Fluorescent Peptides Labeled with Environment-Sensitive 7-Aminocoumarins and Their Interactions with Lipid Bilayer Membranes and Living Cells

The photophysical properties of protected nonnatural amino acids Boc-L-Dap(7DEAC)-OMe (7DEAC: 7-diethylaminocoumarin) and Boc-L-Dap(C343)-OMe (C343: coumarin 343) were investigated to evaluate the suitability of these amino acids as fluorescent units in peptide-based fluorescent biosensors. The absorption and fluorescence spectra of Boc-L-Dap(7DEAC)-OMe and Boc-L-Dap(C343)-OMe exhibited significant red shifts with increasing solvent polarity. The fluorescence quantum yield and lifetime of Boc-L-Dap(7DEAC)-OMe solutions decreased remarkably with increasing solvent polarity, whereas those of Boc-L-Dap(C343)-OMe were slightly affected by the solvent polarity. Fluorescent peptides H-Dap(7DEAC)-LLA-OMe (1), H-Dap(7DEAC)-KLA-OMe (2), and H-Dap(7DEAC)-ELA-OMe (3) labeled with environment-sensitive 7DEAC were synthesized to examine the interactions of these labeled peptides with lipid membranes and living cells. Neutral 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and anionic 1,2-climyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) liposomes were used to investigate peptide-membrane interactions. Hydrophobic peptide 1 exhibited high affinities to both DMPC and DMPC/DMPG mixed membranes. Of the three peptides, cationic peptide 2 exhibited the strongest affinity to DMPC/DMPG membranes, whereas anionic peptide 3 showed a much lower affinity to DMPC/DMPG membranes. These results could be interpreted based on hydrophobic and electrostatic interactions between the peptides and membranes. Peptide 1 was efficiently internalized into HeLa cells, whereas peptides 2 and 3 showed a much lower intracellular delivery.