Examples

In Chapter 3 an example was given of the demonstration that isobutyric acid (and methacrylic acid) in an insect was derived from valine by deuterium labelling (Figure 3.13). In that case the isobutyric acid produced was converted to a less volatile ester of pentafluorobenzyl alcohol in order to show by mass spectrometry that the carbon chain was derived intact from valine. Analysis by gas chromatography and mass spectrometry gave a molecular ion seven mass units greater than 268, so seven deuterium atoms were still present in the isobutyric acid (Figure 5.7)

Figure 5.7 The ester of isobutyric acid made from fully deuterated valine had a mass of 275, seven units greater than the unlabelled ester, therefore seven deuterium atoms were present in the isobutyric acid

We can set up an imaginary experiment to show that the methyl ester group of methyl 6-methylsalicylate, the trail pheromone of the ant Tetramorium impurum is derived from S-adenosylmethionine (Figure 2.16). First [2H3]-S-adenosylmethionine (indirectly from S-adenosylhomocysteine and [2H3]-methyl iodide) is prepared, added to a tissue culture of the excised poison glands of the ant and the two incubated together. There is always a judgement to be made between adding enough labelled compound to get good incorporation of the label, and adding too much so that the metabolism of the tissue is altered. It is often easier to show that a compound can serve as a precursor to the target compound than to show that it is the normal, natural intermediate in the biosynthetic path to the target. The products from the incubation is extracted with hexane and examined by gas chromatography-mass spectrometry Part of the mass spectrum of the normal pheromone (Figure 5.8) shows the molecular ion at m/z 166 and the daughter ion at m/z 134 formed by loss of methanol. This is known to occur from the methyl ester and the phenol groups together. The product after incubation with deuterated S-adenosylmethionine shows a new peak at m/z 169 (M + 3), and the peak at m/z 166 is correspondingly reduced in abundance. The ion at m/z 134 is unaffected, showing that all the deuterium is lost with the methyl ester group. Note that the molecules with three deuterium atoms will elute slightly earlier from the gas chromatograph. This is because the C-D bond is slightly shorter and stronger than the C-H bond, causing the deuterium-containing molecules to be slightly more volatile (an isotope effect).

Figure 5.7 The ester of isobutyric acid made from fully deuterated valine had a mass of 275, seven units greater than the unlabelled ester, therefore seven deuterium atoms were present in the isobutyric acid pentafluorobenzyl isobutyrate mol. mass 268

pentafluorobenzyl heptadeuterylisobutyrate mol. mass 275

pentafluorobenzyl isobutyrate mol. mass 268

pentafluorobenzyl heptadeuterylisobutyrate mol. mass 275

Figure 5.8 Part of the mass spectrum of the pheromone methyl 6-methylsalicylate, and the spectrum after incubation with deuterated S-adenosylmethionine, showing molecular ions at 166 and 169 in a ratio of 85:15

The tri-deuterated S-adenosylmethionine is almost 100% pure. Essentially all the hydrogens in the methyl group have been replaced by deuterium. How much of the deuterated methyl group finds its way into methyl 6-methylsalicylate may be very small. Isotopic enrichment refers to the change in isotope content in the biosynthesized compound above the natural abundance and is usually expressed as atom % excess. The iso-topic enrichment of the methyl group in the pheromone can be estimated from the relative abundances of the peaks at m/z 166 and 169 (Figure 5.8). The ratio is 1:0.18. The enrichment is then 15%.

Alternatively, the experiment can be followed by 13C NMR spectroscopy. We can observe 13C-2H coupling in the proton-decoupled spectrum. The appearance of the total spectrum is shown in Figure 5.9 (a). The effect of substitution of one, two or three hydrogens in a methyl group by deuterium is shown at (b), and the methyl ester portion of the spectrum after incubation with deuterated S-adenosylmethionine is given at (c). As 85% of the molecules are unlabelled the original peak is strong with a weaker deuterium-coupled septuplet beside it, showing that three deuterium atoms are incorporated into 15% of the molecules. The NMR experiment requires rather more material because 13C spectra are naturally weak (natural abundance of 13C is 1.1%), but if the shifts of all the carbon atoms of the molecule are known, deducing the position of labelling can be quite simple.

An interesting example of the power of 13C NMR spectroscopy to solve a biosynthesis problem is found in the study of virginae butanolide A (VBA) (Figure 5.10 (a)), one of a series of similar signalling compounds produced by the bacterium Streptomyces antibioticus (S. Sakuda and Y. Yamada, Comprehensive Natural Products Chemistry, Vol. I, Pergamon Press, Oxford, 1999, p. 139). First the culture conditions for the Streptomyces to produce a good yield of VBA had to be studied, then

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