We have recently isolated new di- and tripeptides (rhizocticins A-D) from cultures of the bacterial strain Bacillus subtilis ATCC 6633. These phosphono-containing active substances exhibit, inter alia, fungicidal properties. This inspired us to undertake a systematic search for new hydrophilic antibiotics from the genus Bacillus subtilis. The antifungal activity, which was found in culture filtrates of B. subtilis BGSC 1E2, was competitively antagonized by di- and tri-peptides. The active compound was isolated from the culture mixture (after 84 h fermentation at 27 °C, complex culture medium) using methods suitable for the isolation of hydrophilic microbial metabolites. Thus, after removal of proteins by precipitation with alcohol and enrichment by adsorption on active charcoal, purification was carried out by gel chromatography (CM-Sephadex C-25, Sephadex G-10). The final purification of the antibiotic was performed on cellulose (yield 5 mg per liter culture filtrate). This amphoteric compound was identified as chlorotetain 1 [(S)-alanyl-(S)-3-[(R)-3'-chloro-4'-oxo-2'-cyclohexenyl]alanine (Ala-CCAla)], which contains the previously unknown, chlorine-containing amino acid CCAla. An amino acid determination showed the presence of the components L-alanine and L-tyrosine. The determination was carried out as follows: after acid total hydrolysis (6 N HCl, 18 h, 110°C) the resulting hydrolyzate was esterified with n-propanol, acylated with trifluoroacetic anhydride, (tfa)₂O, and analyzed by gas chromatography on Chirasil-Val. The FD mass spectrum of 1 showed a peak due to [M+H]⁺ at m/z 289 accompanied by an isotope peak at m/z 291 (Table 1). From this mass difference of 2 and the fragment ion at m/z 253 ([M-Cl]⁺), the presence of chlorine in 1 could be deduced. UV and NMR spectra showed that L-tyrosine is not a natural component of 1, but rather the decomposition product from the acid-sensitive C-terminal amino acid. GC-MS analysis (capillary quartz column SE 52) of the N-tfa-dipeptide methyl ester showed (at m/z 363) the peak of highest mass corresponding to [M-Cl]⁺. The fragment ions at m/z 140 and 141 are typical for N-terminal alanine, the presence of which was confirmed by dinitrophenylation. To explain the formation of tyrosine a six-membered ring system with a 1,4-arrangement of the peptide group and the keto-functionality is required. That the chloro-substituent occupies position 2 in the 2-cyclohexenone system was shown by the NMR chemical shifts of the olefinic proton (δ=7.3) and of the carbon atom to which it is bound (δ=156.3). The C-terminal amino acid contains the cyclohexenone chromophore, with an asymmetric C-atom in the 1'-position, in its side chain. Since, for steric reasons, only an equatorial position for the peptide group need be considered, only one half-chair configuration for each of the two diastereomers is conceivable, in which the 6'-CH₂ group is twisted out of the plane, either downwards [(R)-configuration at C-1'] or upwards [(S)-configuration]. The configuration at the C-1' atom was determined by measurement of the circular dichroism (Table 1). The reversed octant rule can be applied to the almost planar cyclohexenone ring system, as for α,β-epoxy- and α,β-cyclopropylketones. According to this, the observation of a negative n→π*-Cotton effect, which in this case can be attributed to the negative contribution of the 6'-CH₂ group, requires an (R)-configuration. Chlorotetain 1 is stable in aqueous solution at pH 5 at room temperature. In more strongly acidic solutions and also under alkaline conditions the biological activity rapidly decreases, particularly upon warming. It is known that amino- and amidoenones readily undergo a 1,4-addition to form 6-oxo-octahydroindoles. A similar reactivity could well be partly responsible for the instability of 1. 1 inhibits the growth of various fungi and, in higher concentrations, gram-positive and gram-negative bacteria. The fungi Aspergillus fumigatus, Candida albicans, and Paecilomyces variotii are particularly sensitive (MIC < 1 µg). 1 is the first chlorine-containing peptide to be isolated as a metabolite from B. subtilis. Other chlorine-containing amino acids and peptides are known to be metabolites of bacteria, fungi, plants, and sponges. The bacterial strain produces another antibiotic substance, which is structurally related to 1. This was identified as the tetain (bacilysin) (S)-alanyl-(S)-3-[(R)-2',3'-epoxy-4'-oxocyclohexyl]alanine 2. The C-terminus, which 2 was also isolated as anticapsin, irreversibly inhibits the glucosamine-6-phosphate synthetase of bacteria and yeasts after intracellular hydrolysis of the dipeptide. Since 1 and 2 are structurally similar it is conceivable that 1 is formed from 2 during isolation. However, the fact that it was possible, prior to isolation, to correlate the biological activity with the HPLC detection via UV spectroscopy, refutes this suspicion. We were able to show that bacilysin 2 occurs relatively frequently in Bacillus subtilis and related species. In contrast, only one of the investigated cultures of B. subtilis strains, which produced bacilysin, namely B. subtilis BGSC 1E2, was found also to contain chlorotetain 1. Therefore, 1 represents a new chlorine-containing secondary metabolite, which, like 2 and other oligopeptides, can enter the bacterial cell via the dipeptide transport system. The aromatization, which was found after the acid total hydrolysis of CCAla, is also exhibited by other peptide antibiotics containing a labile six-membered alicyclic ring. Thus aromatization of the 4-aminocyclohexadienyl system of the amiclenomycin peptides with elimination of ammonia leads to the formation of L-homophenylalanine, and aromatization of the epoxide-containing six-membered ring of 2 yields L-tyrosine. Because of this tendency to form stabilized aromatic rings, the ring systems of 1 and 2 are only accessible with difficulty by chemical synthesis.