*2.2. Peptide Synthesis, Purification*

Peptides dyed with carboxyfluorescein (5(6)-FAM) were synthesised in-house following conventional solid-phase peptide synthesis (SPPS). After synthesis, the peptides were purified to show a minimum purity of >95%. Peptides without the dye exhibited a purity of >98% and were purchased from ProteoGenix, Schiltigheim, France. All the amino acids used in SPPS (i.e., Fmoc–Ala–OH, Fmoc–Asp(OtBu)–OH, Fmoc–Glu(OtBu)–OH, Fmoc–Phe–OH, Fmoc–Gly–OH, Fmoc–Ile–OH, Fmoc–Lys(Boc)–OH, Fmoc–Leu–OH, Fmoc–Asn–OH, Fmoc–Gln–OH, Fmoc–Pro–OH, Fmoc–Arg(Pbf)–OH) were purchased from AGTC Bioproducts Ltd., Itlings Lane Hessle, UK, with a purity of >98 % purity, which were side-chain protected where appropriate. Preloaded Wang resin with Fmoc-protected amino acids (Fmoc–Arg(Pbf) Wang, Fmoc–Glu(OtBu) Wang, Fmoc–Lys(Boc)–OH) with peptide substitutions in the range of 0.21–5.3 mmol/g and o-benzotriazole–N,N,N ,N –tetramethyl–uronium–hexafluoro-phosphate (HBTU) of >98% purity were purchased from NovaBiochem, Merck Life Science UK Limited, Watford, UK. Anhydrous N,N dimethylformide (DMF), anhydrous dichloromethane (DCM), 5(6)-FAM, piperidine/DMF 20% mix (*v*/*v*), ninhydrin, trifluoroacetic acid (TFA), and triisopropylsilane (TIPS) were all HPLC grade with >99% purity, which were purchased from Sigma Aldrich, Merck Life Science UK Limited, Dorset, UK. Anhydrous diethyl ether of HPLC grade used for precipitation of peptides was purchased from Fisher Scientific UK Ltd., Loughborough, UK. SPPS reactions were carried out in Aldrich® System 45™ vessels with caps and fritted discs purchased from Sigma Aldrich, Merck Life Science UK Limited, Dorset, UK. Acetonitrile and TFA used for peptide purification and for purity analysis with high-pressure liquid chromatography (HPLC) were of HPLC grade and were purchased from Sigma Aldrich, Merck Life Science UK Limited, Dorset, UK.

Once ready for cleavage, the dried Wang resin beads were weighed and transferred from the reaction vessel into a 50 mL round-bottom flask equipped with a magnetic stirrer. A fresh cleavage mixture made of TFA, dH2O, and TIPS (*v*/*v*/*v* = 95/2.5/2.5) was then prepared and 10 mL per 0.1 g of dried resin beads was added to the Wang resin beads in the round-bottom flask. The flask was then carefully closed and wrapped with aluminium foil to prevent photo-bleaching. The reagents were left to react under gentle and continuous stirring for 3–5 h. Subsequently, the Wang resin beads were removed by vacuum filtration and the peptides were dissolved in the cleavage mixture. The peptides were then precipitated by adding the solution dropwise to ice-cold diethyl ether. To ensure maximum precipitation, the diethyl ether was kept overnight at −20 ◦C. The precipitate was then separated from the supernatant by centrifugation at 13,500 rpm and air-drying overnight at −20 ◦C. The dried crude peptide powder was dissolved in dH2O and then freeze-dried using a LyoDry Midi freeze dryer (MechaTech Systems Ltd., Bristol, UK).

Labelling of the synthesised peptides with the fluorescence dye 5(6)-FAM was performed while the molecules were still attached to the Wang resin. The reaction is similar to the amino acid coupling. A mixture of 5 equiv 5(6)-FAM and 4.9 equiv HBTU dissolved in DMF (≈3 mL) was placed in a reaction vessel containing a known amount of washed Wang resin with a previously deprotected N-terminal peptide sequence. The reaction vessel was wrapped with aluminium foil to prevent photo-bleaching and was placed on an orbital shaker. The reaction mixture was left to react overnight under continuous shaking (≈200 rpm). It was then washed three times with DMF (6 mL) and three times with DCM (6 mL). After the washing procedure, the resin beads were dried under vacuum until all of the DCM was removed.
