C2

70 60 50

Figure 5.11 (a) The 13C NMR spectrum of VBA, with the carbon atom numbering. Unnumbered lines are solvent or impurities, (b) The spectrum after a labelling experiment with [1,3-13C2]glycerol. Labelling of atoms 2 and 7 (less than that of 4 and 5) was caused by degradation of glycerol to acetate and incorporation of that, (c) Part of the spectrum showing coupling between adjacent13C atoms that shows that the fi-ketoester was incorporated intact (Adapted from S. Sakuda and Y. Yamada, Comprehensive Natural Products Chemistry, Vol. I, p. 139. Pergamon, Oxford 1999)

atoms 2 and 7 (4.5 and 3.7% respectively). This was attributed to breakdown of glycerol to acetate and incorporation of this into VBA. Two adjacent atoms, as in the (3-ketoester, gives, on incorporation intact, 13C-,3C coupling, and in the NMR spectrum of the resulting VBA this coupling could be seen, with the doublets superimposed on the unenriched singlets (Figure 5.11 (c)). The researchers even went on to show that the glycerol was incorporated as dihydroxyacetone, and other finer points.

5.3.2 Carpophilus Beetle Pheromone

The biosynthesis of the branched acetogenin (2£,4£,6is)-5-ethyl-3-methyl-2,4,6-nonatriene from Carpophilus freemani beetles (Chapter 4 and Figure 4.11) provides another good example of deuterium labelling. Biosynthesis of this compound was studied initially by mass spectrometry, and required deuterated acetic, propionic and butyric acids, as well as synthetic model compounds deuterated in selected methyl groups to understand the mass spectrometric fragmentation of the pheromone (Petroski, Bartelt and Weisleder, Insect Biochemistry and Molecular Biology, 1994, 24, 69). They found the beetles were able to tolerate relatively large amounts of the deuterated acids in their diet (up to 5% of the wet weight of the diet) and produce as much as 100-250 ng of pheromone per beetle per day. This was collected by trapping from the air, and gave them sufficient labelled pheromone to be able to use and 13C NMR spectroscopy as well. They were able to show that acetic, propionic and butyric acids were all incorporated into the pheromone compound. By mass spectrometry as much as 26 to 49% of the pheromone was labelled with CD3 when propionic acid was used. The picture was more complicated with butyric and acetic acids. Their experiments with feeding [13C]acetic acid were not as successful because much of the label was scrambled by being broken down and re-synthesized into a number of compounds.

5.3.3 13C-13C Coupling

In 13C NMR spectra of normal abundance, or even for moderately enriched compounds, the probability of two 13C atoms being next to each other in a molecule is extremely small, so no coupling between 13C atoms is detectable. The 13C nucleus has a spin of 1/2, so it couples like ]H nuclei. Two adjacent 13C atoms each appear as doublets in the (proton-decoupled) 13C spectrum. Taking advantage of 13C-13C coupling can be a neat way of showing that a precursor is incorporated intact in a more elaborate molecule, and that the label has not been scrambled. The example of virginae butanolide A (VBA, above) illustrates this. In Figure 5.10 (e) a P-keto-thioester was synthesized with C-2 and C-3 labelled with 13C, used in biosynthetic experiments and the VBA isolated. The NMR spectrum of the resulting VBA (Figure 5.11 (c)) showed by the coupled carbon atoms at 47 ppm and 73 ppm that these two labels were still together in the VBA. If the intermediate thioester had been degraded to acetate and then re-incorporated into VBA, there could have been 13C atoms in it but the probability of the two still being in the same molecule and still next to each other would be very small and no 13C-13C coupling would have been visible. Doubly labelled acetic acid, 13CH313COOH, also gives 13C-13C coupling, so whenever it is used and an acetate unit is incorporated intact, this coupling will be seen. It is a useful technique to detect bond-breaking and re-arrangement of the carbon chain.

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