## 4 Discussion
### cisTetraDOTA Synthetic Pathway
The second most abundant fragment was the peak of 1137.7 m/z belonging to the dimer (C\({}_{58}\)H\({}_{96}\)N\({}_{12}\)O\({}_{11}\)\({}^{\bullet+}\)), and finally the base ion peak at 589.4 m/z belonged to the monomer fragment (C\({}_{30}\)H\({}_{49}\)N\({}_{6}\)O\({}_{6}\)\({}^{+}\)). In order to obtain a clear picture of the experimental NMR, some DFT simulations were carried out in the present work at the B3 LYP/cc-PVDZ level using the basis set function in the Gaussian 09 package [43]. Molecular optimizations and NMR shifts were computed in the gaseous phase and also by applying a gauge-invariant atomic orbital (GIAO) approach for the NMR [44]. The input structure was modified by the tert-butyl groups exclusion to save computational time. Two different structures were considered: an open and close macrocycle trimer (Figure 6). The theoretical \({}^{1}\)H chemical shifts were calculated for the optimized geometries; the results showed that the range of the \({}^{1}\)H chemical shifts of the typical organic molecules were usually 100 ppm. [45; 46; 47]. The accuracy ensured the reliable interpretation of the spectroscopic parameters. Spectra S19-S24 are available in the Supplementary Information. Two significant peaks were analyzed among the experimental (400 MHz Bruker NMR) and predicted \({}^{1}\)H-NMR results; the shift at \(\delta\) 3.50 ppm (d, 24H) belonging to the \(\alpha\)-carbons bonded to the tertiary macrocycle amine, which showed that equivalent protons were the evidence of a symmetrical adduct, and the shift at \(\delta\) 3.90 ppm (s, 2H) pertaining to the existence of a single methylene bromide (CH\({}_{2}\)-Br). Both signals had a clear difference in the possible outcomes: the \(\alpha\)-carbons for the open trimer predicted \({}^{1}\)H-NMR reported a wide signal distribution between 3.0-5.0 ppm (S20 spectrum), an expected output due to the low symmetry. On the other hand, the closed trimer showed a less pronounced peak distribution  
Figure 6: Optimized structures of the two considered system. Closed trimer, (**left**) and open trimer, (**right**) (C = gray, O = red, H = white and N = blue). between 3.5-3.8 ppm (S23 spectrum), similar to the one obtained experimentally. The second signal was relevant to this study because its presence denoted an incomplete ring formation. Protons H-129 and H-130 belonged to methylene bromide in the open predicted \({}^{1}\)H-NMR with chemical shifts at 4.3 ppm and 4.85 ppm (S20 Spectrum); one of these signals was found at 4.65 ppm in the experimental spectra integrating with two protons (S18 spectrum). The presence of both signals in the experimental \({}^{1}\)H-NMR supported by the predicted \({}^{1}\)H-NMR showed the incomplete reaction between compounds 11 and 17 with the formation of an open and closed trimer, detected by NMR and MS. Despite the efforts in the purification step, both products could not be effectively isolated (Figure 7); however, further analysis was not possible due to impurities and the minimal amount of product obtained. The low yield of this reaction can be explained through the partial solubility of compound 17 in organic solvents, decreasing the effective collisions with compound 12. For that reason, several solvents were used with different polarities, i.e., protic and non-protic, mixed and pure, but it did not affect the course of the reaction. DFT calculations showed that the sp3 HOMO-LUMO from the secondary amines of compound 17 had a different orientation, with one of the HOMOs looking inside the tetraazacyclododecane ring (Figure 8 atom 15). This out-of-phase HOMO decreased the number of effective collisions in the Sn2 reaction with the N,N\({}^{\prime}\)-(1,4-phenylene) diacetamide dibromide, besides increasing the steric hindrance for the N-alkylation. Figure 8: DFT calculations compound 17. Figure 7: Potential synthons obtained instead of G4–CisTetraDOTA following the spectral information.