# Synthesis, Characterization and Biological Investigation of New N-Modified Spinorphin Analogs
###### Abstract  
The emergence of diverse peptide derivatives has been due to constant efforts to find a specific peptide with pronounced biological activity for effective application as a therapeutic. Spinorphin-peptide products have been reported to possess various applications and properties. In the present study, spinorphin peptides with a rhodamine residue and a modification in the amino acid backbone were synthesized by a solid-phase method using Fmoc chemistry. The results obtained from the spectral and electrochemical techniques used: Scanning electron microscopy (SEM), UV-vis, fluorescence, infrared spectroscopy (IR), and voltammetry were used to elucidate the structural characteristics and some physicochemical properties to gain insight into their behavior in the solid state and in aqueous solutions with different pHs. Both Rh-S5 and Rh-S6 had compound anticonvulsant effect comparable to Rh-S against psychomotor seizures at the highest dose of 20 \(\upmu\)g. Furthermore, Rh-S6 showed a strong ability to inhibit seizure propagation and had a similar threshold to Rh-S against the intravenous pentylenetetrazol induced clonic seizure in mice; one of the three hybrid spinorphin analogs tested when screened for anticonvulsant activity. Biological tests against several bacterial pathogens such as _Stanphylococcus aureus_, _Escherichia coli_, and _Bacillus cereus_ showed similar results to negative control of the new peptide derivatives. The compounds also showed weak activity against _Candida albicans_ fungus. The antioxidant testing results revealed more than 50% activity by reviewing the radical deterrence capabilities of 2,2-diphenyl-1-picrylhydrazyl (DPPH). The results are indicative of the ongoing search for universal antimicrobial agents with pronounced synergism when used simultaneously as anticonvulsant, antibacterial, and antifungal agents. spinorphin analogs; rhodamine peptides; biological activity; anticonvulsant activity; electrochemistry; fluorimetry 2  
cytometry, fluorescence correlation spectroscopy, and ELISA. Its application in practice is due to its ability to bind covalently to biomolecules and thus can be used as a marker in bioassays [7; 8]. Therefore, Rhodamine B derivatives are essential for studying more complex biochemical processes and activities. Such kinds of noncytotoxic fluorescence-based probes of bioactive molecules possess desirable features and excellent spectral characteristics and relatively facile syntheses [9; 10; 11]. Recently, [PERSON] et.al. synthesized and studied a series of new hemorphin peptide derivatives to find more potent anticonvulsants and analgesics with no side effects [12; 13; 14]. In the present study, further attempts were carried out to synthesize and fully characterize three new N-modified analogs of spinorphin with rhodamine B and their structure-property relationships in biological action examined. Given the above mentioned properties of biopeptide spinorphin and fluorescent rhodamine dye, it was expected that the newly obtained hybrid peptide compounds would possess a synergistic effect. However, no data are available in the literature or are scarce for spinorphin and its analogs. Therefore, the synthesis, characterization, neuropharmacological evaluation, acid-base properties, and electrochemical behavior of new hybrid spinorphin analogs modified at the N-terminus with rhodamine B are presented in this study with the aim to discover a new type of bioactive signaling molecule. Spinorphin has shown a wide range of biological effects, but it is still not well developed and has few prepared synthetic analogs to date. As already mentioned, spinorphin belongs to the hemoglobin-derived peptides and, more specifically, to the non-classical opioid peptides of the hemorphin family [15; 16; 17]. Our group is one of the leading pioneers in designing, synthesizing, and characterizing these novel biopeptides. The synthesis and detailed survey of new analogs of spinorphin were conducted with several modifications in the peptide skeleton.