The assignment of absolute configuration (AC) is a crucial step in the structural characterization of natural products, especially for those subjected to biological assays. Methods such as X-ray crystallography, stereocontrolled organic synthesis, nuclear magnetic resonance (NMR), and chiroptical spectroscopies are commonly used to determine the AC of chiral natural compounds. Even with these well-established techniques, however, unambiguous stereochemical assignments of natural products remain a challenge, resulting in an increasing number of structural misassignments being reported every year. Herein, we will present the main techniques that have been used in AC reassignments of natural products over the last 10 years, along with some selected examples. Special attention will be paid to the strengths and weaknesses of each approach. With this, we expect to provide the readers with critical information to help them to choose the appropriate methods for correct AC determinations.

This work describes the first determination of the absolute configuration (AC) of (−)-cubebin by means of electronic circular dichroism (ECD), supported by quantum chemical calculations. The comparison of experimental ECD with the corresponding quantum chemical prediction for the proper diastereoisomer resulted in the definitive assignment of the AC of the naturally occurring (−)-cubebin as (8R,8aR,9S). The challenging determination of the relative configuration (RC) of cubebin based only on experimental nuclear magnetic resonance (NMR) methods is stressed. Computation of the 13C and 1H NMR chemical shifts for all the possible diastereoisomers leads to values of mean absolute error and root mean square deviation that do not allow distinguishing among them. Thus, errors in stereochemical determination can easily occur even when using two-dimensional methods, which clearly demonstrate the complexity of this special case. To determine the RC of this bioactive natural compound with high level of confidence, it was necessary to combine the DP4+ method with X-ray crystallography. Therefore, employing the commonly used empirical methods to determine the AC of (−)-cubebin can easily lead to misassignment of its stereochemistry.