Ligand-observed in-tube NMR in natural products research: A review on enzymatic biotransformations,protein–ligand interactions,and in-cell NMR spectroscopy

Natural product-observed NMR methods have considerably expanded the potentialities for in-tube NMR monitoring of complex enzymatic biotransformations and investigation of protein-natural product interactions even in living cells. We review, herein, the significant advantages of ligand-observed in-situ NMR monitoring of enzymatic biotransformations without restoring to laborious and time-consuming chromatographic methods. Emphasis will be given to the potentialities of the use of the NMR bioreactor: (i) to investigate through saturation transfer difference (STD), the capacity of natural products to serve as enzyme substrates, (ii) to monitor multiple biotransformation products of natural products with the use of immobilized enzymes and (iii) to investigate interactions of biotransformed products with protein targets. The use of STD and its variants, transfer effect Noes for PHArmacophore Mapping (INPHARMA) NMR, in conjunction with computational methods, can provide excellent tools in investigating competitive binding modes even in proteins with multiple binding sites. The method has been successfully applied in the study of unsaturated free fatty acids (UFFAs)-serum albumin complexes in which the location and conformational states of UFFAs could not be determined accurately, despite numerous X-ray structural studies, due to conformational averaging. This combined method, thus, may find promising applications in the field of protein-natural product recognition research. The emerging concept of in-cell NMR and recent applications will be discussed since they can provide atomic level insights into natural product-protein interactions in living cells without the need of isotope labelled techniques.