3.5.1. Frontier Molecular Orbital Energy of Peptides

To convince the antioxidant performance of four active amino acids, 22 synthesized dipeptides were prepared, and their quantum chemical parameters were obtained by DFT calculations [38]. As shown in Table 2, the frontier molecular orbital energy of each dipeptide, expressed by EHOMO (Energy of highest occupied molecular orbital) and ELUMO (Energy of lowest unoccupied molecular orbital), represents the active level of 22 synthetics [26]. Theoretically, a higher EHOMO means more unstable electrons, which are more likely to scavenge free radicals as hydrogen donors. It was seen from Table 3 that the free radical-scavenging ability of these dipeptides, according to their calculated EHOMO, ranked from high to low in the order WH > WP > WY > WC > WD > MW > MH > YH > MY > YP > MP > CH > MD > MC > YD > IR > YC > KP > KD > AH > CP > CD. Moreover, a lower energy gap (E-gap) represents a higher chemical reactivity [39]. It means that the antioxidative activity of 22 dipeptides, based on their energy gap, ranked from strong to poor in the order WH > WC > WY > WP > WD > MW > MC > MH > MD > MY > YH > YC > YD > YP > CH > KD > CD > IR > AH > MP > KP > CP. To evaluate the real antioxidation of the 22 peptides, their inhibition towards linoleic acid oxidation was examined (see Figure 1b and Table 3). As a result, the best-synthesized dipeptide which retards the oxidation of linoleic acid was WY, followed by peptides MY, MW, YH, MH, MC, WC, YC, MD, WD, CP, YD, CH, CD, YP, MP, WH, KD, WP, AH, IR, and KP. It was noted that the dipeptides including KP, KD, IR, and AH with low EHOMO and high E-gap values had a low antioxidant capacity, whereas the dipeptides like WC, WY, MW, MC, MY, MH, and YH which have high EHOMO and low E-gap values showed a higher inhibition towards the oxidation of linoleic acid (see Table 3). Although several dipeptides had a poor activity in inhibiting the oxidation of linoleic acid, most of the synthesized dipeptides showed good antioxidant activity, especially these dipeptides containing W, M, or Y residue. It means that there is a corresponding relationship between the antioxidant activity of a dipeptide and its frontier molecular orbital energy.


**Table 3.** Frontier molecular orbital energy(eV) and GE (mM/mM) values of the dipeptides against the oxidation of linoleic acid.

To clarify the possible active site of each peptide, we constructed the dimensional structures of 22 synthesized dipeptides as well as FSEY. It is well known that HOMO always acts as the active site of any organic compound [40]. As shown in Figures 2 and 3a, the HOMOs of all dipeptides as well as FSEY are located at their active amino acid residues, that is, C, W, M, or Y. It indicates that these amino acids will firstly lose their electrons once when interacting with free radicals [41]. Clearly, data from the analysis of the HOMO site and linoleic acid oxidation inhibition confirmed the significance of active amino acid

residues in determining the activity of antioxidant peptides. The results from Hougland also et al. supported our findings [42].

**Figure 2.** HOMO distribution of the dipeptides. Note: red ball represents the oxygen atom; blue ball represents the nitrogen atom; dark gray ball represents the carbon atom; yellow ball represents the sulfur atom; light gray ball represents the hydrogen atom.

**Figure 3.** Quantum chemical parameters of FSEY. (**a**) shows HOMO distribution of peptide FSEY; (**b**) stands for Fukui functions; (**c**) means the predicting sites of FSEY more prone to a nucleophilic, electrophilic or radical attack. Note: red ball represents the oxygen atom; blue ball represents the nitrogen atom; dark gray ball represents the carbon atom; light gray ball represents the hydrogen atom.
