## 4 Discussion
### cisTetraDOTA Synthetic Pathway  
Our final study was the direct synthesis of a macromolecular contrast agent using DOTA as a building block. This final complex was designed using the crowding effect, reducing the distances via a rigid linker to increase rotational movement while limiting translational movement by steric hindrance [39]. This will affect the rotational correlation \(\tau_{R}\) and the hydration number in the second sphere and outer sphere, due to the interception of these coordination spheres of each complex in the macromolecule. Besides the high molecular weight, this will balance the \(\tau_{R}\) of the outer sphere since the reorientational correlation time divides into local and global movements. According to the free model of Lipari-Szabo, if both movements are sluggish, the macro complex will have better relaxivity [10; 40; 41; 42]. The synthesis of this compound was planned over several strategies, with compound 12 as a molecular scaffold and the Cis-DO2A, compound 17, as the tetramer's body. The different synthetic routes used are shown in Table 1. Every single reaction was performed in duplicate and purified by Flash Column Chromatography (FCC) and preparative TLC. In order to purify the samples by FCC and TLC, two solutions were used as eluents. To obtain a low RF for unreacted starting materials with a high relative polarity, polar eluents (DCM (90%):MeOH (9.9%):TEA (0.1%)) were used, while nonpolar eluents (toluene (30%):hexane (69.9%):TEA (0.1%)) were used to separate multiple products with significantly lower polarity after each reaction. This candidate had a higher theoretical relaxivity due to its high molecular weight, rigidity, and multiple Gd\({}^{3+}\) ions, which increase the relaxivity on the inner, second, and outer spheres, as was detailed through Equations (1)-(9). In addition, the higher anisotropy and symmetry provided by the N,N\({}^{-}\)-(1,4-phenylene) diacetamide bridge make it suitable for optical image complexes based on Eu\({}^{3+}\) and Tb\({}^{3+}\). Nonetheless, the results were inconclusive given the limited time available to fully isolate and characterize the proposed molecule. The multiple efforts and alternative pathways are listed in Table 1.  
Self-assembling behavior in this compound was expected through a double N-alkylation over compound 17 to produce cisTetraDOTA, but the evidence provided by the \({}^{1}\)H-NMR and mass spectrometry did not support this hypothesis (spectrum S18 and chromatogram S7, Supplementary Information). On the other hand, these results showed initial evidence of a possible trimer with a molecular weight of 1765.09 Da with an m/z on chromatogram S7 of 1718.220 Da (3.92%), corresponding to the possible \(\alpha\)-cleavage of one tert-butoxy radical (73.07) from the esters with one Na+ ion (C\({}_{86}\)H\({}_{135}\)NaN\({}_{18}\)O\({}_{7}\))\({}^{+}\). A second pattern was found on 1282.823 Da (22.62%), probably belonging to the \(\alpha\)-cleavage of the 66.7% of the tertbutyl esters and one N,N\({}^{\prime}\)-(1,4-phenylene) diacetamide bridge (C\({}_{64}\)H\({}_{98}\)N\({}_{16}\)O\({}_{126}\))\({}^{+}\). The final m/z peak placed on 574.409 Da (22.41%) was assigned to the cisDO2A bonded to one N,N\({}^{\prime}\)-(1,4-phenylene) diacetamide, both with an \(\alpha\)-cleavage fragmentation (C\({}_{29}\)H\({}_{47}\)N\({}_{6}\)O\({}_{6}\))\({}^{+}\). However, at 1848.191 Da (1.68%), the chromatogram showed what seemed to be an open trimer with the bromide still attached to the N,N\({}^{\prime}\)-(1,4-phenylene) diacetamide (1845.02 Da) (C\({}_{90}\)H\({}_{145}\)BrN\({}_{18}\)O\({}_{18}\))\({}^{+}\). Chromatogram S8 showed more evidence about this reaction and the possible compound obtained. At 1801.2 m/z, it was possible to see the molecular ion peak, which fit with the dihydrate trimer (C\({}_{90}\)H\({}_{144}\)N\({}_{18}\)O\({}_{18}\)\({}^{\bullet+}\)-2 H\({}_{2}\)O, Figure S1).