# Synthesis, Characterization and Biological Investigation of New N-Modified Spinorphin Analogs
## 2 Results and Discussion
### Physicochemical Characterization
#### 2.2.1 Determination of Partition Coefficient
As can be seen, the structure of the studied compounds contains two of the known fluorescent amino acids, tryptophan and tyrosine [27; 28; 29]. With both amino acids present in the indicated complex mixture, the fluorescence in the 300-400 nm range is more likely to originate from the indole group of the tryptophan residue, which is known to emit at \(\sim\)350 nm at an excitation wavelength of 280 nm [28]. Moreover, spectra were recorded at different pHs of the medium to determine the study compounds' acid constants and isoelectric points. All compounds show similar behavior of fluorescence properties with minimal difference in wavelength where the compound absorbs in the visible region (1-2 nm). A strong decrease in the intensity of the emission maximum at \(\lambda^{\text{em}}\) = 585 nm is observed with an increase in the pH of the medium, which is due to the structural changes associated with the closure of the spirolactam ring at rhodamine [30]. pH also affects the fluorescence emission intensity of the tryptophan/tyrosine spectral line [31]. The fluorescence emission intensities of tryptophan and tyrosine were highest when the pH ranged from 6.5-7.5 and sharply decreased at pH \(>\) 7.5 [30]. In our case, we observed a sharp decrease in emission intensity occurs at pH \(>\) 9, as shown in Figure 5, which may be influenced by the presence of \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Compounds**} & \multirow{2}{*}{\(\varepsilon\), [L/mol.cm)]} & \multirow{2}{*}{\(\varepsilon\), [L/mol.cm)]} & \multirow{2}{*}{\(\lambda^{\text{em}}\) [nm]} & \multirow{2}{*}{\(\lambda^{\text{em}}\) [nm] at [cm\({}^{-1}\)] against \(\lambda^{\text{ex}}\) = 560 nm} & \multirow{2}{*}{\(\lambda^{\text{ex}}\) = 560 nm} & **Stokes shift** \\  & & & & & [cm\({}^{-1}\)] against \(\lambda^{\text{ex}}\) = 560 nm & \(\lambda^{\text{abs}}\) = 557 nm \\ \hline
**Rh-S** & 3.73 \(\times\) 10\({}^{6}\) & 2.14 \(\times\) 10\({}^{6}\) & 356 & 585 & 859 \\ \hline
**Rh-S5** & 6.19 \(\times\) 10\({}^{6}\) & 2.28 \(\times\) 10\({}^{6}\) & 356 & 584 & 860 \\ \hline
**Rh-S6** & 6.54 \(\times\) 10\({}^{6}\) & 3.22 \(\times\) 10\({}^{6}\) & 356 & 585 & 859 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Photophysical characterizations of rhodamine-peptide derivatives. Figure 4: The UV–Vis absorbance spectra of equal concentration of Rh-S, Rh-S5, and Rh-S6 at pH 7.413 (phosphate buffer solution) against water. a rhodamine moiety. The obtained emission spectra at different pHs helped us to determine the acidity constants of the obtained compounds. pK values were obtained by selecting the inflection points of the I/pH curve at two wavelengths: 275 nm, where tryptophan emits, and 575 nm for the rhodamine part, respectively (Figures 5 and 6, inserted graphs). At these points the concentrations of conjugated acid and base are equal, i.e., [HRh-S] = [Rh-S-], and according to the known relationship pH = pKa + [Rh-S-]/[HRh-S] at this point pKa = pH. Equations (1) and (2) given in the experimental part were also used to calculate the acid constants to confirm the values. Table 1 shows the average values of the constants from the repeatable results. As can be seen, the acid properties increase in the order: for pKa1: Rh-S < Rh-S6 < Rh-S5 and for pKa2: Rh-S5 < Rh-S6 < Rh-S, and in general the values for pKa2 are close to those under basic conditions and lower than those of similar rhodamine-peptide compounds [24]. The proven higher values of the constants given in [24] are due to the influence of the -OH group of tyrosine, located near the spirolactam ring of rhodamine and leading to its more effortless opening. Here, significantly, the tyrosine residue is in a more distant position, and its influence on \"ring Open-ON\" is attenuated. Based on the obtained values of the protolytic constants, the isoelectric points were calculated where the compounds would show zero charge and reduced solubility (Table 1).