Fluorescence labeling of penetratin
After coupling the last amino acid, arginine, its Fmoc protecting group was replaced by Fmoc-6-aminohexanoic acid in order to introduce a 6-carbon linker followed by removing the Fmoc group. Half of penetratin, still on the resin, was treated with 1.5 equivalents of AFDye532 N-hydroxysuccinimide ester (molecular weight 723.77 g/mol, Fluoroprobes, Scottsdale, AZ) and 1.5 equivalents of DIPEA in DMF overnight. The other half of penetratin was reacted with 1.5 equivalents of 5(6)-carboxynaphthofluorescein N -succinimidyl ester (molecular weight 573.51 g/mol, Darmstadt, Germany) in the presence of DIPEA in DMF. Completion of the coupling was assessed by Kaiser test. The labeled peptides were deprotected and released from the resin by treatment with 95:2.5:2.5 (v/v) trifluoroacetic acid (TFA)/triisopropylsilane/water and dithiothreitol for 3 hours. The solutions were filtrated, and the peptides were precipitated with a 15-fold volume of cold diethyl ether. After filtration the crude products were purified by preparative reversed-phase HPLC (JASCO, Victoria, Canada) on a C18 column and lyophilized. The purity of the products (>95%) was assessed by reversed-phase HPLC (JASCO) equipped with an analytical C18 column. The authenticity of each peptide was confirmed by Bruker electrospray ionization mass spectrometry. The predicted and measured molecular mass of the (M+H)+ form of AFDye532-penetratin was 2983.59 and 2983.548, respectively, whereas the predicted and measured molecular mass of the (M+H)+variant of NF-penetratin was 2833.33 and 2832.5, respectively.