A phytotoxic cyclotetrapeptide containing an unusual amino acid, 2-amino-8-oxo-9-hydroxydecanoic acid, was isolated from the fungus Verticillium coccosporum. Structurally, the peptide is closely related to the known peptide chlamydocin. Chlamydocin (1), the first cyclotetrapeptide containing the unusual amino acid 2-amino-8-oxo-9,10-epoxydecanoic acid (aoe), was initially isolated as a cytostatic compound from the soil fungus Diheterospora chlamydosporia.1 Since then, four other groups of cyclotetrapeptides containing aoe have been isolated from different sources with biological activity including host-specific phytotoxicity such as HC-toxin.2 Based on structural analogy, it was inferred and experimentally demonstrated that 1 shows phytotoxic activity albeit not as host-specific as HC-toxin.3 Based on structure-activity data on 1 and its congeners, it appears that the presence of the keto-epoxy unit in the molecule is a prerequisite for the biological activity which is a result of alkylation of nucleophilic substrates by these compounds. In this communication we report the isolation of a novel phytotoxic chlamydocin analogue 2 from the fungus Verticillium coccosporum,4 which has 2-amino-8-oxo-9-hydroxydecanoic acid (aoh) as one of the residues. 2 is cyclo(aib-S-phe-R-pro-S-aoh). The methylene chloride extract from the culture filtrate of V. coccosporum showed phytotoxic activity in Lemna minor assay.5 Bioassay-guided separation resulted in the isolation of an active component6 that was identified as 2 based on the following data. The high resolution FAB mass spectrum of 2 showed a protonated molecular ion at m/z 529.3028 that was in agreement with the elemental composition C28H41N4O6. IR spectrum showed absorptions at 3301 (NH), 1710 (CO), 1676, and 1664 (peptide CO) cm-1. Acid hydrolysis (6N HCl, 110°C, 20 h) followed by chiral TLC (Machery Nagel, Chiralplate) and HPLC (Sumichiral OA5000) analysis showed the presence of S-phe and R-pro, respectively. The presence of aib was also confirmed by hydrolyzate. The structure elucidation was based mostly on NMR data.7 The salient features of the 1H NMR spectrum were the presence of one methyl doublet at 1.04 ppm (aoh), two methyl singlets at 1.22 and 1.73 ppm (aib), the α-protons at 4.29 (aoh), 4.45 (pro), and 5.36 (phe) ppm, and amide protons at 6.17 (aib), 7.46 (phe), and 7.83 (aoh) ppm. Multiplicities were confirmed by a HOM2DJ experiment. COSY analysis confirmed the presence of the spin systems a-f as shown for 2. A LRCOSY experiment revealed the long range connectivities as shown in Figure 1, thus confirming the presence of aoh, phe, and pro units in the molecule. The 13C NMR analysis including DEPT showed the presence of 3 methyls, 9 methylenes, 9 methines, and 7 quaternary carbons (including 5 carbonyls) in the molecule. All the 1J proton-carbon connectivities were established by a HETCOR experiment and a LRHETCOR experiment showed long range connectivities (Figure 1), confirming the assigned structure. The solution conformation of the molecule was studied by a NOESY experiment in C6D6 and the observed connectivities are shown in Figure 2. The absence of spatial proximity between the adjacent α-protons (accompanied by connectivities between the NH protons or substituent protons and the α-proton of the previous amino acid) indicated the presence of trans peptide bonds in the molecule. The observed connectivities also confirmed the relative positions of the amino acid residues. This is in accordance with the reported structure of 1. The absolute configuration at the chiral carbon (C-9) of the aoh unit bearing the secondary hydroxyl functionality was established by chiral derivatization with R- and S-O-methylmandelic acids.8 The relevant 1H NMR data from the two esters are shown in Figure 3. In particular, the upfield shift for the C-9 methyl (0.09 ppm) in the R-ester and the shift for the C-5 methylene (0.09 ppm) in the S-ester established the chirality as R at C-9 of aoh. The spectral assignments for both the esters were confirmed by COSY analysis. The absolute configuration at the α-carbon of the aoh unit was not determined directly but was derived to be S based on the presence of all trans peptide bonds in the molecule and structural analogy with 1. That 2 is a natural product and not an artifact of the isolation procedure was confirmed by the absence of related compounds in the extract (as determined by the LC-MS analysis of the partially purified fractions) and by the use of ethyl acetate for extraction. 2 exhibited activity in L. minor assay at 0.3 μM and at 1.7 μM in the Brachypodium junceum assay. In a preliminary greenhouse experiment, 2 showed activity against velvet leaf (Abutilon theophrasti) (70% control) and B. junceum (80% control) at 2 kg/hectare when applied postemergence. 1 was reported to show phytotoxic activity against maize. Interestingly, related compounds such as dihydrochlamydocin (3) and diol 4 were reported to be inactive when tested for cytostatic properties.9 The proposed mechanism for the activity for 1 and related compounds is that they efficiently alkylate nucleophilic substrates (proteins) as has also been shown in the case of some highly active synthetic analogues containing halogens or alkylating substituents α to the C-8 ketone (lysine analogues) in the aoe unit.10 2 shows high phytotoxicity in spite of the presence of a hydroxyl α to the ketone functionality.