Novel Synthesis Methods For the Production of Human Circulating Metabolites of Natural Products

Doyle, Bradley

Abstract

Upon entering the body, any given xenobiotic can undergo metabolism which facilitates its excretion from the body. When metabolised, a compound typically has the same effects of the initial drug/nutraceutical, however this is not always the case and can significantly differ. The determination of these beneficial and toxicological effects is vital to allow for effective drug development and to increase current knowledge of nutraceuticals. This is because contradictory knowledge of their pharmacological effects is often found. Currently there is no method available that allows for the synthesis of these compounds in useable quantities (mg). This study aimed to provide a method that can be used to simply synthesise these metabolic products in sufficient amounts for further testing.
Three different enzymes families (UGT1a1, SULT1a1 and CYP1a1) were immobilised via a silanization followed by a glutaraldehyde functionalization and tested. These were compared to a variety of different controls being excluding co-factor or enzyme from the system or immobilising an alternative unreactive enzyme towards the substrate. In each of the chapters it was determined that metabolite formation was only observed when both the correct enzyme and co-factor was available within the system. The true run for each enzyme was optimised at two different parameters: flow rate and temperature. For all three of the enzymes used the optimal temperature depicted in their recommended instructions was 37 °C.
The UDP-glucuronosyl transferase immobilised device showed no significant difference at any of the three tested flow rates. However, temperature showed a significant difference oppositely to expected in which 37 °C yielded almost no product at all (97.9 ± 38.5 μM substrate remaining), and both room temperature and 30 °C yielded significant conversion (11.0 ± 8.0 μM and 0.0 ± 0.0 μM remaining respectively).
The Sulfotransferase immobilised device also showed no significant difference between any of the three tested flow rates. Temperature also yielded the contrary results to that which was expected and almost no product at all was formed (81.3 ± 21.8 μM substrate remaining) and both room temperature and 30 °C yielded significant conversion (92.9 ± 7.2 and 92.2 ± 10.2 μM remaining respectively).
The cytochrome P450 based device showed no significant difference between any of the three tested flow rates, the further parameters were not tested due to fluorescence interference issues and further testing is needed.
The UGT and SULT devices were then compared to directly incubating both the substrate and co-factor with the enzyme. A 2-hour period for both methods yielded comparable results (0.22 ng in static conditions and 0.24 ng in flow conditions) but the formation of a complex biological matrix is not formed. Alongside this allowing the reaction to occur over a longer period of time (4 hours) the immobilised enzyme reactor continued to yield product in which the incubation method plateaued; leading to significantly higher metabolite formation (0.2 ng in static conditions and 0.47 ng in batch conditions). This data was not observed in the case of the CYP device due to a fluorescence interference observed in the effluent of the device preventing comparable measurements.
With further optimisation or scaling up of these devices they will likely be viable for the synthesis of sufficient quantities of metabolite to allow for pharmacological testing, with an improvement on the currently available methods by bypassing the necessary complex separation, high costs and commonly observed low yields.

Citation

Doyle, B. (2023). Novel Synthesis Methods For the Production of Human Circulating Metabolites of Natural Products. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4587910

• Thesis Type: Thesis
• Deposit Date: Mar 12, 2024
• Publicly Available Date: Mar 12, 2024
• Keywords: Chemistry
• Public URL: https://hull-repository.worktribe.com/output/4587910
• Additional Information: Department of Chemistry; University of Hull
• Award Date: Feb 21, 2024