## 3 Results and Discussion
### SPPS of Aib-Enkephalin and Aib-ACP  
After testing the green solvent, DMM, in all stages of SPPS, we evaluated the effectiveness of the solvent during the synthesis of a pentapeptide, Aib-enkephalin (H-Tyr-Aib-Aib-Phe-Leu-OH/NH\({}_{2}\)). This sequence represents a difficult step due to the presence of two Aib (2-Aminoisobutyric acid) residues, which are sterically hindered amino acids and often lead to incomplete or slow-rate coupling reactions. In fact, the misincorporation of Aib residues (des-Aib) could easily happen during the synthesis [5]. The amount of des-Aib measures the efficiency of DMM compared to DMF. Aib-enkephalin was synthesized using the microwave-assisted synthesis technique on different resins, particularly those that had shown good swelling: Wang PS and Rink amide CM. The new green protocol previously selected was applied to the synthesis of the pentapeptide, and DMM was used in all stages, including washing. Purities of pentapeptide were calculated by HPLC and are shown in Table 7.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Entry** & **Solvent** & **Time** & **T** (\({}^{\circ}\)**C**) & **DL**\({}^{\text{b}}\) **(\%)** \\ \hline
6 & DMF & 2 h & rt & 48.78 \\
7 & DMM & 2 h & rt & 65.80 \\
8 & DMF & 10 min & 75 (MW) & 89.02 \\
9 & DMM & 10 min & 75 (MW) & 35.29 \\ \hline \hline \end{tabular} \({}^{\text{a}}\) Racemization was calculated by HPLC. \({}^{\text{b}}\) Defined as (Fmoc-D-Phg-Pro-NH\({}_{2}\)/Fmoc-L-Phg-Pro-NH\({}_{2}\)) \(\times\) 100.  
\end{table}
Table 6: Racemization ratio (%) in the synthesis of Fmoc-Phg-Pro-NH\({}_{2}\)\({}^{\text{a}}\).  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Entry** & **Solvent** & **Ceoupling Agents** & **Eq.** & **Time** & **T** (\({}^{\circ}\)**C**) & **Purity (\%)** \\ \hline
1 & DMF & HObt/DIC (1:1) & 3 & 2h & rt & 79.68 \\
2 & DMM & HObt/DIC (1:1) & 3 & 2h & rt & 91.14 \\
3 & DMF & OxymaPure/DIC (1:2) & 3 & 2h & rt & 33.65 \\
4 & DMM & OxymaPure/DIC (1:2) & 3 & 2h & rt & 83.57 \\
5 & DMM & OxymaPure/DIC (1:2) & 3 & 10 min & 75 (MW) & 78.90 \\ \hline \hline \end{tabular}
\end{table}
Table 7: HPLC purities of Aib-enkephalin pentapeptide assembled on different resins.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Entry** & **Solvent** & **Ceoupling Agents** & **Eq.** & **Time** & **T** (\({}^{\circ}\)**C**) & **Purity (\%)** \\ \hline
1 & DMF & HObt/DIC (1:1) & 3 & 2h & rt & 79.68 \\
2 & DMM & HObt/DIC (1:1) & 3 & 2h & rt & 91.14 \\
3 & DMF & OxymaPure/DIC (1:2) & 3 & 2h & rt & 33.65 \\
4 & DMM & OxymaPure/DIC (1:2) & 3 & 2h & rt & 83.57 \\
5 & DMM & OxymaPure/DIC (1:2) & 3 & 10 min & 75 (MW) & 78.90 \\ \hline \hline \end{tabular}
\end{table}
Table 5: Synthesis of NH\({}_{2}\)-Tyr-Ala-OH dipeptide with different approaches.  
As the Aib-aib coupling is considered the key step of the synthesis, the percentage of des-Aib was calculated. When the synthesis was performed on a Wang resin, using DMM (entry 11), a lower percentage of pentapeptide was obtained compared to DMF, but it gave a much higher des-aib rate than the one in DMF. Interestingly, results obtained on a Rink amide CM resin (entry 13), using DMM, are better than those obtained in DMF (entry 12). For both syntheses, no amount of des-Aib was observed. As consequence, the Rink amide CM was selected for further studies. The same protocol was applied to the synthesis of a longer and more difficult sequence, Aib-ACP decapeptide (H-Val-Gln-Aib-Aib-Ile-Asp-Tyr-Ile-Asn-Gly-NH\({}_{2}\)), used as a case study due to its tendency for aggregation and folding during SPPS [16]. HPLC purities of Aib-ACP are shown in Table 8.  
In this case, the synthesis of the decapeptide in DMM (entry **15**) yielded purity values compared to DMF (entry **14**), with a small amount of des-Aib observed for entry **15**. These excellent results, obtained for both the synthesis of Aib-enkephalin and the decapeptide Aib-ACP, make DMM an ideal greener alternative to DMF.