An inhibitor of phosphatidylinositol-specific phospholipase C, pholipeptin, was purified from the culture broth of Pseudomonas sp. Structural determination by 2D NMR spectroscopy with addition of a small amount of trifluoroacetic acid revealed that it was a novel cyclic lipodepsipeptide (1) consisting of 11 amino acids and a 3-hydroxydecanoic acid moiety. Phosphatidylinositol-specific phospholipase C (PI-PLC) is the rate-limiting enzyme of PI turnover, which is considered to be one of the major pathways of cellular signal transduction. For isolation of pholipeptin, an inhibitor of PI-PLC, the culture filtrate (2 L) of Pseudomonas sp. was applied on a Diaion HP-20 column. The active fraction was eluted with MeOH, and extracted with EtOAc at pH 2.0. The extract was concentrated and applied on a silica gel column, and the active fraction was eluted with CHCl₃/MeOH (5/1). The active fraction was again purified with silica gel column and eluted with BuOAc/BuOH/MeOH/H₂O (4/4/1/1). Pholipeptin (20 mg) was finally purified by Sephadex LH-20 column chromatography using MeOH. Pholipeptin is a white powder with mp of 220-224 °C (decomposed). Its IR spectrum revealed absorptions at 3300, 1650, and 1550 cm⁻¹. The FAB-MS spectrum afforded m/z 1376 (M+Na+H) and 1374 (M+Na-H), but fragmentation peaks commonly observed for acyclic peptides were not noted. Therefore, a cyclic rather than linear oligopeptide was suggested for this compound. Pholipeptin also gave positive responses to the Rydon-Smith reaction, and negative responses to a ninhydrin test. Prior to NMR studies, the usual amino acid analysis of pholipeptin had revealed the constituents as Asp (and/or Asn), Thr, Ser, Ile, and Leu in the molar ratio of 2:1:2:1:5-6, respectively. The addition of a small amount of trifluoroacetic acid to the DMSO-d₆ solution afforded relatively sharp NMR signals at pH 4, which allowed us to elucidate the structure. The presence of 5 residues of Leu was primarily confirmed by observing the 5 separate signals for the α-carbons in the ¹³C NMR spectrum. From COSY and HOHAHA experiments, proton spin systems were separately established for each amino acid residue. The existence of 2 residues of Asp was clarified by the lack of ¹H-signals for CONH₂. From HOHAHA, HMQC, and HMBC experiments, the structure of 3-hydroxydecanoic acid (OHDa) was corroborated. The presence of a free hydroxyl proton at C-3 was proved by the saturation transfer signal between the H-3 of OHDa and a water signal in the ROESY experiment. The sequence of the residues of amino acids and OHDa was determined by interpretation of HMBC and ROESY spectra. Because of spectral crowding, the HMBC experiments only allowed construction of three partial structures of OHDa-Leu-4-Asp-1, Thr-Leu-2-Leu-5-Ser-2, and Ser-1-Leu-1-Ile-Asp-2. These sequences were also supported by strong interresidue ROE correlations. Furthermore, the ROESY spectrum showed a strong cross peak between the α-H of Asp-1 and the NH of Thr, which could combine the first two partial sequences to give the sequence of OHDa-Leu-4-Asp-1-Thr-Leu-2-Leu-5-Ser-2. A strong ROE correlation of the α-H of Leu-3 with the NH of Ser-1 indicated that the Leu-3 residue was linked to the third substructure. Although α-carbonyl carbons of both Asp-1 and Ser-2 appeared at δ 170.20, the linkage of Asp-1-Thr enabled us to connect Ser-2 with Leu-3 by the HMBC correlations to establish the whole peptide sequence as OHDa-Leu-4-Asp-1-Thr-Leu-2-Leu-5-Ser-2-Leu-3-Ser-1-Leu-1-Ile-Asp-2. The ester linkage between the residues of Thr and Asp-2 was confirmed by the HMBC correlations of the β-carbonyl carbon at δ 169.55 with the γ-H of Thr and with the α- and βa-H of Asp-2. The absence of the NMR signal for the β-hydroxyl proton of Thr also supported this structure. The addition of trifluoroacetic acid described above not only improved the signal resolution but also caused the downfield shift (0.3 ppm) of the signal of the α-H of Asp-2, whereas the chemical shifts of β-protons of Asp-2 were hardly affected. Therefore, the α-carboxylic acid of Asp-2 was considered to be free as shown in structure 1. Thus, based on the NMR spectroscopic studies, pholipeptin was determined to be cyclic lipodepsipeptide 1. The chiralities of the amino acids were determined by chiral HPLC column as shown in 1. The chiralities of OHDa and Asp residues are under study. In vitro PI-PLC inhibiting activity of pholipeptin was measured as previously described, and the IC₅₀ value was 7.8 μg/ml. The pattern of inhibition was non-competitive with the substrate from Lineweaver-Burk kinetic analysis.