# Synthesis, Characterization and Biological Investigation of New N-Modified Spinorphin Analogs
## 2 Results and Discussion
### Physicochemical Characterization
#### 2.2.3 Voltamperometric Characterization  
A cyclic voltammetric study to demonstrate the preservation of the integrity of the rhodamine-spinorphin molecule in contact with charged surfaces was performed in a phosphate buffer solution medium with a pH close to the physiological pH. Our study revealed that in an electrochemical environment spinorphin derivatives are reduced/oxidized leading to the production of two reduction peaks located at Ep\(\sim\)1.70 and \(-\)1.00 V (Table 3, Figure 7). The peak at the more negative values of the potential can be attributed to the reduction of the rhodamine part, as the difference between the potentials of the anodic and cathodic peaks is \(<\)10 mV (\(\Delta\)Ep = Epc \(-\) Epa) [33] as well as the ratio of peak heights: Ipc/Ipa \(>\) 1 suggesting the quasi-reversible nature of the electrode process. The reversibility of the electrode reaction is also evidenced by the fact that the current intensity increases with the increase of scan rate from 0.2 to 1.60 V.s\({}^{-1}\) (Figure 7). The second peak is due to the electrochemical exchange of electrons from the amino acid sequence, most likely occurring from the tyrosine moiety. The regression equations of the function Ipc = nFACoksh (Reinmuth expression, where: Ipc is peak current, n--the number of electrons in the redox reaction of a molecule, A--the electroactive surface area, C\({}_{0}\)--the concentration of analyte) were used to evaluate the heterogeneous electron transfer rate constants at the rhodamine moiety. The obtained value with the order of 10\({}^{-6}\) cm s\({}^{-1}\) (higher than 5.0 \(\times\) 10\({}^{-6}\) cm s\({}^{-1}\)) characterizes the rhodamine reduction process as quasi-reversible (Table 4). The diffusion coefficients (D) of all compounds (Table 4) were determined using the Randles-Scek equation and the number of electrons, n = 2 [34]. The (D) values of peptide molecules varied in the sequence: Rh-S > Rh-S5 > Rh-S6 demonstrating the hypothesis that bulky molecules move at a lower speed towards the charged electrode surface. Figure 6: Emission spectra of spinorphin derivatives at \(\lambda^{\mathrm{ex}}\) = 275 nm and different pHs. The inset plots represent I(emission)/pH obtained from the emission spectra of the respective compounds. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Compound** & **Regression Equation of ipc, A = f(C, mol L\({}^{-1}\))** & \(\mathbf{k^{o}}_{\mathbf{sh}}\times\mathbf{10}^{-6}\) cm s\({}^{-1}\)** & \(\mathbf{D\times 10}^{-3}\) cm\({}^{2}\) s\({}^{-1}\)** \\ \hline
**Rh-S** & Ip = 2.50 \(\times\) 10\({}^{-8}\)+ 0.0348(\(\pm\)0.0023) \(\times\) C, R\({}^{2}\) = 0.991 & 6.01 & 3.23 \\ \hline
**Rh-S5** & Ip = 8.03 \(\times\) 10\({}^{-8}\)+ 0.0511(\(\pm\)0.0010) \(\times\) C, R\({}^{2}\) = 0.998 & 8.82 & 4.28 \\ \hline
**Rh-S5** & Ip = 8.67 \(\times\) 10\({}^{-8}\)+ 0.0533(\(\pm\)0.0022) \(\times\) C, R\({}^{2}\) = 0.995 & 9.21 & 4.34 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Equation of regression of Ipc vs. concentration (C, mol L\({}^{-1}\)); heterogen rate constant and diffusion coefficient of Rh-S, Rh-S5, and Rh-S6 peptide derivatives at HMDE electrode (with electrode area, A = 0.030 cm\({}^{2}\)).