# Synthesis, Characterization and Biological Investigation of New N-Modified Spinorphin Analogs
## 2 Results and Discussion
### Physicochemical Characterization  
#### 2.2.1 Determination of Partition Coefficient  
The effectiveness of biomolecules is mainly due to specific structural and physico-chemical properties. In principle, the properties of peptides/proteins are very diverse and directly related to their bioactivity. So for example, solubility is an important factor when preparing solutions for introducing peptides into the body, as some peptides dissolve in water, others in dilute salt solutions, and there are also entirely insoluble ones, exhibiting more lipophilicity. With the manifestation of hydrophilic properties, the occurrence of a hydration process is possible; swelling, increasing their mass and volume, while it is possible to partially limit the solubility of the molecule, in which case the peptide molecule becomes more unstable. At the isoelectric point, peptides have the least ability to bind water, i.e., to hydrate; the hydration shell around the peptide molecules breaks down so that they combine to form, for example, aggregates. When the pH of the medium changes, the peptide macromolecule is charged, and its hydration capacity changes. The rhodamine peptide derivatives showed properties as neutral molecules with partition coefficient values from 0.54 to \(-\)0.2, determined by the \"shake-flask method\" in a 1-octanol/buffer system (pH 7.4) (Figure 3). Due to the obtained values, the studied compounds can be arranged in the following sequence from hydrophilic to hydrophobic (lipophilic): Rh-S > Rh-S5 > Rh-S6. This corresponds to the data from the morphology of the compound, where an analogous order of increasing porosity/hydroophilicity was found (Figure 3). It can be seen that the replacement of natural proline with unnatural steric restricted amino acids such as 1-aminocyclopentancarboxylic acid (Ac5c) and 1-aminocyclohexancerboxylic acid (Ac6c) (neuropeptides Rh-S5 and Rh-S6) leads to an increase in lipophilicity, which would assist passage through the brain barrier and increase the efficiency of the molecule. This can be seen in the study of anticonvulsant activity at the subsequent point in this manuscript. 2.2 Determination of Acid-Base Constant (pK\({}_{\text{a}}\)) and Isoelectric Point (pJ) by Spectral Methods  
The amino acid sequences in the spinorphin derivatives and the rhodamine moiety show identical absorption characteristic bands in the UV-Vis region for chromophoric groups present in similar peptide compounds (Figure 4) [24]. The spectra related to the absorption of electromagnetic radiation of the spinorphin derivatives have four well-shaped absorption peaks in phosphate buffer solution, with different intensities depending on the structure of the compounds; located at \(\lambda\)max \(\approx\) 233 and \(\approx\)275 nm, 315 nm and 557 nm, respectively (Figure 4). As known in the literature these are the intrinsic absorption maxima for the \(\pi\)-\(\pi\)\(\ast\) transitions of >C=O in peptide bonds (180 to 230 nm), the absorp  
Figure 3: Logarithmic values of the partition coefficient (log P) of the investigated compounds: Rh- S, Rh-S5, and Rh-S6. tion of the \(\pi\)-electron systems of the aromatic groups of the indole side chains of Trp, the phenolic rings of Tyr and rhodamine (\(\approx\)300 nm), and n-\(\pi\)\(\ast\) transitions in rhodamine (561 nm) [25; 26]. The molar extinction coefficients at the long-wavelength absorption maximum are, respectively: Rh-S (\(\varepsilon\) = 2.14 \(\times\) 106 cm.mol.L\({}^{-1}\)); Rh-S5 (\(\varepsilon\) = 2.28 \(\times\) 106 cm.mol.L\({}^{-1}\)); Rh-S6 (\(\varepsilon\) = 3.22 \(\times\) 106 cm.mol.L\({}^{-1}\)) (Table 2). As can be seen, the Rh-S6 exhibits the highest sensitivity. When studying the fluorescence properties, the emission spectra were recorded upon excitation at two wavelengths: \(\lambda^{\text{ex}}\) = 275 nm where tryptophan/tyrosine fluoresces, and at \(\lambda^{\text{ex}}\) = 560 nm where excitation of the rhodamine moiety is possible (Figures 5 and 6).