Heavy Isotope Labelling

Developments in mass spectrometry and NMR spectroscopy have made it more convenient in many cases to study biosynthetic processes with heavy isotopes. The most information can be obtained from a combined use of these methods. The position of labelling can then be deduced from intact molecules, without the degradation process illustrated with griseofulvin in Figure 5.3. Labelling with heavy isotopes however requires rather higher incorporation of the label for secure identification (Table 5.1).

The use of lsO in the initial stages of the study of photosynthesis provides a simple example how labelling can be informative. When C1802 was supplied to a plant in sunlight, none of the lsO was found in the 02 evolved, but was recovered as H2lsO. When H2lsO was used in the experiment, 1802 was produced. The oxygen in the atmosphere is therefore derived from splitting water, not C02 (Figure 5.6).

C»*02 + 2H2180 ch;;ophy„ ■ (CH216o) + -o2 + H216O

Figure 5.6 The oxygen released in photosynthesis is from the splitting of water, demonstrated by using water containing the heavy isotope of oxygen. The formula (CH20) represents carbohydrates

Deuterium labelling is the most useful for mass spectrometry. A single 2H atom incorporated increases the intensity of the M+l ion. There must be sufficient 2H present to make the ion intensity greater than that due to the natural abundance of 13C (1.1%) and any other isotopes. Natural 2H (0.015%), 15N (0.36%) and 170 (0.04%) add little to the strength of the M+l ion, and lsO (0.2%) adds little to the M+2 ion. Incorporation of an intact C2H3 group will produce a new ion at M+3 that can be readily seen and its intensity measured to give the degree of incorporation.

A thorough knowledge of the capabilities of mass spectrometry and NMR spectroscopy is necessary to make good use of them in biosynthetic studies with heavy isotopes. It is very useful that 13C, 15N and lsO all have nuclear spins of n/2 (2H has a spin of 1, see Table 5.1), which means they can all be studied by NMR methods. Some examples only can be given here. A basic knowledge of mass spectrometry and 13C NMR spectroscopy is assumed; for background in this subject D. H. Williams and I. Fleming Spectroscopic methods in organic chemistry 5th Edition, 1995 is recommended.

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