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A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol

Significance The extraction of tramadol from the roots of Nauclea latifolia excited great interest worldwide. This was the first time that a widely marketed synthetic drug was found as an apparently natural product at high abundance. In this article, we describe the position-specific distribution of 13C in this tramadol. As a conventional approach to study its probable biosynthetic precursors, enzymatic steps, and intermediate metabolites is not currently feasible, we herein propose the concept of a retro-biosynthesis by examining the position-specific isotope distribution within the molecule and rationally interpreting the data in terms of known plant biochemical processes that may be involved in a biosynthesis of tramadol. Thus, clear guidance is given for future labeling studies. Tramadol, previously only known as a synthetic analgesic, has now been found in the bark and wood of roots of the African medicinal tree Nauclea latifolia. At present, no direct evidence is available as to the biosynthetic pathway of its unusual skeleton. To provide guidance as to possible biosynthetic precursors, we have adopted a novel approach of retro-biosynthesis based on the position-specific distribution of isotopes in the extracted compound. Relatively recent developments in isotope ratio monitoring by 13C NMR spectrometry make possible the measurement of the nonstatistical position-specific natural abundance distribution of 13C (δ13Ci) within the molecule with better than 1‰ precision. Very substantial variation in the 13C positional distribution is found: between δ13Ci = −11 and −53‰. Distribution is not random and it is argued that the pattern observed can substantially be interpreted in relation to known causes of isotope fractionation in natural products. Thus, a plausible biosynthetic scheme based on sound biosynthetic principals of precursor–substrate relationships can be proposed. In addition, data obtained from the 18O/16O ratios in the oxygen atoms of the compound add support to the deductions made from the carbon isotope analysis. This paper shows how the use of 13C NMR at natural abundance can help with proposing a biosynthetic route to compounds newly found in nature or those difficult to tackle by conventional means.