Enriching the biological space of natural products and charting drug metabolites, through real time biotransformation monitoring: The NMR tube bioreactor

Background: Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps. Methods: Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets. Results: Herein, we developed a simple and time-effective process, the NMR-tube bioreactor, that is able to: (i) predict which component of a mixture of natural products can be enzymatically transformed, (ii) monitor in situ the transformation efficacy and regioselectivity in crude extracts and multiple substrate biotransformations without fractionation and (iii) simultaneously screen for interactions of the biotransformation products with pharmaceutical protein targets. Conclusions: We have developed a green, time-, and cost-effective process that provide a simple route from natural products to lead compounds for drug discovery. General significanse: This process can Speed up the most crucial steps in the early drug discovery process, and reduce the chemical manipulations usually involved in the pipeline, improving the environmental compatibility.