Prajinamide, a new modified peptide from a soil-derived Streptomyces

Abstract

Actinomycetes are well recognized as the richest source of bioactive compounds, including clinically important antibiotics, antitumor agents and cell function modulators, and hence of high pharmacological and commercial interest.1 Amongst this group, members of the genus Streptomyces are the most prolific producers of secondary metabolites, accounting for up to 80% of the bioactive small molecules discovered from actinomycetes.2 Meanwhile, it is quite notable that further discovery of unknown metabolites from Streptomyces is predicted by the genome analysis: the number of metabolites actually isolated is far more below the number of secondary metabolite biosynthetic gene clusters identified in the whole genomes of S. avermitilis and S. coelicolor.3,4 As a part of our chemical investigation on microbial secondary metabolites, we reported plant-growth promoting spiroacetals of polyketide origin,5 a linear polyketide with a dlactone terminus with cytotoxic activity,6 a polycyclic tetronate with antiinvasive activity7 and a biosynthetically unprecedented heterocyclic polyketide with antibacterial and antiinvasive activities8 from Streptomyces. During the course of our continuing effort to discover structurally unique secondary metabolites from these organisms, a new modified peptide was isolated from the culture broth of a soilderived actinomycete strain Streptomyces sp. SPMA113 collected in Thailand. The strain was cultured in A-11M liquid medium, and the whole culture broth was extracted with 1-butanol. The HPLC/UV analysis of the extract using our in-house metabolite database indicated the presence of an unknown compound showing a UV absorption maximum at 260nm, along with geldanamycins9 and elaiophylins.10 Guided by HPLC/UV, several steps of chromatographic purification resulted in the isolation of a new compound, prajinamide (1, Figure 1). Compound 1 was obtained as a pale yellow oil that analyzed for a molecular formula of C16H25N3O3 (6 degrees of unsaturation) by interpretation of HR–ESI–TOF–MS (observed [M+Na]+ at m/z 330.1788, calculated [M+Na]+ 330.1788). This molecular formula was corroborated by 1H and 13C NMR spectral data (Table 1). Analysis of the combined 1D and 2D NMR data established that 1 possessed three carbonyl, four olefinic methine, two sp3 methine, five sp3 methylene and two methyl carbons, in addition to three exchangeable protons. The IR absorptions at 1647, 1599 and 1538 cm 1 indicated the presence of amide functionalities, which was supported by the resonances of carbonyl carbons at d 165.6, 169.9 and 170.1 observed in the 13C NMR spectrum. As three carbonyls and two double bonds accounted for five of six double-bond equivalents, 1 must be a monocyclic compound. Further analysis of 1H-1H COSYand HMBC spectra provided three substructures (Figure 2). The first was an ornithine lactam that was established by the sequential COSY correlations from an NH proton at d 7.61 (2-NH) to another NH proton at d 8.07 (5-NH) through a methine proton (H-2) and three methylene protons (H-3, H-4 and H-5) and HMBC correlations from H-2, H-3, H-5 and 2-NH to a carbonyl carbon C-1 (d 169.9). The second substructure, a b-alanine, was assigned on the basis of COSY correlations between an NH proton at d 7.98 (8-NH) and H-8 and between H-8 and H-7 and HMBC correlations from H-7 and H-8 to C-6 (d 170.1). Two and three-bond C–H correlations from 2-NH and H-2 to C-6 allowed the b-alanine residue being connected to the ornithine lactam through an amide linkage. COSY correlations between four olefinic protons H-10, H-11, H-12 and H-13 provided a conjugated diene, which was then extended to include a carbonyl carbon C-9 (d 165.6) at C-10 on the basis of HMBC correlations from H-10 and H-11 to C-9. A vinyl methine H-13 showed a COSY correlation to H-14, which showed in turn correlations to two equivalent methyl protons H-15 and H-16, thereby establishing an isopropyl terminus attached to the diene fragment. The geometries of C-10–C-11 and C-12–C-13 double bonds were assigned as Z and E, respectively, on the basis of the vicinal coupling constants (JH10,H111⁄411.3Hz, JH12,H131⁄415.5Hz). The third substructure was thus