Reaction product analysis by high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance: Application to the absolute configuration determination of naturally occurring polyyne alcohols

The absolute configuration of secondary hydroxy functions of seven natural occurring polyyne derivatives has been elucidated by the application of Mosher method of diastereomeric methoxy-2-trifluoromethyl-phenylacetyl (MTPA) ester formation. High-performance liquid chromatography with diode array detection (HPLC-DAD) of the reaction mixture using a water/acetonitrile gradient allowed monitoring the reaction progress. Coupling of high-performance liquid chromatography to solid-phase extraction combined with nuclear magnetic resonance (HPLC-SPE-NMR) was utilized to generate highly reproducible 1H and 19F NMR data needed as input for the absolute configuration determination based on the analysis of relative shift differences. Chromatographic peaks of reaction substrates and reaction products bearing less 10 μg analyte were trapped on SPE cartridges with the aid of water as makeup solvent. Deuterated chloroform was used to elute and transfer the peak content from the SPE to the 60 μl flow cell of a 500 MHz NMR spectrometer. For each analyte 1H NMR spectra were obtained within 15 min. Additionally 19F NMR spectra were recorded for selected analytes in the same timeframe. Based on the obtained NMR data, the absolute configuration of all polyynes under investigation was successfully designated.