CPP (5[6]-carboxyfluorescein-RRRRRR-COOH) synthesis was performed on a 1-mmol scale on preloaded Fmoc-Nw-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine Wang resin (Sigma-Aldrich, Poole, UK) (loading rate 0.62 mmol g
−1) using standard Fmoc–amino acid solid-state peptide synthesis protocols. The initial Fmoc group was removed by deprotection with 20% piperidine in
N,N-dimethylformamide (DMF) (3 × 20 minutes), followed by coupling with Fmoc-Nw-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine in the presence of
O-(6-chlorobenzotriazol-1-yl)-
N,
N,
N′,
N′-tetramethyluronium hexafluorophosphate (HCTU), N,N-diisopropylethylamine (DIPEA), and DMF in a 1:5:5:10 molar ratio. The reaction was agitated at room temperature for 8 hours, and some of the resin was tested for reaction completion using the ninhydrin test following standard protocols.
25 The coupling and deprotection steps were repeated until the peptide sequence reached a total of six arginine residues. The final Fmoc group was removed, the resin washed with DMF and dichloromethane (DCM), and the free N-terminus was coupled with 5(6)-carboxyfluorescein in the presence of HCTU and DIPEA in a 1:5:5:10 molar ratio in DMF for 8 hours, to give an inbuilt fluorescent tag. After washes with DMF, DCM, and diethyl ether, the resin was air dried for 1 hour. The peptide was cleaved from the resin using trifluoroacetic acid (TFA) (27 mL), thioanisole (1.5 mL), 1,2-ethanedithiol (0.9 mL), and anisole (0.6 mL) under N
2 with agitation in the dark for 3 hours, with simultaneous removal of side-chain protecting groups. Resin was removed by filtration and the crude peptide precipitated out of solution in cold diethyl ether and storage at −20°C for 8 hours. The precipitate was isolated by centrifugation at 2164
g for 10 minutes and removal of the diethyl ether. The peptide was purified by preparative reversed-phase HPLC on a Phenomenex Luna C12 column (250 × 21.2 C12 [2] 10-μm Jupiter Proteo 90-Å Axia Packed [Phenomenex, Macclesfield, UK]) with a solvent mixture altered with a linear gradient from 0.1% TFA in water to 0.1% TFA in CH
3CN over 40 minutes. The isolated peptide was lyophilized to yield a yellow solid (258 mg, 19.6% yield at 97% purity), which was identified by electrospray ionization mass spectrometry. Stock solutions of the peptide were prepared in sterile PBS and stored at 4°C until use.