Novel arginine-containing peptides MBJ-0173 and MBJ-0174 from Mortierella alpina f28740

We have constructed a library of isolated natural products obtained from microbial sources in order to perform efficient screenings.¹ During the investigation of rare microbial products with promising biological and pharmacological properties, we have developed an advanced system for compound identification based on accumulated HPLC-MS profiling data and strain information designated as ‘MBJ’s special selection’. Using this screening method, we have already succeeded in discovering novel eremophilane derivatives MBJ-0009 and MBJ-0010 from Nectria sp. f26111,² MBJ-0011, MBJ-0012 and MBJ-0013 from Apiognomonia sp. f24023,³ cytotoxic chaetoglobosin derivatives MBJ-0038, MBJ-0039 and MBJ-0040 from Chaetomium sp. f24230,⁴ and a cytotoxic hydroxamate MBJ-0003 from Micromonospora sp. 29867.⁵ During our continuous search for new substances, two metabolites named MBJ-0173 (1) and MBJ-0174 (2) were isolated together with plactin B,⁶ from the culture broth of Mortierella alpina f28740 (Figure 1a). In this paper, the fermentation, isolation, structure elucidation and preliminarily biological activities of 1 and 2 are described.

Figure 1

(a) Structures of 1 and 2. (b, left) Structure determination of 1. COSY, HMBC (¹H to ¹³C) and ROESY correlations are shown as bold lines, arrows and dashed arrows, respectively. (b, right) Structure determination of 2. COSY and HMBC (¹H to ¹³C) correlations are shown as bold lines and arrows, respectively.

The producing fungus Mortierella alpina f28740 was isolated from a soil sample collected in Ise, Japan. The strain was cultivated 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 shaken 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 and 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 the n-BuOH layer was evaporated in vacuo, the resulting residue was suspended in brine (350 ml) and then extracted with EtOAc (350 ml × 3) and n-BuOH (300 ml × 2), successively. The n-BuOH extract (3.4 g) was subjected to reversed-phase medium-pressure liquid chromatography (Purif-Pack ODS-100, size: 60 (39 g), Shoko Scientific Co., Ltd., Yokohama, Japan) by using an H₂O-MeOH stepwise solvent system (20%, 40%, 60%, 80% and 100% MeOH). The 40% and 60% MeOH fractions were combined (1.5 g) and chromatographed by preparative HPLC on an XSelect CSH C18 column (20 i.d. × 150 mm; Waters, Milford, MA, USA) with a linear gradient from 20 to 60% aqueous CH₃CN containing 0.1% formic acid over 20 min (flow rate: 10 ml min⁻¹) to afford crude 2 (59.3 mg). Further purification was achieved by preparative HPLC on the CSH column with an aqueous CH₃CN containing 0.1% formic acid linear gradient system (20–30% CH₃CN, 20 min; flow rate: 10 ml min⁻¹) to give 2 (4.0 mg, retention time 14.7 min). On the other hand, the 80% and 100% MeOH fractions were combined (0.54 g) and subjected to preparative HPLC on the CSH column eluted with a 20-min linear gradient from 20% to 60% aqueous CH₃CN containing 0.1% formic acid (flow rate: 10 ml min⁻¹) to obtain semi-purified 1 (41.2 mg, retention time: 12.3 min). Final purification was performed using HPLC (linear gradient, 20–50% aqueous CH₃CN containing 0.1% formic acid, 20 min, flow rate: 10 ml min⁻¹) to afford pure 1 (23.5 mg, retention time: 12.9 min).

MBJ-0173 (1) was isolated as a colorless amorphous solid: [α]²³_D −32 (c 0.04, MeOH); UV λ_maxnm (log ɛ): 274 (4.5), 281 (4.5) and 289 (4.4) in MeOH; IR (ATR) ν_max 1635 cm⁻¹ (carbonyl). The molecular formula of 1 was established as C₄₄H₆₂N₁₀O₈ by HR-ESIMS (m/z 859.4852 [M+H]⁺, calcd for C₄₄H₆₃N₁₀O₈: 859.4830). The planar structure of 1 was determined by a series of 2D NMR analyses, including double quantum filtered COSY (DQF-COSY), heteronuclear single quantum coherence (HSQC) and constant-time heteronuclear multiple bond correlation⁷ (CT-HMBC). The ¹³C and ¹H NMR data of 1 is listed in Table 1. The ¹H and ¹³C NMR data suggested that 1 was a peptidic compound, with five deshielded α-methine protons (δ_H≈4), seven NH protons (δ_H 9–7) and seven carbonyl carbons (δ_C 175–162). Further analyses of 2D NMR data revealed that 1 was composed of an N-terminal acetic acid endcapped peptides consinting of 5 amino acid residues: arginine, phenylalanine, tryptophan and two leucine residues (Figure 1b). Additionally, the presence of a dehydrobutyrine (DHB) moiety was revealed by HMBC correlations from an olefinic methine proton H-DHB-β (δ_H 6.52), which was also ¹H spin coupled to a doublet methyl proton H₃-DHB-γ (δ_H 1.53), to an olefinic quaternary carbon C-DHB-α (δ_C 130.7) and an amide carbonyl carbon of DHB (δ_C 162.8). The connectivity among the amino acid units was determined by ¹H-¹³C long-range couplings from NH-Arg (δ_H 7.95), NH-Leu2 (δ_H 8.04), NH-Phe (δ_H 8.51), NH-DHB (δ_H 9.13) and NH-Trp (δ_H 7.10) to carbonyl carbons of Leu1 (δ_C 172.13), Arg (δ_C 171.5), Leu2 (δ_C 172.09), Phe (δ_C 170.5) and DHB, respectively. Furthermore, HMBC correlations from an amide proton of Leu1 (δ_H 8.00) to an acetic carbonyl carbon (δ_C 169.3) indicated that N-terminus of 1 was acetylated. Thus, the planar structure of 1 was established as shown in Figure 1b. The stereochemistry of the trisubstituted double bond was established as Z by means of a J-resolved HMBC-2 spectrum.⁸ The smaller ¹H-¹³C long-range coupling value of 4.8 Hz between H-DHB-β and the carbonyl carbon of DHB indicated that these two nuclei were cis to each other, thus concluding the Z geometry for the double bond.⁹,¹⁰,¹¹

Table 1 The ¹³C (125 MHz) and ¹H (500 MHz) NMR spectroscopic data for MBJ-0173 (1) and MBJ-0174 (2)

The absolute configurations of the amino acid residues in 1 were determined using Marfey’s method.¹² A portion of 1 (0.2 mg) was hydrolyzed in 6 N HCl (0.5 ml) at 110 °C for 12 h. After drying the reaction solution under an air flow, 0.1 M NaHCO₃ (0.2 ml) and 10 mM N-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide (FDAA) in Me₂CO (0.1 ml) were added. The mixture was then reacted at 40 °C for 30 min. The resultant products were analyzed by HPLC-MS (Capcell Pak C₁₈ MGII column (4.6 i.d. × 150 mm; Shiseido, Tokyo, Japan)) developed with aqueous CH₃CN containing 0.1% formic acid linear gradient systems (A: 10–30% CH₃CN in 15 min; B: 30–60% CH₃CN in 15 min, flow rate 1.0 ml min⁻¹). FDAA derivatives of target amino acids were detected by absorption at 340 nm and MS analyses Retention times of authentic FDAA-amino acids (min, solvent system): L-Arg (12.1, A), D-Arg (11.7, A), L-Trp (9.8, B), D-Trp (10.6, B), L-Phe (10.2, B), D-Phe (11.6, B), L-Leu (10.3, B) and D-Leu (12.1, B). The hydrolysate of 1 contained D-Arg (11.7, A), D-Trp (10.6, B), L-Phe (10.2, B) and D-Leu (12.1, B). Hence, the structure of 1 including the absolute configuration was determined as shown in Figure 1a.

MBJ-0174 (2) was obtained as a colorless amorphous powder: [α]²⁴_D +32 (c 0.04, MeOH); UV λ_maxnm (log ɛ): 277 (3.9) in MeOH; IR (ATR) ν_max 3300 and 1633 cm⁻¹ (hydroxy and carbonyl, respectively). The molecular formula of 2 was determined to be C₃₁H₅₀N₈O₆ on the basis of the HR-ESIMS data (m/z 631.3959 [M+H]⁺, calcd for C₃₁H₅₁N₈O₆: 631.3932) in conjunction with the HSQC data. Analyses of a series of 2D NMR spectra revealed the presence of arginine, leucine, tyrosine and two valine moieties in 2 (Figure 1b). The amino acid sequence of 2 was determined by HMBC correlations from α-methine protons of Leu (δ_H 4.69), Val1 (δ_H 4.52), Arg (δ_H 4.30), Tyr (δ_H 4.99) and Val2 (δ_H 4.38) to carbonyl carbons of Val1 (δ_C 171.1), Arg (δ_C 172.6), Tyr (δ_C 172.4), Val2 (δ_C 171.5) and Leu (δ_C 172.1), respectively. Therefore, 2 is a cyclic pentapeptide as shown in Figure 1b.

The absolute configuration of 2 was determined using the Marfey’s method by the same procedure as that of 1. The obtained FDAA derivatives of 2 were analyzed using the same column and HPLC system as above with following aqueous CH₃CN (0.1% formic acid) linear gradient systems: (A) 10–30% CH₃CN in 15 min, (B) 30–60% CH₃CN in 15 min and (C) 20–50% CH₃CN in 15 min, flow rate 1.0 ml min⁻¹. Retention times of the standard FDAA derivatives were as follows (min, solvent system): L-Val (12.9, C), D-Val (14.8, C), L-Tyr (11.3, C) and D-Tyr (12.0, C). The hydrolysate of 2 contained D-Arg (11.7, A), D-Val (14.8, C), L-Tyr (11.3, C) and L-Leu (10.3, B). Therefore, the absolute configurations of 2 were established as shown in Figure 1a.

The obtained structure of MBJ-0173 (1) shows significant similarity to EM-f2368 isolated as a fungal opportunistic pathogen inhibitor.¹³ MBJ-0174 (2) is structurally related to plactins, isolated as stimulators of cellular fibrinolytic activity.⁶ The cytotoxic activity and antibacterial activity of 1 and 2 were tested. Compounds 1 and 2 did not display any significant cytotoxicity against human ovarian adenocarcinoma SKOV-3 cells, human malignant pleural mesothelioma ACC-MESO-1 cells and T-lymphoma Jurkat cells up to a compound concentration of 100 μM, respectively. Compounds 1 and 2 did not exhibit antibacterial activity against Micrococcus luteus (IC₅₀>100 μM) and Escherichia coli (10 μg per disk), respectively.