## 2 Results and Discussion  
### Synthesis and Tyrosine Inhibition of Mimosine Dipeptides  
It has been reported that the attachment of amino acids to kojic acid has generated compounds that strongly inhibit tyrosinase [14]. Like kojic acid, mimosine is a tyrosine analog and could potentially be used for designing more potent tyrosine inhibitors [12]. Thus, we developed mimosine dipeptides and assessed their ability to inhibit tyrosinase. Our general strategy for the design of these compounds was based on the conjugation of mimosine and an amino acid through solid-phase synthesis using Fmoc chemistry. We synthesized ten mimosine dipeptides using enantiomeric amino acids, including phenylalanine (Phe), alanine (Ala), valine (Val), tryptophan (Trp), and proline (Pro). As shown in Figure 1, l- or d-amino acids were attached to Wang resin support. Mimosine was converted to Fmoc-mimosine which was then coupled to the amino acid to obtain the desired mimosine dipeptides. The chemical structures of mimosine and mimosine dipeptides are shown in Figure 2.  
The _in vitro_ assay for tyrosinase inhibition was performed using l-tyrosine as a substrate. As expected, the synthesized mimosine dipeptides inhibited tyrosinase more potently than mimosine (Table 1). In particular, conjugation of tryptophan, valine, and proline or of a d-form amino acid to mimosine led to stronger tyrosinase inhibition. Of the four most potent inhibitors, the IC\({}_{50}\) values of Mi-l-Pro and Mi-d-Trp were 13 and 17 \(\upmu\)M, respectively. The IC\({}_{50}\) of Mi-l-Val and Mi-d-Val against tyrosinase was 12 and 10 \(\upmu\)M, marginally lower than that of the positive control, kojic acid (14 \(\upmu\)M). Figure 1: General route of mimosine dipeptide synthesis. (**AA**) l- or d-amino acid; (**A**) l-mimosine isolation from _Leucaena leucocephalala_ leaves using ion-exchange resin; (**B**) Preparation of Fmoc-mimosine; (**C**) Attachment of Wang resin to Fmoc-amino acid; (**D**) Deprotection of Fmoc using 25% piperidine; (**E**) Coupling of Fmoc-mimosine and amino acid-resin mixture along and the Kaiser test; (**F**) Deprotection and cleavage using 95% trifluoroacetic acid (TFA) to afford desired mimosine dipeptides. Figure 2: The chemical structures of mimosine and mimosine dipeptides. Because of their potent inhibition of tyrosinase, we carried out a kinetic analysis of these four compounds. Lineweaver-Burk plots of 1/[V] versus 1/[S] are shown in Figure 3. The kinetic study of tyrosinase inhibition by these selected compounds revealed that the K\({}_{\mathrm{m}}\) value was increased as the concentration of inhibitors was increased, while the V\({}_{\mathrm{max}}\) values remained unchanged. Therefore, like immosine, these compounds are considered to be competitive inhibitors of tyrosinase [15]. The K\({}_{\mathrm{i}}\) value of these compounds are shown in Table 2. The inhibition constant of Mi-d-Val (0.02 mM) was less than that of Mi-l-Val, Mi-l-Pro, Mi-d-Trp, suggesting that Mi-d-Val has the most potent inhibitory effect. \begin{table}
\begin{tabular}{l c} \hline
**Compound** & **IC\({}_{50}\) (\(\upmu\)M) Mushroom Tyrosine Inhibition** \\ \hline Mi-l-Phe & \(24.1\pm 3.8\)\({}^{\mathrm{ab}}\) \\ Mi-D-Phe & \(19.7\pm 0.8\)\({}^{\mathrm{bc}}\) \\ Mi-l-Ala & \(23.1\pm 2.2\)\({}^{\mathrm{b}}\) \\ Mi-D-Ala & \(17.7\pm 3.1\)\({}^{\mathrm{bc}}\) \\ Mi-l-Pro & \(13.1\pm 3.3\)\({}^{\mathrm{bc}}\) \\ Mi-d-Pro & \(17.2\pm 2.1\)\({}^{\mathrm{bc}}\) \\ Mi-l-Val & \(11.5\pm 0.5\)\({}^{\mathrm{c}}\) \\ Mi-D-Val & \(10.3\pm 0.4\)\({}^{\mathrm{c}}\) \\ Mi-L-Trp & \(19.2\pm 1.5\)\({}^{\mathrm{bc}}\) \\ Mi-D-Trp & \(16.9\pm 2.1\)\({}^{\mathrm{bc}}\) \\ Mimosine & \(32.4\pm 1.1\)\({}^{\mathrm{a}}\) \\ Kojic acid & \(13.7\pm 1.5\)\({}^{\mathrm{bc}}\) \\ \hline \end{tabular}
\end{table} 
Table 1: IC\({}_{50}\) values of immosine and their dipeptides for mushroom tyrosinase inhibition. Figure 3: Effect of Mi-l-Pro, Mi-l-Val, Mi-d-Val, and Mi-d-Trp on tyrosinase activity. Tyrosine activity was assayed as described in the text.