HPLC-NMR ONLINE COUPLING INCLUDING THE ROESY TECHNIQUE - DIRECT CHARACTERIZATION OF NAPHTHYLISOQUINOLINE ALKALOIDS IN CRUDE PLANT-EXTRACTS

Directly coupled 600-MHz HPLC-H-1 MMR was used for the first time to i nvestigate the potential of this technique in the screening of tropica l plants for naphthylisoquinoline alkaloids. A leaf extract of Ancistr ocladus guineensis was analyzed both in the on-now mode and in the sto p-flow mode using reversed-phase isocratic and gradient compositions o f acetonitrile and D2O on a C-18 column. On-now experiments proved the occurrence of naphthylisoquinoline alkaloids in the extract. Stop-now measurements confirmed the presence of ancistrotectorine and two new diastereomeric alkaloids. With 2D TOCSY experiments performed in the s top-flow mode, it was possible to deduce their constitution, For the e lucidation of the relative configuration, a 2D HPLC-NMR ROESY experime nt was developed by supplying a conventional ROESY sequence with a dou ble presaturation block during the relaxation delay. Application of th e optimized ROESY method confirmed the constitution of the two new alk aloids and showed relative cis and trans configurations of the methyl groups at the isoquinoline moieties. These results underline the value of this approach for a rapid and reliable phytochemical screening of plant extracts for naphthylisoquinoline alkaloids. The identification of known alkaloids as well as the characterization of new structures i s possible without any purification step. The results were used as the basic information for the selective isolation of the new structures.