## 3 Results and Discussion
### Solubility Test  
To assess the efficiency of the selected solvents during SPPS, we evaluated their capability to solubilize amino acids and coupling agents. Furthermore, to avoid the formation of side products, we used amino acids protected both in the side chain and on the \(\alpha\)-amino function. The protecting groups, therefore, constitute an important obstacle to solubility. During this test, we solubilized amino acids protected on the amino function with the base-labile Fmoc groups and in the side chain with the acid-labile groups. Starting from the standard amino acid concentration for SPPS of 0.1 M in DMM, we carried out dilutions in case of incomplete dissolution or concentrations up to the formation of precipitate. Table 2 lists the solubilities of the amino acids in DMM and DMF; only Fmoc-His(Trt)-OH showed solubility in DMM at a lower concentration than the standard concentration for SPPS.  
A solvent mixture was used to address the issue of poor solubility of histidine. Mixing solvents can improve certain chemical and physical properties, such as viscosity and density, thereby enhancing the efficacy of solubilization. Unfortunately, it is not always possible to create completely green mixtures. In fact, in most cases, one of the two solvents does not meet any green chemistry parameters. To improve the solubility of histidine in DMM, we tested different concentrations, ranging from 0.01 M to 0.1 M, with varying percentages of NMP (Table 3). At the standard SPPS concentration of 0.1 M, histidine is soluble with a 40% NMP/DMM mixture.  
\begin{table}
\begin{tabular}{l c c c c c c c c c c c c c c c c} \hline \hline \multicolumn{1}{c}{**Solvent**} & \multicolumn{11}{c}{**Fmooc-aa(PG)-OH**} \\ \hline  & **Gly** & **Ile** & **Leu** & **Val** & **Ala** & **Phe** & **Pro** & **Met** & **Tyr** & **Thr** & **Ser** & **Asp** & **Glu** & **Cys** & **Asn** & **Gln** & **His** & **Lys** & **Trp** & **Arg** \\ \hline
**DMF** & & & & & & & & & & & & & & & & & & & & & \\ \hline DMM & & & & & & & & & & & & & & & & & & & & & \\ \hline \hline \end{tabular}  
* \({}^{\mathrm{a}}\) Solubilization monitored at 0.1 M concentration. Legend: green = soluble; red = insoluble. PG = protecting group.  
\end{table}
Table 2: Solubilization efficacy of Fmoc-AA(PG)-OH amino acids in DMF and DMM \({}^{\mathrm{a}}\).  
Figure 1: Swelling results of several resins in DMF and DMM.  
Table 4 also shows the solubility values of the coupling agents in DMM and DMF. Most of the coupling reagents exhibit good solubility in DMM comparable to the standard concentration (0.1 M), except COMU and HATU, which are insoluble. Then, Fmoc-Val-OH and Fmoc-His(Trt)-OH were mixed with coupling agents (HOBt/DIC and Oxyma/DIC), and the solubility was evaluated (Table 4). As shown in Table 4, Fmoc-His(Trt)-OH showed better solubility in combination with coupling agents, reducing the percentage of NMP (from 40% to 30%). In regard to the coupling agents, different chemical classes lead to the formation of the amide bond. The most used are derivatives of benzotriazole (hydroxybenzotrazole or HOBt and 2-1H-Benzotriazole-1-yl-1,1,3,3-tetramethylluoronium hexafluorophosphate or HBTU) and are usually coupled to carbodiimides. Benzotriazole derivatives are unsafe for humans and the environment. The derivatives of ethyl cyanoacetate, such as the oxime of ethyl cyanoacetate (OxymaPure), are activators that neutralize the basicity of carbodiimides, thanks to their acidic character (pka 4.6), thus preventing the catalyzed base reactions, in particular the racemization. Furthermore, OxymaPure, used as additive in the carbodiimide (DIC) approach, allows the desired product with quantitative yields and is fully compatible with microwave-assisted peptide synthesis [25].