Novel siderophore, JBIR-16, isolated from Nocardia tenerifensis NBRC 101015

Nocardia is the causative microorganism of a human infection called nocardiosis. It is also known to produce a variety of compounds with antitumor,^{1, 2} antimicrobial^{3, 4} and immunosuppressive activity.^{5, 6} To take up iron, which is an essential element for all organisms, into cells under iron-deficient conditions, microorganisms produce iron chelators, namely, siderophores. Five types of siderophores have been isolated: hydroxamates, catecholates, salicylates, nitrosophenols and carboxylates.⁷ Some siderophores, such as brasilibactin A,⁸ asterobactin,⁹ nocobactin NA¹⁰ and nocardamine,¹¹ are produced by Nocardia spp. In the course of our screening program for biologically active compounds of microbial origin, we isolated a novel heterobactin¹² analog, JBIR-16 (1), containing hydroxamate and catecholate, from the culture broth of Nocardia tenerifensis NBRC 101015 (Figure 1). In this paper, we report the production, isolation, structural determination, and briefly the biological activity of 1.

Figure 1

Structure of 1 (a), and ¹H–¹H (bold lines) and main ¹H–¹³C (arrows) correlations in the 2D NMR of 2 (b).

The producing strain N. tenerifensis NBRC 101015 was cultured on a rotary shaker for 6 days in a 50-ml Erlenmeyer flask containing 10 ml of Brain Heart Infusion broth (Difco Lab., Detroit, MI, USA), including 2% glucose as a seed culture. The seed culture was transferred into 500-ml Erlenmeyer flasks containing 100 ml of a producing medium consisting of a twice concentrated nutrient broth (Difco Lab.), including 1% glucose and 1% glycerol, and incubated on a rotary shaker at 250 r.p.m. for 6 days.

After centrifugation of the culture broth, the supernatant (5 l) was applied on a HP-20 column (Mitsubishi Chemical, Tokyo, Japan), the column was washed with water and then eluted with 100% MeOH to obtain the fraction containing 1 (833 mg). The R_f value of 1 showed 0.6 on a TLC analysis using chloroform–MeOH (10:1) as mobile phase. The eluate was evaporated to dryness, applied on a normal-phase medium-pressure liquid chromatography column (Purif-Pack SI-60, Moritex, Tokyo, Japan) and eluted with a chloroform–MeOH gradient (0–80% MeOH). The main fraction (43.8 mg) was finally separated by preparative HPLC using an XBridge Prep C₁₈ column (5 μm OBD, 20 i.d. × 150 mm, Waters, Milford, MA, USA: mobile phase; 35% aqueous MeOH, flow rate; 10 ml min^–1, detection; 254 nm) to obtain 1 (3.6 mg; retention time 22 min).

Compound 1 was obtained as a white powder ([α]_D²⁵ −9.3, c 0.1, in MeOH, UV (MeOH) λ_max (ɛ) 205 (50 800), 249 nm (15 085). The IR spectrum of 1 revealed the characteristic absorptions of amide carbonyl (ν_max 1643 cm⁻¹) and amide N–H (ν_max 1542 cm⁻¹) groups. The HR electron spray ionization-MS of 1 resulted in the (M+H)⁺ ion at m/z 574.2149, consistent with a molecular formula of C₂₆H₃₂N₅O₁₀ (calculated for C₂₆H₃₂N₅O₁₀, 574.2133).

Although the partial and putative structure of 1 was elucidated by the analyses of a series of NMR techniques, such as constant time heteronuclear multibond correlation¹³ and double-quantum filtered (DQF)–COSY, their connectivities could not be confirmed because of the lack of signals of exchangeable protons. Therefore, the structure of 1 was determined by spectral analyses of a pentamethylated derivative 2, which was prepared by the treatment of 1 with methyl iodide. Compound 2 gave the (M+H)⁺ ion at m/z 644 in the positive mode. In addition, ¹H and ¹³C chemical shifts of these methyl residues (Table 1) indicated the presence of five hydroxyl groups in 2. The direct C–H connectivity was established by heteronuclear single quantum coherence (see Table 1). The analyses of DQF–COSY and heteronuclear multibond correlation spectra of 2 established five substructures as follows: In the DQF spectrum, the sequence from methylene proton 1-H₂ (δ_H 3.52, 3.60) to an amide proton 4-NH (δ_H 7.00) through methylene protons 2-H₂ (δ_H 1.95, 2.01), 3-H₂ (δ_H 1.63, 2.37) and an α-methine proton 4-H (δ_H 4.30, δ_C 51.5) was observed. ¹H–¹³C long-range couplings in heteronuclear multibond correlation spectrum from 1-H₂ and 4-H to a carbonyl carbon C-5 (δ_C 167.1), together with a ¹³C chemical shift of C-1 (δ_C 49.7), revealed a 3-amino piperizin-2-one moiety. The proton spin coupling between an amide proton 7-NH (δ_H 7.31) and methylene proton 7-H₂ (δ_H 3.91, 4.08), which in turn ¹H–¹³C long-range coupled to an amide carbonyl carbon C-6 (δ_C 169.2), established a glycine residue. The sequence from an amide proton 9-NH (δ_H 8.66) to another amide proton 12-NH (δ_H 8.13) through an α-methine proton 9-H (δ_H 4.82, δ_C 53.2), methylene protons 10-H₂ (δ_H 1.85, 2.06), 11-H₂ (δ_H 1.77) and 12-H₂ (δ_H 3.40, 3.80), and an ¹H–¹³C long-range coupling from the α-methine proton 9-H to an amide carbonyl carbon C-8 (δ_C 172.5), revealed an ornithine moiety. An aromatic proton 5′-H (δ_H 7.03) was ortho- and meta- coupled to the aromatic protons 6′-H (δ_H 7.13) and 7′-H (δ_H 7.63), respectively, supporting the presence of a 1,2,3-trisubstituted benzene ring. In addition, ¹H–¹³C long-range couplings from the methoxyl protons 3′-OCH₃ (δ_H 3.90) and 5′-H to an oxygenated aromatic quaternary carbon C-3′ (δ_C 147.7), from the methoxyl protons 4′-OCH₃ (δ_H 3.88) and 6′-H to an oxygenated aromatic quaternary carbon C-4′ (δ_C 152.8), and 7′-H to a carbonyl carbon C-1′ (δ_C 166.0) were observed. These results established a 2,3-dimethoxybenzoate moiety as shown in Figure 1. In the same manner, another 2,3-dimethoxybenzoate moiety was determined.

Table 1 ¹H- and ¹³C-NMR data of 1 and 2

The connection of these substructures was elucidated by ¹H–¹³C long-range couplings from 4-NH to C-6, from 7-NH to C-8, from 9-NH to C-1′ and from 12-H to C-1″ (δ_C 166.0), as shown in Figure 1b. In addition to the molecular formula of 2, no cross signal was observed with the remaining methoxyl group 1-NOCH₃ (δ_H 3.68, δ_C 61.3), indicating that this methoxyl group is attached to the nitrogen of the piperidin-2-one moiety. Thus, the structure of 1 was established as shown in Figure 1a. A compound related with 1 is heterobactin A,¹² in which one 2,3-dihydrobenzoate moiety in 1 is replaced by a benzoxazole residue. In addition, rhodobactin,¹⁴ which possesses both hydroxamate and biscatecholate moieties, has been reported as a siderophore. To our knowledge, this is the first report with regard to secondary metabolites from N. tenerifensis.

Compound 1 showed a red color in 10% aqueous FeCl₃ and the ESI–MS of the complex resulted in the (M+Fe+H)⁺ ion at m/z 627 in the positive mode. The formation of 1–iron complex was also confirmed by the changes in UV spectrum (absorption maxima at 205 and 249 nm were changed to 245 and 364 nm, respectively). However, 2 did not form a complex with ferric ion. The hydroxamate involved in trichostatin A is known to chelate metal ions.¹⁵ Mycobactin¹⁶ and exochelin¹⁶ have been reported as siderophores that lack catechol units. In contrast, azotochelin,¹⁷ which possesses 2,3-dihydrobenzoate moieties but not hydroxamate functional groups, has also been reported to show siderophore activity. Thus, both the hydoxamate and catechol functional groups in 1 are considered to have a significant role in chelating properties. The lack of the siderophore activity of 2, by which hydroxamate and catechols are methylated, also supports the role of these functional groups. Further biological activities are under investigation.