The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 9-10 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.

Lipopeptides, carrying the N-terminal lipoamino acid 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid (Pam3Adh-OH, 1), were obtained by solid-phase synthesis and by synthesis in solution. 2-Amino-6,7-dihydroxyheptanoic acid (Adh) can be regarded as a methylene analogue of S-glycerylcysteine, the N-terminal amino acid of lipoprotein from the outer cell membrane of Escherichia coli (a methylene group substitutes for the sulfur atom). The lipopeptides Pam3Adh-Ser-Ser-Asn-Ala 2a-d, in which the four possible stereoisomers of Pam3Adh-OH (2S,6S)-1 (1a), (2S,6R)-1 (1b), (2R,6S)-1 (1c), and (2R,6R)-1 (1d) are linked to the naturally occurring sequence Ser-Ser-Asn-Ala of the N-terminus of lipoprotein, and also Pam3Adh-Ser-(Lys)4 [2S,6S)-3), with a peptide part rendering the molecule water soluble, were capable of stimulating murine splenocytes polyclonally in vitro, as determined in a proliferation assay and in a hemolytic plaque assay against trinitrophenylated sheep erythrocytes. The diastereomers (2S,6S)-2 and (2R,6S)-2 with S-configurated C-6 were more active than the diastereomers (2S,6R)-2 and (2R,6R)-2 with R-configurated C-6; a change of the configuration at C-2 had less effect on the stimulatory activity. (2S,6S)-2 and (2S,6S)-3 are potent immunoadjuvants. A significantly enhanced primary immune response against trinitrophenylated sheep erythrocytes was obtained in vitro at lipopeptide concentrations of about 5 micrograms/mL and an immunization dose of 10(7) sheep erythrocytes/mL. Balb/c mice, which were immunized with a mixture of ovalbumin and (2S,6S)-2 or (2S,6S)-3, respectively, had a substantially higher antiovalbumin titer 28 days after immunization than mice which had received ovalbumin, (2S,6S)-2 or (2S,6S)-3 alone. Finally, the novel lipopeptides constitute potent macrophage activators: (2S,6S)-3 was able to induce tumor cytotoxicity against the tumor cell line L929 in bone marrow derived macrophages.

We demonstrate in a quantitative in vitro induction assay that tetracycline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor-tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe(2+)-dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in the presence of the drug, whereas about 1% cleavage is obtained after 30 min in the presence of Fe2+ without tetracycline. Cleavage is not inhibited by several radical scavengers, suggesting a highly localized reactivity of the redox-active oxo intermediates in the proximity of the Fe(2+)-tc chelater where they are generated. The products can be separated by HPLC only after denaturation, indicating that the complex is not disrupted by cleavage. Residues at which the cleavage takes place are identified using the masses of the fragments determined by electrospray mass spectrometry and their N-terminal sequences. The major cleavage site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cleaved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycline-Mg2+ complex. We discuss potential reaction mechanisms leading to protein cleavage.

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