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I are reported here for first time. We have also used I are reported here for first time. We have also used two positive controls, the parent compstatin (Peptide II) and the most active peptide with natural amino acids, W4/A9 (Peptide III), and a negative control, linear compstatin with sequence IAVVQDWGHHRAT (Peptide I). Herein, we use the terms Peptides I referring to their amino acid sequences (positions 1?13) irrespective of the presence, or not, of blocking groups at the termini or pegylation andNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptChem Biol Drug Des. Author manuscript; available in PMC 2012 June 1.L ez de Victoria et al.Pagebiotinylation extensions at the C-terminus for the SPR experiments. The specifics of each sequence per experimental study is given in Tables (vide infra).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptA main aspect in the design of efficacious compstatin analogs is the balance between hydrophobicity and polarity. Enhanced hydrophobicity at key sequence positions can provide enhanced binding, whereas increasing the polarity at key sequence positions can enhance solubility of the free peptides. Solubility is important for storage and delivery of biopharmaceuticals administered in solution. The newly designed active peptides presented here pave the way for new combinations of natural and non-natural amino acids, which will have improved balance between hydrophobicity and polarity.Materials and MethodsSurface Plasmon Resonance (SPR) Compstatin peptides were synthesized by Abgent Inc. (San Diego, CA). The peptide sequences and mass spectral analysis are shown in Table 1. Three peptides served as known controls. Two were positive controls, parent compstatin (Peptide II) and W4/A9 (Peptide III), and one was negative control, linear compstatin (Peptide I). At the carboxy-terminus, the peptides were pegylated with an 8-mer PEG (polyethylene glycol) block spacer, followed by a lysine, and biotinylated, followed by an NH2 block. Four peptides were acetylated at the amino-terminus (Peptides VII ). Binding of compstatin peptides to C3 was determined by SPR with Biacore X100 (GE healthcare, Piscataway, NJ) according to previous studies23?6 with modifications. The PEG spacer in the peptides increases mobility, solubility, and accessibility, and also decreases non-specific interactions. Streptavidin sensor chip SA was used to immobilize biotinylated compstatin peptides as the ligands. PBS containing 0.05 tween-20 was used as the running buffer and the assay was carried out at 25 . Human C3 was purchased from Complement Technology Inc. (Tyler, TX). Approximately 300 of C3 were placed in a Microcon MWCO 100 kD (Millipore, Billerica, MA) and centrifuged at 10000 rcf for 15 minutes at 4 . After discarding the flow-through, 100 of running buffer was added to the C3 and the solution was centrifuged again. This process was repeated 3 times. C3 was recovered after the last run by adding 100 of running buffer at a time, for 5 times. Concentration was determined using the Beer-Lambert Law with an extinction coefficient of 1 at 280 nm of 10.3 (g/100mL)-1 cm-1 (obtained from the Complement Technology Inc. report). The compstatin peptides were immobilized onto the chip through their carboxy-terminus, as previously suggested.23 That study had shown that binding of compstatin to human C3 was not possible if the peptide was immobilized through the amino-terminus, thus revealing the importance of a free amino-terminus for t.