A new antibiotic leucinostatin A was isolated from the culture filtrate of Paecilom yces lilacinus A-267 and its structure was elucidated by mass spectrometric and degradative methods.The peptide antibiotic leucinostatin isolated from Paecilonzyces lilacirzus A-267 has aroused considerable interest owing to its antitumour activity on Ehrlich solid carcinoma and antibacterial activity against Gram-positive bacteria and a wide range of fungi. A structural study revealed leucinostatin to be a new basic peptide composed of unusual amino-acids: cis-4 methyl-L-proline (MePro),2 L-threo-P-hydroxyleucine (Hy-Leu),3 and x-aminoisobutyric acid (Aib). In an independent study, Kenner et al.4 reported the isolation of antibiotic I.C.T. No. 13959 which contains the same amino-acids as leucinostatin but which has not yet been characterized.Leucinostatin is a mixture of several components which were separated by alumina column chromatography to give mainly leucinostatin A and B. We report here the structure of leucinostatin A. Leucinostatin A (l), C,,H,,,N,,013; m.p. 98-101 "C; [cc]~ -11.0" (c 0.1, MeOH); Amax (EtOH) 202 and 220 (sh) nm; Vmax (CHC13) 3280 (NH), 1705 (CO), and 1645 (amide CO) cm-l; lH n.m.r. (CDCI,) 6 3.10 (N,N-dimethyl); 13C n.m.r.(CDCI,) 6 21 1.0 (s, CO), 180-1 60 (cn. 8 x s, amide CO), and 150.6 and 120.9 p.p.m. (each d, C=C), has a molecular weight of 1217 from its field desorption mass spectrum [f.d.m.s. m/z 1218 (MH+)] and showed a negative reaction for ninhydrin, but a positive Dragendorff reaction. These data indicated that (1) is a basic peptide antibiotic with one ketone carbonyl, one conjugated double bond, and dimethylamino-groups.Acid hydrolysis (6~ HCI, 110 "C, 20 h) of (1) followed by amino-acid analysis gave the following results : (HyLeu), (Aib),-, (Leu),-, (P-Ala), (MePro),. Cellulose column chromatography of the hydrolysate gave (S)-N1,N1-dimethylpropane-1 ,Zdiamine (2)-2HC1, m.p. 11 5-1 17 "C: [MI: +9-8" (c 0.12, MeOH); chemical ionization (c.i.) m.s. m/z 103 (MH+); IH n.m.r. (CDCI,) 8 1.50 (3H, d, J 6 Hz), 3.00 (6H, s), 3.25-3.65 (2H, m), and 3.95 (lH, m), and an unidentified amino-acid (3). The S-configuration of (2) was established by comparison with an authentic sample prepared from Boc-Ala by successive treatment with i, CIC02Et, ii, HNMe,, iii, CF,CO,H, and iv, LiAIH4. The IH n.m.r., i.r,, and mass spectra of the amino-acid (3)t revealed that (3) is 4-methyl-6- (2-oxobutyl)-2-piperidinecarboxylic acid whose stereochemistry was established by proton spin-decoupling experiments. The structure of (3) corresponds to trichoponamic acid obtained by the hydrolysis of trichopolyns.6From the diethyl ether extract of the hydrolysate was isolated (S)-(E)-4-methylhex-2-enoic acid (4), [a]: + 49.7" (c 0.25, CHCI,); m/z 128 (M'.); Amax (EtOH) 207 nm; Vmax 3600-2400 (OH), 1685 (CO), and 1640 (C=C) cm-l; lH n.m.r. (CDCl,) 6 0.89 (3H, t, J 7 Hz), 1.05 (3H, t, J 7 Hz), 1.43 (2H, q, J7 Hz), 2.26 (lH, m), 5.77 (lH, d, J 16 Hz), and 6.98 (lH, dd, J 16, 8 Hz). Catalytic hydrogenation of (4) afforded a saturated acid, [cc]iO + 7.6" (c 0-15, CHCI,), which is identical to (S)-4-methylhexanoic acid (lit.,s [ct]ko +7*4"). Since leucinostatin A (1) is negative for ninhydrin and methylation with CH2N2 recovered the starting material the C- and N-termini of (1) could be protected by the diamine (2) and the fatty acid (3), respectively. The U.V. absorptions and 13C n.m.r. chemical shifts of (1) at 150.6 and 120.9 p.p.m. are, therefore, ascribed to the N-terminal a$-unsaturated amide structure. Partial hydrolysis (6~ HCl, room temp., 40 h) of (1) gave mainly two peptides (5) and (6), and the diamine (2). Hydrolysis with 2N HC1 (reflux, 2 h) afforded fragments (7) and @), the one containing the N-terminal fatty acid and the other the C-terminal diamine. Sequences of the fragments (5)-(8) were determined by dansylation, dansyl-Edman degradation,c.i.m.s., andlH n.m.r. spectroscopy. The results are summarized in Figure 1. As the C- and N-termini of leucinostatin A were blocked with X[=(2)] and FA [=(4)], respectively, the amino acid (3) should be placed between the fragments (5) and (7). The above-mentioned components constitute a peptide, C,2H109N11012, which corresponds to the dehydration product of leucinostatin A (1). Alumina treatment of the diacetyl compound obtained by acetylation of (1) gave the 0-monoacetyl derivative, f.d.m.s. m/z 1264 (M + Naf) and 1242 (MH+); vmax (CHCI,) 1745, 1680, and 1660 cm-l; 13C n.m.r. (CDCl,) 6 119.3 (d), 13 1-6 (d), 144.2 (d), 153.9 (d), and 200.8 (s) p.p.m. The chemical shifts at 131-6, 144.2, and 200.8 p.p.m. can be ascribed to the newly formed a$-unsaturated ketone system. It is interesting that the amino-acids contained in leucinostatin A are unusual and that the C-terminal linkage of the propanediamine (2) of the antibiotic has not been found previously.