*2.8. Peptide Synthesis*

All the peptides derived from alignments with proteins of bovine milk proteome were subjected to synthesis, employing a solid-phase technique performed on a fully automated peptide synthesizer (Syro II, MultiSynTech Gmbh, Witten, Germany). Wang resins preloaded with the first Nα-Fmoc-protected amino acid were utilized for stepwise assembly of the entire peptide chain. This assembly was done according to the Fmoc standard strategy and was based on the use of HATU as the coupling reagen<sup>t</sup> [23,24]. The side-chain protected amino acid building blocks utilized in these peptide syntheses were Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Thr(tBU)-OH. A step of deprotection of the final peptides was conducted, followed by cleavage from the resin with a mixture of 88% (*v*/*v*) trifluoroacetic acid (TFA) with 5% phenol ( *w*/*v*), 5% H2O (*v*/*v*) and 2% (*v*/*v*) of triisopropylsylane via shaking at RT for 2.5 h. A step of vacuum filtration allowed to remove the resin from the assembled peptide chains. Then, the peptides were precipitated with cold diethyl ether and transformed into pellet by a centrifugation procedure. Two washes with cold diethyl ether were performed on the precipitated peptides. At the end, purification of the crude peptides was done through flash chromatography (SP1, Biotage, Uppsala, Sweden) on a Biotage SNAP Ultra C18 12 g cartridge packed with Biotage HP-Sphere C18 25 μm spherical silica. A final step of molecular mass confirmation was performed by mass spectroscopy on a MALDI-TOF/TOF mass spectrometer (ABI 4800, AB Sciex, Framingham, MA, USA). The mechanism of action analysis was conducted on four selected peptides, **N-15-M**, **E-11-F**, **Q-14-R** and **A-17-E.**
