygenase
- molecular docking