Synthesis and Purification of Surfactant Peptides

MB (34 amino acid sequence: NH2-CWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCSCOOH; see Fig. 2B), S-MB (41 amino acid sequence: NH2-FPIPLPYCWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS-COOH; see Fig. 2A) and SP-B(1–8) [8 amino acid sequence: NH2-FPIPLPYC-CONH2] were prepared with either a ABI 431A solid phase peptide synthesizer (Applied Biosystems, Foster City, CA) configured for FastMoc™ chemistry [54] (link), a Symphony Multiple Peptide Synthesizer (Protein Technologies, Tucson, AZ) using standard Fmoc synthesis, or a Liberty Microwave Peptide Synthesizer (CEM Corp., Matthews, NC) configured for standard Fmoc synthesis. A low substitution (0.3 mmole/gm) pre-derivatized Fmoc-serine (tBu) Wang resin (NovaBiochem, San Diego, CA) or H-Ser(OtBu)-HMPB Nova PEG resin (NovaBiochem, San Diego, CA) were used to minimize the formation of truncated sequences with the MB and S-MB peptide, while a Rink Amide MBHA resin (NovaBiochem, San Diego, CA) was employed for synthesis of the SP-B(1–8) peptide. All residues were double-coupled to insure optimal yield [48] (link). After synthesis of the respective linear sequences, peptides were cleaved from the resin and deprotected using a mixture of 0.75 gm phenol, 0.25 ml ethanedithiol, 0.5 ml of thioanisole, 0.5 ml of deionized water and 10 ml trifluoroacetic acid per gram of resin initially chilled to 5°C, and then allowed to come to 25°C with continuous stirring over a period of 2 h to insure complete peptide deprotection [48] (link). Crude peptides were removed from the resin by vacuum-assisted filtration, and by washing on a medium porosity sintered glass filter with trifluoroacetic acid and dichloromethane to maximize yield. Filtered crude peptides were precipitated in ice cold tertiary butyl ether, and separated by centrifugation at 2000×g for 10 min (2–3 cycles of ether-precipitation and centrifugation were used to minimize cleavage-deprotection byproducts). Reduced crude peptides from ether-precipitation were verified for molecular mass by MALDI-TOF spectroscopy, dissolved in trifluoroethanol (TFE):10 mM HCl (1∶1, v∶v), freeze dried, and purified by preparative HPLC [48] (link). Final folding of HPLC-purified peptides was facilitated by air-oxidation for at least 48 h at 25°C in TFE and 10 mM ammonium bicarbonate buffer (4∶6, v∶v) at pH 8.0 [55] (link). Final oxidized MB and S-MB were re-purified by reverse phase HPLC, verified in molecular mass via MALDI-TOF, and disulfide connectivity was confirmed by mass spectroscopy of enzyme-digested fragments (trypsin and chymotrypsin digestion).