R. B. Merrifield,* L. D. Vizioli, and H. G. Boman abstract: Cecropin A (l-33) was synthesized by an improved stepwise solid-phase method. The synthesis was designed to give high coupling yields and minimal amounts of byproducts. All coupling steps were monitored for completion by a new ninhydrin procedure, and the fully protected peptide-resin was analyzed for deletion peptides by the solid-phase Edman preview technique. Both methods indicated that the average coupling yield was> 99.8%. The unpurified peptide mixture resulting from HF cleavage and extraction into 10% acetic acid was analyzed by reverse-phase high-pressure liquid chromatography, and93% of the total product was shown to be the desired [Trp (For) 2] cecropin A (l-33), indicating an average yield per synthetic cycle of 99.8%. Removal of the formyl group at pH 9, followed by ion-exchange chromatography, gave the purified product. Cecropin A (l-33) showed antibacterial activity against both Gram-positive and Gram-neg-ative bacteria. Against Escherichia coli, the activity was only slightly lower than that of the natural 37-residue cecropin A when tested over a 100-fold concentration range; the minimum inhibitory concentration was approximately 1/xM. The formyl derivative was somewhat less effective in killing E. coli than the free 1-33 peptide. The antibacterial activity was discussed in terms of an amphipathic a-helix structureand the binding of the peptide to bacterial membranes. e cecropins are a newly discoveredclass of antibacterial peptides produced by the humoral immune response of certain insects (Hultmark et al., 1980; Boman & Hultmark, 1981; Boman & Steiner, 1981). Cecropins and about 10 other im-mune proteins are induced in the hemolymph of the pupae of the giantsilk moth Hylophora cecropia following injection of live bacteria. The first cecropins to be purified to homogeneity and for which tentative primary structures are available are the A and B forms (Steiner et al., 1981). They each contain 37 amino acid residues, with a basic N-terminal region and a hydrophobic C-terminal region ending with a blocked car-boxyl groups. Cecropins A andB are strongly homologous but differ significantly in structure and function from other known basic peptidessuch as melittin. The cecropins are antibacterial against a variety of Gram-negative bacteria and therefore differ from lysozyme, which is effective only against certain Gram-positive bacteria. The f From the Rockefeller University, New York, New York 10021 (RBM and LDV), and the Department of Microbiology, University of Stockholm, S-10691 Stockholm, Sweden (HGB). Received March 17, 1982. The work was supported by Grant AM01260 from the US Public Health Serviceand by a grant from the Swedish Natural Science Foundation to HGB (BU2453-108). range of susceptible pathogens is broader than that for melittin, and in addition, the latter is lytic for Chang liver cells whereas cecropins have no effect on these cells or on sheep erythrocytes or insect cells in tissue culture (Steiner et al., 1981). A synthetic program on the cecropins has been undertaken with the objectives of confirming their structure, providing sufficient material to enable more extensive studies on their mode of action, and examining the role of their very interesting structures on their bactericidal activity. This paper describes in detail the solid-phase synthesis of cecropin A (l—33), which at the initiation of the work was believed to represent the complete sequence of the molecule. The structure of the synthetic protected peptide-resin I is Boc-Lys (ClZ)-Trp (For)-Lys (ClZ)-Leu-Phe-Lys (ClZ)-Lys (ClZ)-Ile-Glu (OBzl)-Lys (ClZ)-Val-Gly-Gln-Asn-Ile-Arg (Tos …