Final Report Summary - DOSYMNPS (Novel applications of diffusion NMR spectroscopy in microbial natural products research) Effective dereplication (identification of already known compounds) is essential for the investigation of complex natural mixtures to avoid unnecessary and time-demanding isolation/purification work on already well-studied natural products. Nowadays, thanks to new NMR techniques, it is not always necessary to separate the components of a mixture in order to obtain spectroscopic information from its constituents. This project has contributed to this field developing new diffusion NMR spectroscopy approaches of remarkable application in microbial natural products research.Diffusion NMR spectroscopy constitutes a powerful tool for mixture analysis in which the spectra of the components can be virtually separated according to their apparent diffusion coefficients (dependent on molecular size). In spite of its potential to characterize mixtures by NMR without the need to separate the components, this approach is clearly still undervalued in the field of natural products chemistry.Using designed artificial mixtures of secondary metabolites a proper methodology to reconstruct each component spectrum from the diffusion NMR data has been implemented. Pulse sequence acquisition parameters have been optimized. Raw data have been processed by well established exponential DOSY (Diffusion Ordered Spectroscopy) fitting or direct exponential curve resolution (DECRA) algorithms. Additionally a novel methodology to process diffusion NMR data has been developed complementing the results obtained by DOSY and DECRA processing. A new protocol based on the comparative analysis of the processed data obtained with the different algorithm methods has been employed to identify each component signals. Such identification combined with line fitting spectral deconvolution routines has rendered synthetic component spectra with close resemblance to the actual pure compound spectra. To prove the power of diffusion NMR as dereplication tool, the reconstructed synthetic components spectra or the information contained in them have been used as query in different databases including: an in-house experimental NMR spectral library, two databases of structures containing experimental or calculated chemical shifts and a natural products database which uses as query fields structural features easily recognizable in NMR spectra. This dereplication workflow has been significantly enhanced by incorporating the information obtained from complementary HSQC spectra of the mixtures and the putative molecular formulae of the mixture components determined from additional LC-HRMS analyses. Since diffusion NMR allows an estimation of the molecular weight of the components, a protocol has been developed to correlate the exact mass (and putative molecular formulae) with the estimated molecular weight from diffusion experiments for dereplication purposes. The new methodology works well with simple mixtures. The study of its application to real life samples of microbial origin has revealed that in general crude extract and rough fractions obtained by reversed-phase flash chromatrogaphy are too complex and challenging for deconvolution of their composition by diffusion NMR. However, the developed methodology appears suitable to less complex fractions mixtures obtained by semipreparative HPLC, which typically contain just a few components generally under non equimolar ratio. This strategy has been incorporated to the dereplication workflow followed at the host institution (Fundación MEDINA). The new methodologies developed are expected to find spread use among researchers in natural products chemistry.
