The prediction of isotopic patterns in phenylpropanoids from their precursors and the mechanism of the NIH-shift: Basis of the isotopic characteristics of natural aromatic compounds

The deuterium patterns (the relative abundances of isotopomers) of natural aromatic compounds are determined by that of their precursors in the shikimic acid pathway – erythrose, PEP and NADPH – and by intramolecular (IIE) and kinetic (KIE) isotope effects accompanying the NIH-shift during hydroxylation by monooxygenases. The theoretical 2H-distribution in the aromatic ring of phenylpropanoids can be predicted from that of their precursors – erythrose-4-phosphate, phosphoenolpyruvate and NADPH – and by invoking the mechanism of the NIH-shift and implied deuterium isotope effects. For each position in the non-oxygenated ring, the predicted natural 2H-abundance is in excellent agreement with experimental data obtained from quantitative 2H NMR-measurements on natural compounds, especially concerning the relative 2H-abundances p > o ⩾ m. For the p-hydroxylated derivatives, the experimentally determined 2H-abundance sequence order m > o can also be deduced, assuming an anisotropic migration (intramolecular isotope effect) of the p-hydrogen atom to the two differently 2H-substituted m-positions during the NIH-shift (intramolecular hydrogen transfer) and an in vivo deuterium kinetic isotope effect of ∼1.20 on the final hydrogen elimination from the proposed ketodiene intermediate. The predicted 2H-distribution pattern of methyl salicylate 10, a representative of an o-hydroxylated natural compound, is in excellent agreement with that reported from 2H NMR analyses. However, for salicyl alcohol, minor differences between the theoretical and experimentally determined pattern are found that cannot yet be satisfactorily explained. On the other hand, a very good agreement is found between the theoretical and experimental pattern of coumarin, provided a deuterium kinetic isotope effect of ∼1.30 is assumed for the elimination of the H-atoms from the ketodiene intermediate. The secondary m-hydroxylation of p-coumaric acid in the biosynthesis of vanillin seems to proceed without large isotope effects. Parallel differences are also observed for the 18O-kinetic isotope effects on the corresponding monooxygenase-catalysed reactions. The results demonstrate convincingly that the mechanisms of these general reactions of the phenylpropanoid biosynthetic pathway are identical and follow general principles. Small observed differences between the 2H-patterns of individual natural aromatic compounds originating from the same hydroxylation type can therefore be assigned to differences