Fmoc-Aib-Aib-OH

De novo antimicrobial peptides with the sequences: (KLAKKLA)n, (KLAKLAK)n (where n = 1,2,3), (KALKALK)3, (KLGKKLG)n, and (KAAKKAA)n (where n = 2,3), were prepared as the C-terminus amides. These peptides were designed to be perfectly amphipathic in helical conformations. Peptide antibacterial activity was tested against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Peptide cytotoxicity was tested against human erythrocytes and 3T3 mouse fibroblasts. The 3T3 cell testing was a much more sensitive test of cytotoxicity. The peptides were much less lytic toward human erythrocytes than 3T3 cells. Peptide secondary structure in aqueous solution, sodium dodecylsulfate micelles, and phospholipid vesicles was estimated using circular dichroism spectroscopy. The leucine/alanine-containing 21-mers were bacteriostatic at 3-8 microM and cytotoxic to 3T3 cells at about 10 microM concentrations. The leucine/alanine- or leucine/glycine-containing 14-mers and the leucine/glycine 21-mer were bacteriostatic at 6-22 microM but had much lower cytotoxicity toward 3T3 cells and higher selectivities than the natural antimicrobial peptides magainin 2 amide and cecropin B amide. The 7-mer peptides are devoid of biological activity and of secondary structure in membrane mimetic environments. The 14-mer peptides and the glycine-containing 21-mer show modest levels of helicity in model membranes. The leucine/alanine-containing 21-mer peptides have substantial helicity in model membranes. The propensity to alpha-helical conformation of the peptides in amphipathic media is proportional to their 3T3 cell cytotoxicity.

A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50-80% alpha,alpha-dialkylated glycines (alpha,alpha-Dags) were synthesized on solid-phase resin as the C-terminus amides using N-alpha-fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in alpha,alpha-Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 microm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 microg for melittin, whereas 500 microg doses were nonlethal for many of the other peptides. Several of the alpha,alpha-Dag-rich peptides selectively destroy B. abortus-infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.