Cyanobacteria have been recognized in the last decade as a rich source for biologically active cyclic peptides. Many of these cyclic peptides contain long chain amino-acid residues. The microcystins are by far the most common cyclic peptides associated with strains of freshwater blooms of cyanobacteria. Scytonemin A3, puwainaphycin C4, calophycin5 and faxaphycins6 are other examples of such peptides that have been found in this group of prokaryotic microorganisms. We report here the isolation and structure elucidation of a novel cyclic peptide, Schizotrin A (1), from a terrestrial Schizotrix sp. (TAU strain R-89-2) which possesses moderately strong antibacterial and antifungal activities. The Schizotrix sp. (TAU strain R-89-2) was isolated from a sample collected at a greenhouse in Rehovot, Israel. A pure clonal strain was grown in mass culture in the laboratory. The freeze-dried cells were extracted with 70% ethanol in water and the resulting extract was subjected to repeated reverse-phase (C-18) chromatography, guided by antimicrobial activity-assay. In a typical isolation the freeze-dried cells (30 g from 100 L of culture) were extracted with a 7:3 ethanol/water solution. The crude extract was fractionated on a C18 column. Aliquots of each fraction were assayed for antimicrobial activity (Bacillus subtilis). The active fractions were combined and applied to a preparative C18 HPLC column using 7:3 ethanol/water as the eluant. The final purification by HPLC on YMC RF-18 column with 3:1 ethanol/water afforded 29 mg of 1. Schizotrin A (1) was isolated as an amorphous powder (0.1% of dry cells weight), [α]D 20 -64.7° (c 0.034, MeOH), λmax (ε) 224 (19,750) nm and HRFABMS (thioglycerol matrix): m/z 1490.779297 [(M+H)+, calcd for C72H109N13O21 m/z 1490.778274, Δ -1.0 mDa]. The 1H and 13C NMR spectra of 1 in conventional NMR solvents gave broad signals resulting from the equilibria of different conformers of the peptidic ring. Only when 1 was dissolved in deuteromethanol/water solutions and heated above 310°K could the later signals be recognized as two methyl doublets. Unfortunately, the combination of high temperature and presaturation of the water signal resulted in the disappearance of all of the amide proton signals, which are essential for determining the amino-acid sequence by NMR techniques. When the water in these mixtures was substituted with a water/trifluoroacetic acid (TFA) mixture at pH 3 a sharp proton and carbon spectra was achieved at room temperature. For the structure elucidation of 1, 1:1- and 2:1-methanol-d3/water (with TFA) solutions were used. Assignments of the signals for the thirteen amino acid residues that compose Schizotrin A are based on the analysis of 1H, 13C, DEPT, COSY, TOCSY, HMQC and HMBC spectra. The thirteen residues that were assigned are: N-methyl alanyl (NMeAla), butyryl, didehydroaminobutyryl (Dhb), glutaminyl (Gln), glycyl (Gly), homotyrosinyl methylether (Htm), phenylalaninyl (Phe), two prolyl (Pro) residues, two serinyl (Ser) residues and a novel long chain β-amino acid residue; 3-amino-2,7,8-trihydroxy-10-methyl-5-oxyundecanoyl (Aound). Unusual chemical shifts of some proton signals were noticed. The H-5 signal for Aound (5.03 ppm) appeared at a much lower field than any of the other signals for protons on hydroxyl-bearing carbons of this residue and thus are assumed to be esterified. The signal for proR H-5 of Pro(2) is 0.3 ppm upfield shifted relative to the signals for the other H-5 protons of the two proline residues and the signals of the valine methyls are roughly 0.2 ppm higher field than normal (0.76 and 0.66 ppm). The upfield shift of the latter three signals appeared to be attributed to an anisotropic effect such as diamagnetic shielding by the ring current of a neighboring aromatic moiety. This was confirmed by NOE experiments. HMBC correlations allowed the construction of three residues: (i) N-butyryl-N-methyl Ala, (ii) Pro(1)-Gly and (iii) Htm-Ser(2)-Dhb-Ser(1). The signals for the Gln and Phe carbonyls overlapped, thus the correlations between the Gly α protons and Gln-CO in the HMBC map could not be assigned unequivocally. The cross peaks in the NOESY spectrum, however, were of great help in the assignment of the amino acid sequence. The Htm-Ser(2)-Dhb-Ser(1) fragment could be extended through the NOESY correlations of: (i) Htm H-6/6' with Pro(2) pro-R H-5 and pro-S H-5; (ii) Pro(2) H-2 and Aound NH; (iii) Aound H-2 and Gln NH, and (iv) Gln H-2 and Gly NH, to the Gly-Gln-Aound-Pro(2)-Htm-Ser(2)-Dhb-Ser(1) fragment. The attachment of the Gly to Pro(1) was confirmed by the NOE connectivities between Pro(1) pro-R H-5 and pro-S H-5 and the Gly α protons. Correlations between Pro(1) H-2 and Phe NH, Phe H-2 and Val NH, and Val H-2 and Ser(1) NH, close the peptidic ring. The N-butyryl-N-methyl-Ala fragment was assigned as the esteric group attached to C-5 of the Aound chain on the basis of NOESY correlations between Aound H-3, and the Ala methyl and H-2. The absolute configurations of the common amino acids were analyzed by Marfey's HPLC method. The amino acids were determined as: L-Proline (2), L-Serine (2), L-Phenylalanine, D-Valine, N-methyl-D-Alanine and D-Glutamine, by comparison with authentic D and L standards of each amino acid. Studies are in progress to assign the stereochemistry of the chiral centers in the Htm and Aound residues. Compound 1 was not active against Staphyloccocus aureus, S. albus, and Escherichia coli at 67.1 nM/6 mm disc but gave a 15 mm zone of inhibition against Bacillus subtilis at 6.7 nM/6 mm disc. Compound 1 gave 7, 7, 9 and 8 mm zones of inhibition against Saccharomyces cerevisiae, Candida albicans, C. tropicalis and Rhodotorula rubra at 16.7, 13.4, 13.4 and 13.4 nM /6 mm disc, respectively, but not against C. paropsillosis at 67.1 nM/6 mm disc. Compound 1 also gave 28%, 25%, 37% and 47% reduction in radial growth of the colonies of the fungi Sclerotium rolfsii, Rhizoctonia solani, Fusarium oxysporum and Colletotrichum gloeosporioides at 13.4, 13.4, 33.5 and 13.4 nM/6 mm disc, respectively. Interestingly, the structure of schizotrin A (1) resembles three other cyanobacterial cyclopeptides: scytonemin A3, puwainaphycin C4, and calophycin5. The latter deca- and undecacyclic peptides, although having a different amino acid profile, share a common segment, i.e. a proline residue attached to the amino group of a 2-hydroxy-3-amino-long chain acid residue. This segment may be responsible for the biological activity of these compounds.