## 4 Discussion  
### Click Chemistry Synthetic Pathway  
Our first approach to the synthesis of highly active CA for MRI was the use of the extensively known azide-alkyne Huisgen cycloaddition protocol because it offers a reliable and selective strategy for the rapid synthesis of adducts through heteroatom links. Our goal was to synthesize a linear heterolanthanide (Gd\({}^{3+}\)-Tb\({}^{3+}\)) tetramer using DOTA-derivative blocks and 1,4-phenylenediamine as a rigid molecular scaffold. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Solvent** & **Base** & **Base Eq.** & **T (\({}^{\circ}\)C)** & **Time (h)** & **Eq. C17** & **Eq. C12** & **Result** & **Yield (\%)** \\ \hline
**Chloroform** & TEA & 3 & r.t. & 3 & 1 & 5 & - & - \\ \hline
**Chloroform** & Pyridine & 3 & 0 & 12 & 1 & 1 & - & - \\ \hline
**Chloroform** & Pyridine & 5 & \(-\)78 & 12 & 1 & 1 & - & - \\ \hline
**DMF** & TEA & 10 & r.t & 12 & 1 & 5 & - & - \\ \hline
**DMF** & TEA & 10 & r.t & 12 & 1 & 2 & - & - \\ \hline
**DMF** & TEA & 10 & 50 & 12 & 1 & 1 & - & - \\ \hline
**DMF** & Cs\({}_{2}\)CO\({}_{3}\) & 5 & 50 & 12 & 1 & 2.2 & - & - \\ \hline
**DMF** & Cs\({}_{2}\)CO\({}_{3}\) & 10 & 60 & 12 & 1 & 2.2 & - & - \\ \hline
**Toluene** & Cs\({}_{2}\)CO\({}_{3}\) & 10 & 110 & 12 & 1 & 2.2 & - & - \\ \hline
**Toluene** & Cs\({}_{2}\)CO\({}_{3}\)/1\% & NaI & 10 & 110 & 12 & 1 & 2.2 & - & - \\ \hline
**Toluene** & Cs\({}_{2}\)CO\({}_{3}\)/5\% & NaI & 10 & 110 & 24 & 1 & 2.2 & - & - \\ \hline
**Toluene** & Cs\({}_{2}\)CO\({}_{3}\)/5\% & NaI & 10 & 110 & 48 & 1 & 2.2 & - & - \\ \hline
**Acetonitrile0.1** & Cs\({}_{2}\)CO\({}_{3}\)/5\% & NaI & 10 & 110 & 48 & 1 & 2.2 & - & - \\ \hline
**Acetonitrile** & Cs\({}_{2}\)CO\({}_{3}\)/5\% & NaI & 10 & 82 & 24 & 1 & 2.2 & - & - \\ \hline
**Acetonitrile** & NaHCO\({}_{3}\) & 2 & 82 & 48 & 1 & 2.2 & - & - \\ \hline
**Acetonitrile** & NaHCO\({}_{3}\) & 2 & 82 & 56 & 1 & 2.2 & - & - \\ \hline
**DMF** & TEA & 5 & 80 & 48 & 1 & 2.2 & - & - \\ \hline
**DMF** & NaHCO\({}_{3}\) & 5 & 80 & 48 & 1 & 2.2 & - & - \\ \hline
**Toluene0.1** & Cs\({}_{2}\)CO\({}_{3}\)/5\% & NaI & 10 & 110 & 48 & 1 & 2.2 & * & * \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The reaction between compounds 12 and 17 is a possible way to reach CisTetraDOTA. The NMR and MS results suggest that the reaction produced an opened and closed trimer. The evidence was elusive (spectrum 18 and chromatogram 7); even with HPLC and TLC preparative purifications, it was not possible to remove the presence of starting materials on both results, affecting the identification of the product of these reactions. However, this result could lead to a new kind of synthetic route to make a new closed CA, thus, this finding will be studied in future projects. Not successful: -; undefined: \({}^{*}\). The first complex synthesized (compound 7, Figure 4) was composed of a DOTA derivative with an N-Hydroysuccinamide (NHS) terminal to produce an adduct of high molecular weight by direct coupling with peptides and proteins. The compound was successfully synthesized (73.2%, 0.123 mmol) and characterized using Lu\({}^{3+}\) as an ion to check the NMR shifts due to its diamagnetic behavior and similar ionic radius to Gd\({}^{3+}\). The characterization of this compound can be found in the Supplementary Information. Two significant factors were found at the end of this synthesis: first, the NHS ester had a low lifetime in the complex due to the quick hydrolysis with water, and second, the Cu\({}^{+}\) was retained by the 1,2,3-triazole ring with traces of 0.63-0.83 ppm. Both effects decreased their functionality and jeopardized their use in humans due to the high toxicity of Cu(I) and losing the NHS terminal diminished the possibility of making adducts between the potential CA with biomolecules of high molecular weight such as proteins. To tackle the limiting factors impacting the reaction, we first produced an in situ NHS ester synthesis without further purification, which allowed the addition of nucleophiles directly and the formation of the final adduct [35].