## 2 Results and Discussion
### Qualitative FEYNF-NH\({}_{2}\) Peptide Analysis and Purification  
Reverse-phase high-performance liquid chromatography (RP-HPLC) was used to analyze and separate the crude mixture of synthesized peptide. The HPLC profile of the raw peptide mixture FEYNF-NH\({}_{2}\) is given in Figure 2. To detect the peptide bond specifically, a targeted wavelength of 215 nm was used during the analysis. The initial separation of the raw peptide mixture followed a clearly defined pattern guided by a linear gradient, as clearly depicted in Figure S1 (in Supplementary Materials). This approach facilitated the systematic fractionation of components within the mixture, allowing for discernible identification and characterization of individual compounds based on their distinct retention times.  
The chromatographic analysis within the initial 5 min window revealed the presence of peaks attributed primarily to acetic acid and potential residual solvents employed during the synthesis process. However, a distinct compound, detected at 9.09 min of retention time, was unequivocally identified via mass spectrometry as the FEYNF peptide. The considerable intensity of this peak strongly indicates a successful synthesis process. Notably, a secondary peak appearing at 10.05 min of retention time, albeit with lower intensity, corresponds to a synthesis by-product demonstrating heightened hydrophobicity. This phenomenon, characterized by a marginally extended eluti  
Figure 1: (a) Chemical structure depiction (2D geometry) of the FEYNF-NH\({}_{2}\) pentapeptide (KingDraw V3.0.2.) highlighting \(\Phi\) (Phi) and \(\Psi\) (Psi) torsion angles; where \(\Phi=\mathrm{C}(j-1)-\mathrm{N}(j)-\mathrm{C}\alpha(j)-\mathrm{C}(j)\) and \(\Psi=\mathrm{N}(j)-\mathrm{C}\alpha(j)-\mathrm{C}(j)-\mathrm{N}(j+1)\); \(j\)—atom index in the backbone sequence; (b) schematic representation of the solid-phase peptide synthesis (SPPS) protocol (adapted from [19]).  
observed occurrence in solid-phase peptide synthesis (SPPS) using Rink amide as solid support [28].  
In this investigation, we employed a purification methodology for the FEYNF peptide via RP-SPE chromatography with gradient elution, a technique adapted from [29]. Notably, our protocol differed by introducing a crucial modification: after peptide elution with acetic acid, the mixture underwent adjustment to an optimal intermediate pH of 4.5, in accordance with the column cartridge specifications. This adjustment, within the pH range of 2-8 to prevent denaturation of the stationary phase, served a dual purpose: ensuring the integrity of the stationary phase and eliminating the need for a time-consuming freeze-drying step typically required to remove excess acetic acid. By preventing denaturation and obviating extended drying procedures, this adaptation streamlined our purification process, significantly enhancing efficiency and expediting the overall workflow.  
It should be mentioned that the determined purity of the pentapeptide FEYNF-NH\({}_{2}\) was assessed immediately after synthesis (raw product) and quantified at an impressive 95.47%. Moreover, the compounds eluted within the initial 5 min of the chromatography predominantly stem from the presence of acetic acid used for solubilization/during the washing step and residual solvents originating from the synthesis process, as well as the injection peak [30]. To guarantee the utmost quality of the peptide, a meticulous purification step was executed through solid-phase extraction. This rigorous process yielded a confirmed purity of 99.54% for the pentapeptide used in the experiments. Importantly, this achieved purity underwent thorough validation through subsequent HPLC analysis, as illustrated in Figure S2 (in Supplementary Materials).