Vestaines, novel vasoactive compounds, isolated from Streptomyces sp. SANK 63697

Abstract

We conducted a screening program for vasoactive compounds and detected a potent activity in the cultured broth of Streptomyces sp. SANK 63697. From the cultured broth, two active compounds, vestaine A₁ and B₁, were isolated. The structures of these compounds were elucidated by physicochemical data and spectral analyses, and found to be new compounds.

Introduction

Blood vessels deliver oxygen and nutrition into all the tissues of our body, and collect carbon dioxide and waste products. It is essential to maintain vessels’ integrity, thus our body possesses angiogenesis, which is a process critical to forming new vessels. When tissues are damaged, pro-angiogenic factors stimulate the regeneration of new vessels, and such newly formed vessels are stabilized by means of tightening cell–cell junctions, being covered with mural cells, lined with extracellular matrix and so on.¹ However, excess angiogenesis induces fragile and leaky blood vessel formation, and causes several pathological conditions.^{2, 3}

Vascular endothelial growth factor (VEGF) is a key molecule of both angiogenesis and endothelial permeability.⁴ Excess angiogenesis and increased permeability induced by overproduction of VEGF are observed in patients suffering from several diseases, such as retinopathy and cerebral hemorrhage.^{5, 6} This indicates that vasoactive substances, which not only assist angiogenesis, but also cancel the adverse effects of VEGF, are useful for treating diseases caused by fragile and leaky vessels. To evaluate vasoactive effect, the VS-G assay, a co-culture system of endothelial cells with fibroblasts, had been established.⁷ In the course of screening to obtain vasoactive compounds, the morphological changes of human umbilical vein endothelial cells (HUVECs) were used as an indicator of candidate compounds in the VS-G assay. Potent activity was detected in the cultured broth of Streptomyces sp. SANK 63697, and two active compounds named vestaine A₁ (1) and B₁ (2) were isolated, and their structures were elucidated (Figure 1).

Figure 1

Structures of vestaines.

In this report, we describe the taxonomy of the producing organism, isolation, physicochemical properties and structural elucidations of vestaines. Details of the biological activities will be reported independently.⁷

Results

Taxonomy of the producing organism

The strain SANK 63697 was isolated from a leaf of Terminalia catappa collected in Tokyo, Japan. The strain grew well on yeast extract–malt extract agar (ISP 2) and inorganic salts–starch agar (ISP 4), and the colony color was pale yellow to brown. The mass of aerial mycelia produced on ISP 2, oatmeal agar (ISP 3), ISP 4, glycerol–asparagine agar (ISP 5) and tyrosine agar (ISP 7) was white to gray. The mature spore chains were rectinaculiaperti and each had >20 spores per chain. The spores had a smooth surface (Figure 2).

Figure 2

Scanning electron micrograph of strain SANK 63697 grown on ISP 4 for 2 weeks at 28 °C.

The 16S ribosomal RNA gene sequence (1472 nucleotides) was determined and analyzed using the EzTaxon-e database.⁸ The results indicated that the gene sequences between SANK 63697 and Streptomyces tendae ATCC 19812^T (D63873) showed a 100.0% similarity. On the basis of the morphological and cultural properties, and 16S ribosomal RNA gene sequence similarity, the strain SANK 63697 was classified into Streptomyces sp. SANK 63697 (Figure 2).

Isolation

The cultured broth (5 l) was filtered with the assistance of Celite 545 (Thermo Fisher Scientific, Waltham, MA, USA) and separated into mycelium and supernatant. The active substances were extracted from the mycelium with 80% aq. acetone (1.2 l) and the extract was filtered as described above. After addition of n-butanol (100 ml) as a defoaming agent, the filtrate was concentrated in vacuo to remove acetone. The pH of the concentrate was adjusted to 3.0 with 1 n HCl and then the concentrate was subjected to a Diaion HP-20 column (Mitsubishi Chemical, Tokyo, Japan, 200 ml). The column was washed with 30% aq. acetone (600 ml) and the active substances were eluted with 80% aq. acetone (600 ml). The eluate was concentrated in vacuo to dryness to give an oily substance (418 mg).

The substance was dissolved in 20 ml of 50% aq. ethanol and was subjected to an ODS open column (Cosmosil 140C18-OPN, Nacalai Tesque, Kyoto, Japan, 40 ml). The chromatography was performed by a stepwise elution using 40, 45 and 50% MeCN in 0.1 m aq. NaClO₄ (200, 800 and 400 ml, respectively). These eluates were fractionated into 15 ml portions.

Isolation of vestaine A₁ (1)

Fractions of the 45% MeCN in 0.1 m aq. NaClO₄ eluate containing 1 in the Cosmosil column chromatography were combined and concentrated in vacuo to remove MeCN. For desalting, the residue was subjected to an HP-20 column (10 ml). After washing the column with water (40 ml), 1 was eluted with 80% aq. acetone (40 ml). The eluate was concentrated in vacuo to dryness to give a partially purified substance (24.1 mg). The substance was further purified by preparative HPLC using an ODS column (SYMMETRY C18, 19 i.d. × 100 mm, Waters, Milford, MA, USA). The chromatography was performed with MeCN/0.05  m NaClO₄ (45:55) at a flow rate of 9 ml min⁻¹. The eluate containing 1 was concentrated and subsequently desalted in the same manner as described above, and concentrated in vacuo to dryness to give 1 as a white powder (12.2 mg).

As a result of HPLC analysis, 1 was exhibited as two peaks (peak 1 and peak 2) with the same molecular formula shown in Figure 3.

Figure 3

HPLC and HR-MS data (ESI) of 1. (a) HPLC chart of 1. HPLC conditions: column, Unison UK-C18, 3 μm, 4.6 i.d. × 75 mm; mobile phase, MeCN/20 mm aq. ammonium acetate (1/1); flow rate, 1 ml min⁻¹; sample, 2 mg ml⁻¹, 3 μl injection. (b) UV spectrum of peak 1. Both peak 1 and peak 2 show the same UV spectrum. (c) HR-MS data (ESI) of peak 1. (d) Enlarged view of (c) ranged from m/z 461.25 to 461.40.

Isolation of vestaine B₁ (2)

Fractions of the 45–50% MeCN in 0.1 m aq. NaClO₄ eluate containing 2 on the Cosmosil column chromatography were combined and concentrated in vacuo to remove MeCN. For desalting, it was subjected to an HP-20 column (20 ml). After washing the column with water (80 ml), 2 was eluted with 80% aq. acetone (80 ml). The eluate was concentrated in vacuo to dryness to give a partially purified substance (50.6 mg). The substance was applied to the preparative HPLC using the same ODS column described above. The chromatography was performed with MeCN/0.05 m NaClO₄ (50:50) at a flow rate of 9 ml min⁻¹. The eluate containing 2 was concentrated. It was then desalted as described above and concentrated in vacuo to dryness to give a substance mainly containing 2 (23.7 mg). Finally, it was rechromatographed by the same preparative HPLC using the solvent system of MeCN/0.05 m NaClO₄ (45:55), desalted in the same manner as above and concentrated in vacuo to dryness to give pure 2 as a white powder (12.7 mg). 2 also gave two peaks (peak 3 and peak 4) in the analytical HPLC.

Physicochemical properties

Physicochemical properties of the vestaines are summarized in Table 1. The molecular formula of each compound was determined by positive ion ESIMS. Peaks 1 and 2 showed the same molecular formula of C₂₄H₄₆N₂O₄S, and peaks 3 and 4 also have the same as that of C₂₅H₄₈N₂O₄S. The presence of a sulfur atom was further proved by two characteristic isotopic ions at 461.3211 (C₂₄H₄₇N₂O₄³⁴S) and 461.3297 (C₂₂¹³C₂H₄₇N₂O₄S) for 1 as shown in Figure 3d, and at 475.3368 (C₂₅H₄₉N₂O₄³⁴S) and 475.3455 (C₂₃¹³C₂H₄₉N₂O₄S) for 2.

Table 1 Physicochemical properties of vestaine A₁ and B₁

Structural elucidation

Structural elucidation of vestaine A₁ (1)

The structural elucidation was mainly focused on 1. The ¹H and ¹³C NMR spectral data obtained in methanol-d₄ are summarized in Table 2. As well as the HPLC pattern of 1, a pair of signals was observed in the NMR spectra. Therefore, observed NMR signals of 1 were classified into two categories as 1a and 1b.

Table 2 ¹H and ¹³C NMR signal assignments of vestaines in methanol-d₄

In the ¹H NMR signal set of 1a, signals from the highly overlapped alkyl group (δ 1.22–1.39), including three methyl groups were observed. In addition, two signals (δ 2.80–2.90 and 4.6) disappeared over time. It seemed to indicate that this phenomena was observed in the process when 1b was formed from 1a or vice versa, and it was dependent on tautomerism. In the ¹³C NMR spectrum, 16 assignable signals and some overlapped signals (δ 53.2 and 30.6–30.8) were observed. As the latter of the overlapped signals (δ 30.6–30.8) were all methylene carbon signals, it was suggested that 1a included an alkyl chain moiety. The observed 16 assignable signals were classified into 3 × methyl, 7 × methylene, 3 × methine and three quaternary carbons by DEPT spectrum. Two carbon signals that became hard to observe existed as well as the proton signals (C-2′ and C-4′).

Three partial structures as shown in Figure 4a were elucidated by several NMR techniques, such as DQF-COSY, HSQC, HMBC and ¹H–¹⁵N HMBC. In addition, an alkyl chain that had the partial structure of (c) as a terminal was recognized. Although there was no direct evidence of a sulfur atom-binding position, the appropriate position was between C-3 and C-1′, because of the chemical shift value of H-3 (δ 2.86 and 3.28) and H-1′ (δ 3.02 and 3.30), and the observed ¹H–¹³C long-range coupling from H-3 to C-1′ and H-1′ to C-3 without having ¹H–¹H coupling between H-1′ and H-3. It was also supported by the product ion (m/z 162) in the collision-induced dissociation spectra of 1, as shown in Figure 4b. Accordingly, the partial structure (a) was clarified as acetylated cysteine residue. The position of an alkyl chain was axiomatically determined to be between C-3′ of the partial structure (b) and C-11′ of (c). However, the length of the alkyl chain and the number of methyl groups binding to N-2′ remained uncertain.

Figure 4

NMR and MS analyses of 1. (a) Partial structures obtained from the NMR analyses of 1. Both 1a and 1b had the same partial structures. (b) Product ion observed in coliision-induced dissociation spectra of the protonated molecules of 1. Both 1a and 1b gave the same production.

To elucidate the two unclear points, 1 was dissolved in dimethylsulfoxide-d₆ (DMSO-d₆) and analyzed by several NMR techniques. As a result, it was recognized that 1 was converted to compound 3 in the DMSO-d₆. Compound 3 no longer had tautomerism, and all the carbon signals, including the alkyl chain carbons, were observed clearly. The molecular formula of 3 was determined to be C₂₁H₃₇NO₄S (m/z, found 400.2508 (M+H)⁺, calcd 400.2516 for C₂₁H₃₈NO₄S), which is the de-trimethylamine derivative of 1. By several NMR techniques, the structure of 3 was elucidated and found to be the compound from which N-2′ was eliminated and a new olefin was formed instead, as shown in Figure 5. The results indicated that 1 originally had a trimethylamine group and the Hofmann elimination afforded 3 in DMSO. Thus, the planar structure of 1a was deductively revealed as shown in Figure 1.

Figure 5

Hofmann elimination of vestaines. DMSO, dimethylsulfoxide.

By analyzing all NMR signals derived from the set of 1b, it was proved to have the same partial structures; therefore, the planar structure of 1b was determined to be the same as that of 1a. Namely, 1b was a diastereomer of 1a at C-2′ generated through the keto–enol equilibrium.

Stereochemistry of the cysteine residue of 1 was determined by the advanced Marfey’s method.⁹ Acid hydrolysates of 1 were coupled with L- or D-FDLA (1-fluoro-2,4-dinitorophenyl-5-leucine amide) and analyzed by LC–HR-MS. Almost all DLA-derived cysteine was observed as DLA-derived cystine. By comparing authentic standards, the chirality of cysteine was determined to be L configuration.

Structural elucidation of vestaine B₁ (2)

Compound 2 was also comprised of two forms: 2a and 2b, it is uncertain which peak observed in the HPLC analysis is 2a/2b. As shown in Table 1, the molecular formulae of 2a and 2b had one more methylene than those of 1a and 1b. Together with the IR spectral data, the chemical shifts of ¹H and ¹³C NMR signals of 2 in methanol-d₄ (Table 2) were closely similar to those of 1. Therefore, the structural elucidation of 2 was carried out in the same way as 1, including the usage of the Hofmann elimination product of 2 (4). As a result, 2 was proved to have seco-type endo-terminal as shown in Figure 1.

Biological activities

Both 1 and 2 dose-dependently induced island-shaped morphological changes in the VS-G assay as shown in Figure 6. They showed approximately equal activity. Vestaines induced island-shaped morphology, whereas VEGF induced tube-like morphology in HUVECs. Details of the biological activity of vestaines will be reported independently.⁷

Figure 6

Activities of vestaines in the VS-G assay. (a) Morphologies of GFP-HUVECs treated with DMSO, VEGF (50 ng ml⁻¹), vestaine A₁ (195 nm) or vestaine B₁ (195 nm). (b) GFP-HUVECs were treated at various concentrations of vestaine A₁ or vestaine B₁. The value of treatment with vestaine A₁ is the black line and that of vestaine B₁ is the dotted line. In vitro activity was shown using the number of maximum area value of treatment with vestaine A₁ as a base of 100. Each value is represented as the mean±range (n=2 wells per group). DMSO, dimethylsulfoxide; HUVEC, human umbilical vein endothelial cell; VEGF, vascular endothelial growth factor. A full color version of this figure is available at The Journal of Antibiotics journal online.

Discussion

In the course of our screening program for vasoactive compounds, two active compounds, vestaine A₁ and B₁, were isolated from the cultured broth of Streptomyces sp. SANK 63697. Structures of these were elucidated by physicochemical data and spectral analyses, and they were determined to be new amphoteric compounds. Vestaine A₂, which showed the same molecular formula as A₁, and vestaine B₂, which had the same formula as that of B₁, were also observed in the same cultured broth. As the production of A₂ and B₂ was low, they were not purified.

Vestaines had two typical properties. One is that vestaine A₁ and B₁ both formed two diastereomers through keto–enol equilibrium. The other is that they were unstable under a basic condition. For these properties, we had to devise a method for their purification and structural elucidation.

In the purification, we added sodium perchlorate (NaClO₄) to the HPLC mobile phase. NaClO₄ is a well-used ion-pairing reagent for HPLC analysis of anion surfactants, and among the solvents we tested, this combination gave a good separation not only for vestaines as the amphoteric surfactants, but also for the two diastereomers of each vestaine.

In the structural elucidation, we could classify all the NMR signals into two sets for each diastereomer, although the signal disappearances caused by hydrogen-deuterium exchange existed. Finally, following the structural elucidation studies of the decomposition products, we deduced the structures of the vestaines. To form such decomposition products in DMSO was also one of the characteristic properties of vestaines. We hypothesized that the Hofmann elimination of vestaines is caused by the basicity of trimethylamine eliminated from the vestaines.

In the stereochemistry determination study of the cysteine residue, we applied the advanced Marfey’s method.⁹ Although cysteine residue is known to be racemized easily during acid hydrolysis,¹⁰ we expected it would be hard to racemize in the case of vestaines, because the cysteine residue of the vestaines was S-alkylated. This hypothesis was strongly supported by Jacobson et al.¹¹ By using flash hydrolysis techniques,¹² to prevent excess racemization, we obtained the non-racemized cysteine residue and succeeded at determining the chiral configuration of cysteine as being L-form.

In conclusion, the present study revealed that vestaines are novel compounds that possess unique structures and characteristic biological activities. Although VEGF induced tube-like morphology in the VS-G assay, vestaines induced island-shaped morphological changes in HUVECs at a low concentration, which indicates that vestaines affect endothelial cells in a different manner from VEGF. Although it is necessary to analyze their function and mode of action in more detail, vestaines and their derivatives can be unprecedented, useful molecular probes for investigating the mechanism of angiogenesis.

Experimental procedures

Fermentation

The growth of Streptomyces sp. SANK 63697 on an agar slant was homogenized with 10% glycerin (10 ml) and filtered by a cell strainer (mesh size 100 μm, Thermo Fisher Scientific). A portion of the obtained filtrate (50 μl) was transferred into a 500 ml Erlenmeyer flask containing 80 ml of sterilized primary seed medium composed of soluble starch 4.0%, glucose 1.0%, soybean meal 1.0%, yeast extract 0.45%, corn steep liquor 0.25%, CoCl₂·6H₂O 0.0001%, KH₂PO₄ 0.05%, ZnSO₄·7H₂O 0.001%, Mg₃(PO₄)₂·8H₂O 0.005%, NiSO₄·6H₂O 0.0001% and Disfoam CB-442 (NOF, Tokyo, Japan) 0.005%. The flask was incubated at 28 °C for 48 h on a rotary shaker at 210 r.p.m. The primary seed culture (1 ml) was transferred into a 500 ml Erlenmeyer flask containing 80 ml of sterilized production medium, which was composed of soluble starch 8.0%, glucose 1.0%, soybean meal 1.0%, yeast extract 0.45%, corn steep liquor 0.25% and Disfoam CB-442 0.005%. Each of the 70 flasks were prepared in this way and incubated at 28 °C for 96 h on a rotary shaker at 210 r.p.m.

HPLC analysis of vestaines

Both 1 and 2 are shown as two peaks in the analytical HPLC, respectively. The HPLC conditions are described as follows: column, Unison UK-C18, 3μm, 4.6 i.d. × 75 mm (Imtakt, Kyoto, Japan); mobile phase, MeCN/20 mm aq. ammonium acetate (1:1); flow rate, 1.0 ml min⁻¹. Retention time of each compounds are as follows: peak 1 of 1, 4.2 min; peak 2 of 1, 4.4 min; peak 3 of 2, 5.9 min; peak 4 of 2, 6.2 min.

General experimental procedures

IR spectra were obtained on an FT/IR-6100typeA spectrometer (JASCO, Tokyo, Japan). NMR spectra were recorded on AVC500 spectrometers equipped with DCH and TCI cryogenic probes (Bruker, Billerica, MA, USA). HR-MS were recorded on an LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific) connected with an Agilent 1200 HPLC System (Agilent Technologies, Santa Clara, CA, USA).

N-acetyl-S-[(1E)-14-methyl-3-oxopentadec-1-en-1-yl]-L-cysteine (3)

Compound 1 (1.1 mg) was dissolved in DMSO-d₆ and the progress of the reaction was monitored by NMR at fixed intervals. The Hofmann elimination reaction completed for ~14 days and most of 1 was converted into 3. After analyzed by NMR, the solution was lyophiled and analyzed by LC–HR-MS. HR-MS (ESI): m/z: calcd for C₂₁H₃₈NO₄S: 400.2516, found: 400.2508 (M+H)⁺. ¹H NMR (500 MHz, DMSO-d₆, TMS (internal standard)): δ 0.84 (6H, d, J=6.6 Hz), 1.10–1.15 (2H, m), 1.19–1.27 (14H, overlapped), 1.43–1.50 (2H, m), 1.47–1.54 (1H, m), 1.83 (3H, s), 2.47 (2H, t, J=7.3 Hz), 3.13 (1H, dd, J=7.1, 13.2 Hz), 3.31 (1H, dd, J=4.7, 13.2 Hz), 4.23 (1H, m), 6.17 (1H, d, J=15.5 Hz), 7.75 (1H, d, J=15.5 Hz). ¹³C NMR (125 MHz, DMSO-d₆, TMS (internal standard)): δ 22.4 (two carbons), 22.5, 23.9, 26.7, 27.3, 28.6, 28.7, 28.82, 28.88, 28.95, 29.2, 34.3, 38.4, 39.2*, 52.5, 122.7, 147.0, 168.9, 171.1, 196.1 (*signal was detected by DEPT spectra owing to the overlap with the solvent signals).

N-acetyl-S-[(1E)-14-methyl-3-oxohexadec-1-en-1-yl]-L-cysteine (4)

Compound 2 (1.2 mg) was dissolved in DMSO-d₆ and analyzed in the same manner as 3. HR-MS (ESI): m/z: calcd for C₂₂H₄₀NO₄S: 414.2672, found: 414.2670 (M+H)⁺. ¹H NMR (500 MHz, DMSO-d₆, TMS (internal standard)): δ 0.82 (3H, d, J=7.0 Hz), 0.83 (3H, t, J=7.0 Hz), 1.03–1.10 (1H, m), 1.17–1.32 (18H, overlapped), 1.44–1.53 (2H, m), 1.84 (3H, s), 2.48 (2H, overlapped), 3.12 (1H, dd, J=8.2, 13.5 Hz), 3.31 (1H, overlapped), 4.38–4.44 (1H, m), 6.19 (1H, d, J=15.7 Hz), 7.76 (1H, d, J=15.7 Hz). ¹³C NMR (125 MHz, DMSO-d₆, TMS (internal standard)): δ 11.1, 19.0, 22.3, 23.8, 26.4, 28.6, 28.7, 28.8 (two carbons), 28.89, 28.94, 29.3, 33.3, 33.6, 35.9, 39.2*, 51.6, 123.1, 146.0, 169.2, 171.3, 196.2 (*signal was detected by DEPT spectra owing to the overlap with the solvent signals).

DLA-derived cysteine

Compound 1 (0.2 mg) was hydrolyzed with 5 n HCl (500 μl) at 105 °C for 1 h. The hydrolysates were concentrated to dryness. To the hydrolysates, water (100 μl) and 1 m NaHCO₃ (50 μl) were added, and incubated at 37 °C for 1 h. To the solution (30 μl), 1% D- or L-FDLA/acetone solution (40 μl) was added and incubated at 37 °C for 1.5 h. After the incubation, the reactions were terminated by the addition of 1 n HCl (10 μl). The reactants diluted with 50% aq. methanol were analyzed by LC–HR-MS.

Compound 2 (0.2 mg) was also hydrolyzed, derived with D- or L-FDLA, and analyzed in the same way as described above.

As almost all of the DLA-derived cysteine was observed as DLA-derived cystine and LL-cystine was derived with D- or L-FDLA. In addition, DLA-derived meso-cystine standard was made by the method of Kawasaki et al. ¹³

Detection of the DLA-derived cystine

The DLA-derived cystine were analyzed by LC–HR-MS. LC–HR-MS conditions are described as follows: column, Unison UK-C18, 3μm, 3.0 i.d. × 50 mm (Imtakt); mobile phase, 5 mm aq. ammonium formate in 0.02% formic acid (A): MeCN in 0.02% formic acid (B); gradient rate, A:B (7:3)–A:B (3:7), 10 min; flow rate, 0.6 ml min⁻¹. Retention time of each compounds are as follows: LL-cystine-L-DLA, 6.1 min; LL-cystine-D-DLA, 7.4 min; meso-cystine-L-DLA, 5.8 min; meso-cystine-D-DLA, 5.8 min; 1-L-DLA, 6.1 min; 1-D-DLA, 7.4 min; 2-L-DLA, 6.1 min; 2-D-DLA, 7.4 min.

VS-G assay

The VS-G assay was established by Ishimoto et al.⁷ It is a phenotype assay method using a co-culture system of green fluorescent protein (GFP)-labeled HUVECs with fibroblasts.

In brief, GFP-HUVECs were seeded at 7.5 × 10³ cells onto the confluent fibroblasts’ feeder layers in HuMedia-EG2 (KuRaBo, Tokyo, Japan). After adhesion of GFP-HUVECs to the fibroblasts’ layers, assay compounds in assay medium (M199, 0.5% fetal calf serum, 1% penicillin-streptomycin) were added and incubated at 37 °C for 3 days. Cell images were captured by using an In Cell Analyzer 6000 (GE healthcare Japan, Tokyo, Japan) or ImageXpress ULTRA (Molecular Devices, Sunnyvale, CA, USA). The value of area covered by GFP-HUVECs was analyzed using IN Cell Investigator 1.6 (GE healthcare Japan) or MetaXpress (Molecular Devices), and its value was used as in vitro activity. Details of the assay method will be described in another report.⁷