SYNTHESIS AND CHARACTERIZATION OF INDOLICIDIN, A TRYPTOPHAN-RICH ANTIMICROBIAL PEPTIDE FROM BOVINE NEUTROPHILS

Indolicidin, a novel tryptophan-rich microbicidal tridecapeptide amide isolated originally from granules of bovine neutrophils, has been pre pared by optimized manual and automated protocols of stepwise solid-ph ase synthesis with N-alpha-9-fluorenylmethyloxycarbonyl (Fmoc) amino a cid derivatives. Both stand ard polystyrene (PS) and polyethylene glyc ol-polystyrene (PEG-PS) graft supports were used in combination with h andles that provide C-terminal peptide amides: 5-(4-Fmoc-aminomethyl-3 ,5-dimethoxyphenoxy)valeric acid (PAL) or 5-(9-Fmoc-aminoxanthen-2-oxy )valeric acid (XAL). Final deprotection/cleavage was carried out with reagent K, trifluoroacetic acid-phenol-water-thioanisole-1,2-ethanedit hiol (82.5:5:5:5:2.5), or reagent B, trifluoroacetic acid-phenol-water -tri(isopropyl)silane (88:5:5:2), and related cocktails. Initial purit ies as high as 93% were obtained immediately following cleavage. In th e largest-scale synthesis carried out, 0.8 g of HPLC-purified indolici din(>99% pure) was obtained, representing a 39% overall yield based on C-terminal Arg(Pmc) anchored to PAL-PS-resin. The main synthetic prod uct, and some by-products, were characterized by analytical high-perfo rmance liquid chromatography (HPLC), sequencing, and fast atom bombard ment mass spectrometry (FARMS). The antimicrobial potencies of natural and synthetic indolicidin, as determined by in vitro antibacterial an d antifungal assays, were identical. Further, the reactivities of natu ral and synthetic peptides with anti-indolicidin antibody were indisti nguishable. (C) Munksgaard 1995.