# Synthesis, Characterization and Biological Investigation of New N-Modified Spinorphin Analogs
## 4 Conclusions  
Three new polar solvent-soluble spinorphin analogs modified at the N-terminus with rhodamine B were synthesized in high yields via a traditional solid-phase Fmoc strategy. All compounds are colored and soluble in physiological pH exhibiting absorptions at 557 nm. The modification in the spinorphin amino acid sequence by introducing the two unnatural steric restricted amino acids makes the excitation centers and electrochemical electron transfer more readily accessible. This was proved by an increase in the intensity of absorption and emission lines and current peaks in the spectral and voltametric characterization of the compounds. The data showed that at physiological pH the peptide derivatives were in the ionic form with increasing value of the distribution coefficient in the order: Rh-S6 < Rh-S5 < Rh-S. Based on the screening of the new hybrid spinorphin analogs modified at the N-terminus with rhodamine B in three seizure tests with a different mechanism of action in mice, we can suggest that the modification at the N-terminus with fluorescent dye rhodamine of the bioactive peptide spinorphin Rh-S6 has a strong capability to inhibit seizure spread while having comparable activity to the heptapeptide Rh-S against drug-resistant psychomotor seizures with the potency to elevate the threshold for clonic seizure. Biological tests against some bacterial pathogens such as _Staphylococcus aureus, Escherichia coli_, and _Bacillus cereus_ showed that all peptide derivatives were similar to the negative control results. The compounds also showed weak activity against _Candida albicans_ fungus. The results indicate the ongoing search for universal antimicrobial agents with pronounced synergism when used simultaneously as anticonvulsant, antibacterial, and antifungal agents.  
Conceptualization, P.T., S.G., J.T.; methodology, P.T., S.G., J.T.; software, S.G. and P.T.; formal analysis, P.T., S.G., P.P., J.T., S.S. and H.H.; investigation, P.T., S.G., P.P., J.T., S.S. and H.H.; writing--original draft preparations, P.T., S.G., J.T.; writing--review and editing--P.T., S.G., J.T.; visualization, P.T., S.G., J.T.; project administration, P.T., S.G. All authors have read and agreed to the published version of the manuscript.  
This work was financially supported by the Bulgarian National Scientific Fund project KII-06-J/O-02/3 (title: New opioid peptide-nanoparticle conjugates: synthesis, characterization and neuropharmacological applications) of the Ministry of Education and Science, Bulgaria. The Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia funded the Indonesian counterpart through decree No: 122/E5/PG.02.00.PT/2022 and contract No: 2386/UN36.11/LP2M/222 (title: Konjugasi Peptide Baru-Nanopartikel Berbasis Logam: Sintesis, Karakterisasi, Dan Aplikasinya Pada Bidang Farmakologi).  
All procedures were performed in agreement with the European Communities Council Directive 2010/63/EU. The experimental design was approved by the Institutional Ethics Committee. The experimental design was approved by the Bulgarian Food Safety Agency (# 300/N 5888-0183). There were no human participants.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** Data is contained within the article.  
**Conflicts of Interest:** The authors declare no conflict of interest.