## 3 Results and Discussion  
First, we studied the coupling efficiency of the MW assisted solid-phase reaction between Boc-Phe-OH nanoparticles and H-Gly-Rink amide-PEG grafted resin [26, 27] using several coupling methods. To perform aqueous based solid-phase synthesis, the solid support is required to swell in water. PEG grafted resin was reported to have a high swelling ability in both non-polar and polar solvents including water. Water-dispersible Boc-Phe-OH nanoparticles were produced by dispersion-based processes with wet milling [28, 29, 30]. Boc-Phe-OH was ground with zirconia beads in aqueous 0.2% Triton X-100 soultion using a planetary ball mill for 4 h at room temperature. The beads were removed by filtration to obtain water-dispersible Boc-Phe-OH nanoparticles. The dispersed nanoparticles were found by DLS analysis to have a mass mediam diameter of 327.3 \(\pm\) 14.1 nm (Figure 2). We also prepared several hydrophobic Boc-amino acid nanoparticles, Boc-Leu-OH, Boc-Tyr(\(\mathrm{H}\)Bu)-OH, as a nanoparticle formulation using the same wet milling process. In the case of Boc-Val-OH, the nano-based materials, which the oily substrates were dispersed in aqueous phase uniformly, were obtained after wet milling. In the Boc strategy, the hydrophobic Bzl type groups are generally the first choice for the side chain protection step. We also successfully prepared water-dispersible Boc-Tyr(\(\mathrm{BrZ}\))-OH, which has a hydrophobic BrZ side chain protecting group [11]. \begin{table}
\begin{tabular}{l l l} \hline \hline \multicolumn{1}{c}{**Step**} & \multicolumn{1}{c}{**Reagents**} & \multicolumn{1}{c}{**Time**} \\ \hline Washing & Water & 3 min \(\times\) 10 \\ Coupling reaction & Water-dispersible Boc-amino acids & 3 min \\  & (3 equivalent) DMTMM, NMM, MW; 70 \({}^{\circ}\)C; 70 W & 3 min \\ Washing & 0.2\% Triton X-100 solution & 3 min \(\times\) 5 \\ Washing & EtOH & 3 min \(\times\) 3 \\ Deprotection & TFA & 10 min \(\times\) 2 \\ Washing & 0.5 mol/L NaHCO\({}_{3}\) solution & 3 min \(\times\) 3 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Protocol for solid-phase peptide synthesis using a MW assisted coupling reaction in water. WSCI [31], DMTMMM [32,33] and combinations of an additive sulfo-HOSu [34] were examined [24]. Reaction temperature was 70 \({}^{\circ}\)C using a MW power of 70 W (Figure 1). The results are summarized in Table 1. The coupling reaction using WSCI in combination with sulfo-HOSu was completed within 3 min (Entry 2). Surprisingly, in the case of DMTMM (Entry 6), the reaction was completed in only 1 min. In the previous work using Fmoc-amino acid nanoparticles, we have studied about efficiency of these aqueous MW-assisted coupling conditions. Even when using Fmoc amino acid nanoparticles, DMTMM showed a high reactivity in the aqueous reaction with MW irradiation [24]. These results were suggested that the microwave radiation in highly polar water-based solvent systems leads to very smooth reactions between aqueous nanoparticles and the solid support. To evaluate the effectiveness of our procedure for MW assisted solid-phase synthesis in water using Boc chemistry, the preparation of the model peptide Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH, an endogenous opioid peptide) was investigated. The reaction was carried out in aqueous solution according to the protocol described in Table 2. The synthesis was carried out on an HMBA-PEG grafted resin, which is compatible with Boc chemistry [35]. The first amino acid, Boc-Leu-OH was introduced on the resin using DIC and DMAP in DMF. The water-dispersible Boc-Phe-OH and Boc-Tyr(fBu)-OH nanoparticles were prepared by wet-milling in the presence of Triton X-100. Boc-Gly-OH was dissolved in aqueous 0.2% Triton X-100 solution. Boc-Phe-OH, Boc-Tyr(fBu)-OH nanoparticles and Boc-Gly-OH solution were serially coupled onto H-Leu-HMBA-PEG grafted resin. Coupling reactions were carried out by DMTMMM with MW radiation set to a power of 70 W for 3 min at 70 \({}^{\circ}\)C. The Boc group was removed using TFA.