MBJ-0110, a novel cyclopeptide isolated from the fungus Penicillium sp. f25267

We have constructed an isolated natural compound library designed to facilitate extensive biological screenings.¹ The library consists of over 1000 isolates, including over 140 ‘JBIR compounds’ that were discovered in our laboratory. To enrich this library, we recently initiated a screening program for rare microbial products using the advanced compound-identification system designated as ‘MBJ’s special selection’.², ³ As a result, our program yielded novel compounds named ‘MBJ compounds’, such as a cytotoxic hydroxamate MBJ-0003 from Micromonospora sp. 29867;⁴ cytotoxic eremophilane derivatives MBJ-0009 and MBJ-0010 from Nectria sp. f26111;⁵ MBJ-0011, MBJ-0012 and MBJ-0013 from Apiognomonia sp. f24023;² cytotoxic chaetoglobosin derivatives MBJ-0038, MBJ-0039 and MBJ-0040 from Chaetomium sp. f24230;³ bicyclic depsipeptides MBJ-0086 and MBJ-0087 from Sphaerisporangium sp. 33226;⁶ and aziridine-containing peptide MBJ-0035 from Streptosporangium sp. 32552.⁷ Further screening for novel compounds led to the identification of MBJ-0110 (1) from the culture of Penicillium sp. f25267. Herein we report the fermentation, isolation, structure elucidation and preliminary biological activity data.

Penicillium sp. f25267 was isolated from a soil sample collected in the Shiga Prefecture, Japan. The strain was cultured in 250-ml Erlenmeyer flasks, each containing 25 ml of a seed medium consisting of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural Cooperative Assoc., Hokkaido, Japan), 1% glucose (Junsei Chemical, Tokyo, Japan), 2% soybean powder (SoyPro, J-Oil Mills, Tokyo, Japan), 0.1% KH₂PO₄ and 0.05% MgSO₄·7H₂O (pH 7.4 before sterilization). The flasks were incubated on a rotary shaker (220 r.p.m.) at 25 °C for 3 days. Aliquots (0.5 ml) of the broth were transferred to 500-ml Erlenmeyer flasks containing 50 ml of a production medium of the same composition, which were then cultured on a rotary shaker (220 r.p.m.) at 25 °C for 4 days.

The whole culture broth (2 l) was extracted with an equal volume of n-BuOH. After concentration in vacuo, the extract was successively partitioned between EtOAc (350 ml × 3) and H₂O (300 ml). The aqueous layer was evaporated to dryness and the residue (1.4 g) was fractionated by reversed-phase medium-pressure liquid chromatography (Purif-Pack ODS-30, Shoko Scientific, Yokohama, Japan; 40–100% aq. MeOH with 10% stepwise increments in the MeOH concentration). The fractions were monitored using an ultra performance liquid chromatography-diode array detection-evaporative light scattering-mass spectrometry system and 1 was isolated based on peak-guided fractionation. The 50% MeOH eluate (30.9 mg) was subjected to preparative reversed-phase HPLC using a Capcell Pak C₁₈ MG II column (20 mm inside diameter (i.d.) × 150 mm; Shiseido, Tokyo, Japan) with a solvent system of 20% CH₃CN/H₂O containing 0.1% formic acid (flow rate: 10 ml min^–1), to yield semi-purified 1 (6.9 mg, retention time (Rt)=15.3 min). Final purification was carried out by preparative HPLC using an X-Bridge C₁₈ column (19 mm i.d. × 150 mm; Waters, Milford, MA, USA) with a solvent system of 20% CH₃CN/H₂O containing 0.1% formic acid (flow rate: 10 ml min^–1) to afford 1 (3.5 mg, Rt=13.3 min).

MBJ-0110 (1) was obtained as a colorless amorphous powder: [α]²⁴_D –186 (MeOH; c 0.18); UV end; IR (attenuated total reflectance) ν_max: 3400 (hydroxy) and 1683 (carbonyl) cm⁻¹. The molecular formula of 1 was established as C₂₇H₄₁N₅O₈ by high-resolution (HR)-ESI–MS (m/z 564.3018 [M+H]⁺, calcd for C₂₇H₄₂N₅O₈ m/z 564.3033). Its peptidic nature was evident from the resonances corresponding to α-methine protons (δ_H 4.00–5.08) and the resonances corresponding to the carbonyl carbons (δ_C 169.0–175.9) in the ¹H and ¹³C NMR spectra of 1, respectively. The direct connectivity between protons and carbons was established by a HSQC spectrum; Table 1 summarizes the ¹³C and ¹H NMR spectroscopic data for 1. The ¹H sequences and ¹H−¹³C long-range couplings from α-methine protons to the corresponding amide carbonyl carbons, which were elucidated by double quantum-filtered COSY and constant time-HMBC⁸ spectra, respectively, revealed the involvement of an isoleucine (Ile), a pipecolic acid (Pip), a proline (Pro) and an aspartic acid (Asp) residue, as shown in Figure 1b. In addition to the above-mentioned amino-acid moieties, the presence of a 4-hydroxypipecolic acid (C-22 to C-27) moiety was proved based on a ¹H sequence from an α-methine proton H-23 (δ_H 4.00) to nitrogen-bearing methylene protons H₂-27 (δ_H 3.39 and 2.98) via aliphatic methylene protons H₂-24 (δ_H 2.62 and 2.07), an oxymethine proton H-25 (δ_H 5.17, δ_C 68.9) and aliphatic protons H₂-26 (δ_H 2.19, 2.02), and ¹H–¹³C long-range couplings from H-23 and H-24 (δ_H 2.07) to an amide carbonyl carbon C-22 (δ_C 169.0), and from H₂-27 to an α-methine carbon C-23 (δ_C 55.5).

Table 1 ¹³C and ¹H NMR spectroscopic data for 1

Figure 1

(a) Structure of 1. (b) NMR analysis of 1. COSY, bold line; ¹H–¹³C HMBC, solid arrow; ¹H–¹⁵N HMBC, dashed arrow; ROESY, bidirectional dashed arrow. (c) ESI–MS/MS fragmentation ions of 1.

The amino-acid sequence in 1 was determined by the HMBC correlations from an α-methine proton H-2 (δ_H 4.36) to a carbonyl carbon C-7 (δ_C 173.0), from an α-methine proton H-8 (δ_H 5.08) and ɛ-methylene protons H₂-12 (δ_H 3.97 and 3.00) to a carbonyl carbon C-13 (δ_C 175.9), from an α-methine proton H-14 (δ_H 4.95) to a carbonyl carbon C-18 (δ_C 171.3), from an α-methine proton H-20 (δ_H 4.82) to C-22 and from H-25 to an ester carbonyl carbon C-1 (δ_C 169.7). Although only ¹H–¹³C HMBC information suggested two structural possibilities, α- and β-aspartyl amide linkages, we concluded that the aspartyl acid moiety is linked to adjacent Pro by β-amino bond because of the existence of a ROESY correlation between H_b-17 (δ_H 3.70) and H_a-19 (δ_H 3.10) and ¹H–¹⁵N HMBC correlations from H₂-16 (δ_H 2.08, 1.96), H_b-15 (δ_H 1.80) and H_b-19 (δ_H 2.96) to a nitrogen atom of Pro (δ_N 140). Therefore, the structure of 1 was determined as shown in Figure 1b.

To verify the proposed structure, 1 was treated with 0.1 N NaOH overnight at room temperature, followed by ESI–MS/MS analysis of the alkaline hydrolysate (molecular formula: C₂₇H₄₃N₅O₉; HR-ESI–MS: [M+H]⁺ m/z 582.3159, C₂₇H₄₄N₅O₉ 582.3139). The ESI–MS/MS data showed major fragment ions (m/z 185.0945, 243.0976, 324.1554, 340.1477 and 451.2165) that supported the proposed structure (Figure 1c).

The multiplicity and a large ¹H spin coupling constant value of H-23 (doublet, J_H–H=7.2 Hz) and ROESY correlations between H-23/H_ax-27 (δ_H 2.98) and H-23/H_eq-24 (δ_H 2.62) implied that the piperidine ring is in the chair conformation and the H-23 is axially orientated. In addition, the broad singlet signal of H-25 proved its equatorial orientation. Taken together, the relative configurations of C-23 and C-25 were determined as 23S* and 25S*, respectively.

The absolute configurations of the amino-acid residues were determined to be l-Pro, l-Pip and l-Asp by using Marfey’s method.⁹ A portion of 1 (0.4 mg) was hydrolyzed in 6 n HCl at 110 °C for 12 h and then dried under air flow. The resulting hydrolysate was treated with 0.1 m NaHCO₃ (200 μl) and 1% N-(5-fluoro-2,4-dinitrophenyl)-l-alaninamide (l-FDAA) in Me₂CO (100 μl) at 40 °C for 30 min. Amino-acid standards were derivatized with l-FDAA in a similar manner. The Marfey’s derivatives were analyzed using a HPLC–MS system as follows: a Capcell Pak C₁₈ MG II column (4.6 mmi.d. × 150 mm) was developed with a linear gradient system of water/MeCN with 0.1% formic acid (20–50% MeCN, 15 min; flow rate, 1.0 ml min^–1). FDAA derivatives were detected by absorption at 340 nm, and assignment was secured by ion-selective monitoring. The retention times of the standard FDAA derivatives were as follows: l-Asp, 7.9 min; d-Asp, 8.2 min; l-Pip, 13.6 min; d-Pip, 12.8 min; l-Pro, 10.1 min; d-Pro, 10.7 min; l-Ile, 14.7 min; d-Ile, 16.8 min; l-allo-Ile, 14.7 min; and d-allo-Ile, 16.7 min. The retention times of the FDAA derivatives of 1 were as follows: Asp, 7.9 min; Pip, 13.6 min; Pro, 10.1 min; and Ile, 14.7 min.

The absolute configuration of the Ile residue in 1 was established by HPLC comparison of the 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate (GITC) derivative of hydrolysate of 1 with standard samples.¹⁰ Triethylamine (50 μl) and a GITC solution (250 μl, prepared at 3.9 mg ml^–1 in CH₃CN) were added to the acid hydrolysate of 1 or an authentic amino-acid standard. The reaction mixture was kept at room temperature for 30 min and the reaction was then quenched by adding 40 μl of MeCN–5% AcOH in H₂O (1:1). Analysis of the GITC derivatives was performed on a Capcell Pak ADME column (4.6 mm i.d. × 150 mm; Shiseido) employing an isocratic elution of 40% CH₃CN containing 0.1% formic acid (1.0 ml min^–1). GITC derivatives were detected by absorption at 248 nm, and assigned by ion-selective monitoring. The retention times of the GITC derivatives were as follows: l-Ile, 11.6 min and l-allo-Ile, 11.3 min. The retention time (11.6 min) of the GITC derivative of 1 implied that the Ile residue in 1 is L-Ile.

We evaluated the cytotoxic and antimicrobial activities of 1, but it showed neither cytotoxicity to human ovarian adenocarcinoma SKOV-3 cell lines (IC₅₀>100 μM) or human malignant pleural mesothelioma ACC-MESO-1 cell lines (IC₅₀>100 μM), nor antimicrobial activity against Micrococcus luteus and Bacillus subtilis.

The obtained structure of 1 is very rare in nature; only petrosifungins A and B,¹¹ and JBIR-113, -114 and -115¹² have been isolated as pipecolic acid-containing peptides of fungal origin. To the best of our knowledge, there are no reports in the literature of peptide compounds possessing the 4-hydroxypipecolic acid moiety.