2.1.2. Tetrabromostyloguanidine (**2**)

For the configurational assignment of tetrabromostyloguanidine (**2**) 27 interproton distances from ROESY spectra were used (the complete list of ROEs of **2** is given in the SI, Table S2). Using the same methodology as in case of axinellamine A (**1**), the results for the 1000 generated possible structures of tetrabromostyloguanidine (**2**) are shown in Figure 5a ("best 400") as a graphical representation of the total error (dimensionless) for each structure, ordered according to ascending total errors. As already discussed for **1**, one stereogenic center of **2** was again set as reference and fixed by the application of a chiral volume restraint (C-10). In order to verify and demonstrate that the results of the fc-rDG/DDD calculations do not depend on the choice of the stereogenic center that is fixed, these calculations were also repeated for all eight centers of **2** and are reported in the Supporting Information (see Supplementary Figures S5–S7).

**Figure 4.** (**a**) Plot of the total "*pseudo energy*" of ranked rDG structures of axinellamine A (**1**), showing the first 700 out of 1000 structures generated (*K*NOE = 10.0 Å−2), the minimum energy level is indicated by the green line. The dashed lines at higher energies indicate the differentiability of the best-fit solution with respect to alternate assignments of diastereotopic methylene protons (orange) or wrong (red) configurations, the corresponding Δ*E* values are given on the right. The inset plot shows the first 100 structures with all-correct assignments and their separation into distinct conformational families by smaller energy steps. (**b**) Molecular structures of axinellamine A (**1**) showing the superposition of all DG structures identified up to the first wrong configuration (597 structures); the central fragment (green circle) of the DG best-fit (lowest pseudo energy) structure is plotted below, displaying the correct configuration of **1**.

The first wrong structure with respect to the eight stereogenic centers is No. #378 (red circle in Figure 5). This structure differs from structures #1 to #377 by the configuration of C-20, which is actually the same position (different atom numbering, see Scheme 1) for the first change as observed in case of axinellamine A (**1**). The first "pseudo-configurational" change, i.e., an alternative diastereotopic assignment of methylene protons of the exocyclic methylene group C-19, was already observed at structure No. #99 (orange circle in Figure 5) with a very low energy difference of Δ*E* = 0.14, indicating some ambiguity in the assignment of these CH2-protons. The second and more characteristic "jump" in energy is observed at structure No. #203. This jump in energy includes both either a new conformation of **2** and its side chains, or an alternative assignment of the diastereotopic protons at the endocyclic methylene group C-13, both changes have similar penalties in experimental versus calculated NMR parameters. At structure No. #277 the alternative assignment of the diastereotopic protons at C-13 is observed, and at structure No. #302 both methylene groups are inverted and both changes are manifested in rather small changes in pseudo energy only. The total number of structures with the correct configuration for tetrabromostyloguanidine (**2**) generated is 702/1000. Though all 128 relative configurations of **2** were initially generated by the rDG "metrization" step, only a few "survived" the 4D (36 configuration) and 3D (19 diastereomers) stages of sampling, and of the latter, all 18 wrong configurations appear after structure No. #377, and are ranked in their pseudo energy significantly higher (Δ*E* ≥ 5.32) than the best-fit geometry of **2** with correct configuration (see discussion of **1**).

The diastereotopic assignment of the methylene protons can also be alternatively obtained by a *J* coupling approach (<sup>3</sup>*J*HH and HMBC intensities). Using this information within the fc-rDG/DDD calculation, the results can still be improved (see Supporting Information, Figures S8 and S9). For this calculation the two methylene groups are used with a fixed chiral volume, changing the number of floating centers from nine to seven (C-10 fixed). The first wrong structure for this calculation in respect to the eight stereogenic centers changes from No. #378 to No. #420, and many of the smaller steps in pseudo energy originating from alternate CH2-assignments vanish altogether.

**Figure 5.** (**a**) Plot of the total "*pseudo energy*" of ranked rDG structures of tetrabromostyloguanidine (**2**), showing the first 400 out of 1000 structures generated (*K*NOE = 10.0 Å−2), the minimum energy level is indicated by the green line. The dashed lines at higher energies indicate the differentiability of the best-fit solution with respect to alternate assignments of diastereotopic methylene protons (orange) or wrong (red) configurations. (**b**) Molecular structures of tetrabromostyloguanidine (**2**) showing the superposition of all DG structures identified up to the first wrong configuration (377 structures); the central fragment (green circle) of the DG best-fit (lowest pseudo energy) structure is plotted below, displaying the correct configuration of **2**.

In total, the relative configuration of all stereogenic centers in **2** is unequivocally determined by the ROE data used, although some ambiguities remain on the assignment of diastereotopic protons. However, both the unambiguity of the configurational assignment, as well as the ambiguity of the CH2-assignments is again established by a single rDG simulation, without any other assumptions or restraints used rather than experimental NMR data exclusively.

### 2.1.3. 3,7-*epi*-Massadine chloride (**3**)

For the configurational assignment of 3,7-*epi*-massadine chloride (**3**) 36 interproton distances from ROESY spectra were used (the complete list of ROEs of **3** is given in the SI, Table S3). Results for the 1000 generated structures of 3,7-*epi*-massadine chloride (**3**) are shown in Figure 6a ("best 200") as a graphical representation of the total error for each structure, ordered according to ascending total errors. As already discussed for **1** and **2** one stereogenic center of **3** was set as reference (C-13).

The first wrong structure in respect to the eight stereogenic centers is No. #56 (red circle in Figure 6). This structure differs from the preceding structures by a configurational change of C-3 and C-7, which represents the original massadine configuration. The first "pseudo-configurational" change was again observed earlier at structure No. #25 (orange circle in Figure 6a), which represents the alternative assignment of the diastereotopic protons at the methylene group C-1-'. The results of the calculations for **3** can be improved if a diastereotopic assignment of the methylene protons is carried out prior to the DG/DDD calculations (see discussion of compound **2**). In this case, the first wrong structure becomes No. #123 (see Figures S11 and S12). The diastereomeric differentiability in this case is not as pronounced as for compounds **1** and **2**. This becomes obvious just by looking at the occurrence of the first wrong structure (**1**: #597, **2**: #377, and **3**: #56), but it is still an unambiguous result. Another difference to the first two examples is the energy difference between the different configurations, which is much lower for **3**. This indicates that the extent and certainty with which the experimental data does differentiate between the different structures (diastereomers) is not as pronounced as it was observed for **1** and **2**. For 3,7-*epi*-massadine chloride (**3**), the NMR data set was less well defined because of the longer mixing time of the ROESY experiment.

**Figure 6.** (**a**) Plot of the total "*pseudo energy*" of ranked rDG structures of 3,7-*epi*-massadine chloride (**3**), showing the first 200 out of 1000 structures generated (*K*NOE = 10.0 Å−2), the minimum energy level is indicated by the green line. The dashed lines at higher energies indicate the differentiability of the best-fit solution with respect to alternate assignments of diastereotopic methylene protons (orange) or wrong (red) configurations. (**b**) Molecular structures of 3,7-*epi*-massadine chloride (**3**) showing the superposition of all DG structures identified up to the first wrong configuration (55 structures); the central fragment (green circle) of the DG best-fit (lowest pseudo energy) structure is plotted below, displaying the correct configuration of **3**.

### *2.2. Configurational Assignment with NOEs and RDCs*

The terrestrial alkaloids tubocurarine (**4**) and vincristine (**5**) form the completion of the current investigation. In case of **4** only one stereogenic center relative to a second one needs to be assigned and is therefore a seemingly rather simple model for the described approach, but was chosen for demonstration purposes and its long-range separated stereogenic centers. Vincristine (**5**) is a more complex structure with nine stereogenic centers. In both examples, NOEs involving diastereotopic protons of methylene groups were used as unassigned and averaged restraints only, and in the case of **4**, the *o*/*o'*-protons of the *p*-disubstituted aryl ring were treated similarly (see Supplementary Tables S4 and S5 and Figure S4). As it will be demonstrated later in the manuscript, NOEs are not sufficient for the unambiguous assignment of the relative configuration of compounds **4** and **5**. Further data was necessary for a complete assignment. Due to the lack of experimental RDC data, we have decided to use synthetic RDC data sets for both compounds. Though one might argue that is a general weakness of the method, it is important to know for demonstration purposes that additional NMR parameter may help to solve the structural problem. Where applicable, RDCs involving CH2-groups were also treated as unassigned values, and only the sum of both individual <sup>1</sup>*D*CH methylene RDCs are used as restraining parameters. Though this reduces the quality of the data set that might be experimentally accessible, this method was chosen to reduce the amount of prior information to learn more about the limits of the DG based structure analysis described here. The full data sets of NOEs and RDCs used for **4** and **5** are listed in Supplementary Tables S4–S7. As this data explicitly does not allow to differentiate diastereotopic CH2-protons, we will not ge<sup>t</sup> into a debate on the subject of assignments on prochiral centers in this chapter.
