Mintaimycins, a Group of Novel Peptide Metabolites from Micromonospora sp. C-3509

A group of peptide metabolites (1–4), designated as mintaimycins, were isolated from Micromonospora sp. C-3509. The planar structures of mintaimycins were determined by combination of mass spectrometry, 1D and 2D NMR spectroscopy, and the stereochemistry of mintaimycins were partially resolved by Marfey’s or Mosher’s method. Mintaimycins featured a central β-methylphenylalanine or phenylalanine linked at its amino group with 5-methyl-2-hexenoic acid, and at its carboxyl group with 5-hydroxy-norleucine or leucine that combined a derivative of hexanoic acid or 4-methylpentanoic acid. Mintaimycin A1 (1), the principal component, was found to exhibit the biological activity of inducing pre-adipocyte differentiation of 3T3-L1 fibroblast cells at 10.0 μmol/L.


Introduction
Micromonospora, a genus of actinomycetes, is most famous for producing secondary metabolites of aminocyclitols with strong antibacterial activity and enediynes with severe antitumor activity. Micromonospora echinospora subsp. calichensis, for example, is the producer of the clinical antibiotic gentamicin and payload in antibody-drug conjugate calicheamicin [1]. Micromonospora is also known for producing secondary metabolites with diverse chemical structures and biological activities. Many bioactive polyene macrolactams, aromatic polyketides and peptides have been identified from the genus [2][3][4][5][6][7][8].
We are interested in new secondary metabolites from actinomycetes [9][10][11]. Micromonospora sp. C-3509 as a soil strain isolated from Sheshan in Wuhan of China was previously identified as a calicheamicin producer [12]. To explore whether the strain produced any other secondary metabolites, we performed a microbial chemistry investigation for it. Herein, the discovery of a group of novel peptide metabolites (mintaimycins) from the strain was described. . The 13 C NMR and DEPT spectra (Figures S18 and S19) of 1 showed 29 carbon resonances, which corresponded to the above groups and three additional carbonyl carbons (δ C 166.4 (C-1 ), 172.5 (C-1) and 173.8 (C-12 )).
Compound 3 was a white amorphous powder. Its molecular formula C 28 H 42 N 2 O 5 was determined from the HRESIMS data, which is CH 2 less than 1. Compound 3 showed nearly identical NMR data to 1 except for the absence of a doublet methyl at δ C 20.7 (δ H 1.29, d, J = 7.2 Hz, C-10), and presence of an additional methene (δ C (37.  Figure 1. Compound 3 was designated as mintaimycin A 2 . Its NMR data were assigned in Table 1. Compound 4 was obtained as a white amorphous powder. Its molecular formula was deduced as C 29 H 46 N 2 O 6 by HRESIMS, which is CH 3 OH more than 3. Spectroscopic data showed that 4 was a close homologue of 3 except for the signals of a methoxy (δ C 52.2, δ H 3.63 s, OCH 3 -13 ) in 4. Analysis of 2D-NMR data (Figures S38-S44) suggested that 4 should be the δ-lactone ring-open (hydrolyzed) and then methyl esterified derivative of 3, which was supported by the 1 H-1 H COSY correlations ( Figure 1) of H 2 -4 /H-5 /H-6 and the HMBC correlations from OCH 3 -13 to C-12 . Thus, the planar structure of 4 was depicted as in Figure 1. Compound 4 was designated as mintaimycin A 3 . Its NMR data were assigned in Table 1.

Stereochemistry of Mintaimycins
Mintaimycins have five or six chiral carbons for determination of their configurations. The chiral carbon(s) in β-MePhe/Phe of mintaimycins was determined of configuration by Marfey's method [14,15]. Specifically, the β-MePhe in 1-2 was determined as (2S, 3S)-β-MePhe ( Figure S45), and the Phe in 3-4 was determined as (2S)-Phe (L-Phe, Figures S46 and S47). The chiral carbon C-6 (with a secondary hydroxy group in 1 and 3) and C-10 (with a secondary hydroxy group in 2) were deduced of their configurations by Mosher's method [16]. Specifically, comprehensive analysis of 1 H NMR resonances of Rand S-MTPA ester derivatives ( Figures S48-S95) revealed systematic distribution of ∆δ SR values (δ S -δ R in ppm), thus establishing R configuration for chiral carbon C-6 in 1 and 3 ( Figure 2 and Table S2; Table S3 and Figure S96), and S configuration for chiral carbon C-10 in 2 (Table S4 and Figure S97). According to the plausible pathway proposed for mintaimycins biosynthesis described below, chiral carbon C-6 in 2 and 4 should take the same configuration as chiral carbon C-6 in 1 and 3.

Speculation of Pathway for Mintaimycins (1-4) Biosynthesis
As a group of peptide metabolites with similar or same building/assembling blocks, mintaimycins must have shared the same biosynthetic mechanism. Based on the biosynthetic pathway of jomthonic acids [17], a group of secondary metabolites from Streptomyces with similar structure to mintaimycins, a plausible pathway for mintaimycins biosynthesis was proposed as in Figure 4. In the pathway, fragment 1 as the common start building block may come from condensation, keto-reduction, and dehydration of isovaleric acid and acetic acid (catalyzed by polyketide synthase, PKS). Catalyzed by another PKS, fragment 3 may come from condensation and keto-reduction of 5-hydroxy-norleucine or leucine with a derivative of hexanoic acid or 4-methylpentanoic acid (tetrahydro-6-methyl-2H-pyran-2one, methyl 5-hydroxyhexanoate or methyl 4-methylpentanoate). Fragments 1 and 3 are joined with fragment 2 by two amide bonds catalyzed by non-ribosomal peptide synthase (NRPS). Therefore, mintaimycins belong to the biosynthetic products of NRPS-PKS.
As a group of peptide metabolites with similar or same building/assembling blocks, mintaimycins must have shared the same biosynthetic mechanism. Based on the biosynthetic pathway of jomthonic acids [17], a group of secondary metabolites from Streptomyces with similar structure to mintaimycins, a plausible pathway for mintaimycins biosynthesis was proposed as in Figure 4. In the pathway, fragment 1 as the common start building block may come from condensation, keto-reduction, and dehydration of isovaleric acid and acetic acid (catalyzed by polyketide synthase, PKS). Catalyzed by another PKS, fragment 3 may come from condensation and keto-reduction of 5-hydroxy-norleucine or leucine with a derivative of hexanoic acid or 4-methylpentanoic acid (tetrahydro-6-methyl-2H-pyran-2-one, methyl 5-hydroxyhexanoate or methyl 4-methylpentanoate). Fragments 1 and 3 are joined with fragment 2 by two amide bonds catalyzed by non-ribosomal peptide synthase (NRPS). Therefore, mintaimycins belong to the biosynthetic products of NRPS-PKS.

Biological Activities of Mintaimycins
An antibiotic M 9026 complex from Micromonospora sp. NRRL 15118 with three antitumor and antimicrobial components was disclosed in the US Patent 4, 692, 333, and planar structure of antibiotic M 9026 factor 3 was provided in SciFinder. As mintaimycins
Mintaimycins are also similar to jomthonic acids (a group of secondary metabolites from Streptomyces) that possess the biological activity of inducing pre-adipocyte differentiation [14,18]. We assayed mintaimycin A 1 (1) of this activity. At a concentration of 10.0 µmol/L, mintaimycin A 1 (1) exhibited a prominent activity of inducing pre-adipocytes to mature adipocytes for 3T3-L1 cells ( Figure 5, Figure S98).
Mintaimycins are also similar to jomthonic acids (a group of secondary metabolites from Streptomyces) that possess the biological activity of inducing pre-adipocyte differentiation [14,18]. We assayed mintaimycin A1 (1) of this activity. At a concentration of 10.0 μmol/L, mintaimycin A1 (1) exhibited a prominent activity of inducing pre-adipocytes to mature adipocytes for 3T3-L1 cells ( Figure 5, Figure S98).

General Procedures
UV spectra were acquired with a Cary 300 spectrometer. IR spectra were obtained using a Nicolet 5700FTIR microscope spectrometer. Analytical HPLC was conducted on an Agilent system with a 1260 Quat-Pump and DAD detector. For semi-preparative HPLC, a reverse-phase C18 column (Spursil 5μm C18 column: 250 × 10.0 mm) was used with MeCN-H2O as a solvent system. LC-MS was performed on a 1100-6410 Triple Quad from Agilent or an Agilent 1100 LC/MSD with a G1946D single quadrupole mass spectrometer. High-resolution mass spectrometry was carried out on a XEVO G2-XS QTof from Waters. NMR data were collected using a Bruker-600 or an ADVANCE HD 800 MHz and a Bruker Avance Ⅲ HD 700 MHz spectrometer, where chemical shifts (δ) were reported in ppm and referenced to DMSO-d6 solvent signal (δH 2.49 and δC 39.5), CDCl3 solvent signal (δH 7.26 and δC 77.0) and acetone-d6 solvent signal (δH 2.04 and δC 206.0). 3T3-L1 fibroblast cell (pre-adipocyte) line was purchased from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.

General Procedures
UV spectra were acquired with a Cary 300 spectrometer. IR spectra were obtained using a Nicolet 5700FTIR microscope spectrometer. Analytical HPLC was conducted on an Agilent system with a 1260 Quat-Pump and DAD detector. For semi-preparative HPLC, a reverse-phase C 18 column (Spursil 5µm C 18 column: 250 × 10.0 mm) was used with MeCN-H 2 O as a solvent system. LC-MS was performed on a 1100-6410 Triple Quad from Agilent or an Agilent 1100 LC/MSD with a G1946D single quadrupole mass spectrometer. High-resolution mass spectrometry was carried out on a XEVO G2-XS QTof from Waters. NMR data were collected using a Bruker-600 or an ADVANCE HD 800 MHz and a Bruker Avance III HD 700 MHz spectrometer, where chemical shifts (δ) were reported in ppm and referenced to DMSO-d 6 solvent signal (δ H 2.49 and δ C 39.5), CDCl 3 solvent signal (δ H 7.26 and δ C 77.0) and acetone-d 6 solvent signal (δ H 2.04 and δ C 206.0). 3T3-L1 fibroblast cell (pre-adipocyte) line was purchased from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.

Extraction and Isolation of Mintaimycins (1-4)
Solid state fermentation culture (45 L) of Micromonospora sp. C-3509 was extracted with an equal volume of EtOAc three times. The EtOAc extract was concentrated under reduced pressure at room temperature, which yielded a dark brown residue (25.7 g). The residue was loaded onto a preparative ODS column (Spherical C 18 , 40-60 µm, 61 × 219 mm) and fractionated with a stepwise gradient of MeOH-H 2 O at a constant flow rate of 18 mL/min, which yielded fractions F1-F55.
HPLC analysis indicated that mintaimycins A 3 (4) appeared in F37-F38. These fractions were combined and dried (48.0 mg), then applied on a C 18 column for repeated semipreparative HPLC, which yielded pure preparation of mintaimycins A 3 (4, 0.4 mg).
Mintaimycin    (1R and 1S, 2R and 2S, 3R and 3S). Then, the reaction solutions were evaporated to dryness, and the residues were applied on analytical HPLC column for purification of these ester derivatives. MTPA derivatives (1R and 1S, 2R and 2S, 3R and 3S) were dissolved in acetone-d 6 , CDCl 3 and DMSO-d 6 , respectively, for NMR analysis.

Pre-Adipocyte Differentiation Assay
3T3-L1 fibroblast cells were maintained in high-glucose DMEM medium containing 10% FBS. The cells were cultured in the 12-well plate to reach a confluence state. Two days post-confluency, the assay was initiated by incubating cells with 10% FBS DMEM containing the inducer mixture (0.5 mmol/L of 3-isobutyl-1-methylxanthine, 10.0 µg/mL of insulin, and 1.0 µmol/L of dexamethasone). At the same time, 1.0 and 10.0 µmol/L of mintaimycin A 1 (1) were added, and 2.0 µmol/L of rosiglitazone was used as a positive control. After 3 days, the medium with inducer mixture was replaced by DMEM containing insulin (10.0 µg/mL) and incubated for another 2 days, then the medium was changed with high-glucose DMEM in the presence of 10% FBS every 2 days. After 14 days, the cells were processed for oil red O staining according to standardized protocol [19].