Application of gradient-enhanced nuclear Overhauser effect spectroscopy (GOESY) in the structure elucidation of plant secondary metabolites

Nuclear Overhauser effect (nOe) enhancement plays a vital role in structure determination, especially conformational analysis, by NMR (Neuhaus and Williamson, 1989). A number of one- and two-dimensional NMR methods, notably, nOe-difference spectroscopy (Sanders and Mersh, 1982), NOESY (States et al., 1982; Bodenhausen et al., 1984; Neuhaus and Williamson, 1989) and ROESY (Bothner-By et al., 1984; Bax and Davies, 1985; Neuhaus and Williamson, 1989) Hwang and Shaka, 1992; Hwang et al., 1992) have routinely been used to determine this effect. However, NOESY and ROESY experiments can be quite time-consuming, depending on mixing times (Braun et al., 1996), and in nOe-difference experiments imperfect subtraction often gives rise to a subtraction artefact which makes it difficult to visualise the nOe enhancements (Stonehouse et al., 1994). Therefore there has always been a pressing need for a modified, reliable and quicker nOe experiment. With the advent of gradient technology and subsequent advancement in this field, the use of gradients to take advantage of specific coherence selection in several NMR experiments is now becoming more and more popular (Hurd, 1990). As a result, a number of gradientenhanced versions of many useful high-resolution oneand multi-dimensional homonuclear and heteronuclear NMR experiments have been developed (Hurd, 1990; Hurd and John, 1991; Rinaldi and Keifer, 1994; Bernassau and Nuzillard, 1994; Parella et al., 1995; Mackin and Shaka, 1996; Wagner and Berger, 1996; Uzawa and Koshino, 1996). The major advantages that result from gradient selection of coherence are the elimination of the need for phase cycling and a reduction in coherent t1 artefacts (Hurd and John, 1991). Using this gradient technology a new method of measuring transient nOe enhancements, which avoids the need to compute difference spectra and thus gives spectra containing no subtraction artefact, has recently been developed and the acronym GOESY (gradient enhanced nuclear Overhauser effect spectroscopy) has been assigned for this experiment (Stonehouse et al., 1994; Stott et al., 1995). Since its introduction, to our knowledge, this method has never been applied to the structural determination of plant secondary metabolites. However, GOESY together with ROESY techniques have recently been employed successfully to assign 1H NMR chemical shifts for ampicillin (Tung et al., 2000). We now report on the application of GOESY in the structure determination of five compounds, namely eucalyptin (1), arctigenin (2), 5-geranyloxy-7-methoxycoumarin (3), 2,6-dihydroxy-4-methoxyisovalerophenone (4) and N-feruloyltyramine (5), isolated from plants, and we also evaluate the future of this experiment as a routine method.