Isotope Ratio Monitoring 13C Nuclear Magnetic Resonance Spectrometry for the Analysis of Position-Specific Isotope Ratios

Studies of changes in isotope ratios in a given molecule caused by an enzyme reaction can give considerable insight into mechanism. Similarly, cascades of enzymespathways- can also introduce fractionation that can be used to characterize the type of metabolism being exploited. In both cases, classical studies have used enrichment techniques that have allowed the determination of isotope fractionation. More recent approaches, however, allow such studies to be carried out on substrates in which the isotope ratios are in the natural abundance range. This has the advantage of avoiding potentially demanding synthesis of specifically labeled compounds as well as allowing multipositional isotope ratio determinations. The downside can be that considerable quantities of analyte are required. In this chapter, we present the use of isotope ratio monitoring by NMR spectrometry as a means to access positional isotope ratios, potentially for all positions in the target molecule(s). It should be noted that, while the approach is generic, in that the general conditions for obtaining the required data follow the same protocol, no single protocol exists for all applications. The chapter is therefore split into two parts: general comments pertinent to the approach followed by a number of examples illustrating how different questions can be approached.