Introduction

Terpenoids are the largest family of compounds found in nature with over 24 000 known examples and are mainly produced by fungi and plants. Production of these compounds by actinomycetes including Streptomyces, however, was rather rare, and only limited numbers of compounds were reported to date.1 In view of the excellent ability of Streptomyces to produce many kinds of bioactive secondary metabolites with structural diversity, this phenomenon seemed to be interesting for us.

Being stimulated in this phenomenon, we started screening for terpenoids produced by actinomycetes and succeeded in the isolation of oxaloterpins2 and napyradiomycin analogs.3 Our strategy for screening of terpenoids in these cases was as follows: (1) prepare a lipophilic fraction from the fermentation broth of actinomycetes by EtOAc extraction, (2) analyze by TLC development followed by colorization with vanillin-H2SO4. Select the spots that change to bright purple or violet, (3) analyze fractions corresponding to these spots by NMR and (4) select fractions showing methyl singlets at ca. δH l.0 as candidates for terpenoids.2 This strategy, however, was not efficient as many of the selected fractions did not show the expected NMR features.

To carry out more efficient screening of terpenoids, we started using liquid chromatography NMR (LC-NMR) that enabled to detect compounds with several methyl groups without amenable to tedious procedures. During this screening process, we noticed that this method enabled to detect polyketides and peptides that showed strong methyl doublets at around δH l.0.

Here, we describe isolation aided by LC-NMR of the two new peptides, tumescenamides A and B, produced by Streptomyces tumescens YM23-260, and their structural elucidation.

Results and discussion

Fermentation, extraction and isolation

Several actinomycetes strains were cultured at 28 °C for 7 days by rotary shaking in 500 ml baffled Erlenmeyer flasks each containing 100 ml of the production medium. The filtrate of each fermentation broth was extracted with EtOAc and the residual oily material obtained after removal of the solvent was analyzed by LC-NMR (ODS column, 70% CH3CN and 30% D2O) to check for the presence of methyl signals at around δH 0 in the 1H-NMR spectrum.

A sample prepared from S. tumescens YM23-260 showed an LC peak (tR=5.2 min), which contained a substance showing the presence of nine methyl doublet signals at δH 0.4–1.6 (Figure 1). Additional signals at δH 4–5 ascribed to the α-methine signals of amino acids suggested that this LC peak was because of a peptide. In addition, a pair of aromatic doublet signals at δH 6.6 and 7.1 (each 2H) indicated the presence of a p-substituted aromatic ring presumably owing to a tyrosine residue.

Figure 1
figure 1

LC-NMR spectrum of EtOAc extract of the fermentation broth of Streptomyces tumescens. Retention time=5.2 min. ODS column, 70% CH3CN and 30% D2O. The strong peak at δH 1.9 is because of the residual suppression peak of the solvent.

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These data implied that this LC peak might contain a new metabolite. In addition to this peak, another LC peak (tR=7.9 min) showed similar NMR signals. Thus we carried out their large-scale purification as follows. The crude EtOAc extract prepared as described above from 4 l of the fermentation broth was subjected to silica gel column chromatography and C-18 reverse-phase HPLC yielding a major component (15 mg) and a minor component (0.6 mg).

Two-dimensional NMR studies of these compounds by COSY, heteronuclear single-quantum coherence and constant-time heteronuclear multiple bond correlation (CT-HMBC)4 as well as by HR-MS and IR measurements proved them to be new peptides as expected, and they were designated as tumescenamides A (1) and B (2) (Figure 2).

Figure 2
figure 2

Structures of tumescenamides A (1) and B (2).

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Structure elucidation

Compound 1 was isolated as a white powder ([α]25D+30, c 0.65, MeOH). The HRFAB-MS spectrum of 1 established its molecular formula as C37H57N5O8 (m/z 700.4248 [M+Na]+, calcd. 700.4279). Its peptide structure was clarified by the 1H-NMR, 13C-NMR and heteronuclear single-quantum coherence spectra recorded in acetone-d6 (Table 1) (Supplementary Figures S1–S6). Diagnostic resonances for five carbonyl carbons (δC 176.6 (Leu-1), 171.3 (Thr-1), 171.2 (Val-1), 169.5 (Tyr-1), 165.3 (α-amino-2-butenoic acid (ABA))) and four α-amino methine protons and carbons (δH 3.85/δC 61.7, 4.45/56.7, 4.92/55.5, and 4.29/54.4), indicated the peptide structure of 1. Further structural information on 1 was obtained by analyzing heteronuclear single-quantum coherence, DEPT, CT-HMBC and COSY spectra (Supplementary Figures S7 and S8) resulting in the construction of six partial structures: threonine (Thr), valine (Val), leucine (Leu), tyrosine (Tyr), ABA and a fatty-acid (FA) side chain.

Table 1 NMR data for tumescenamide A (1) (500 MHz, acetone-d6)
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The structure of the FA moiety was straightforwardly determined to be 2,4-dimethylheptanoic acid by analysis of its CT-HMBC spectrum as shown in Figure 3. Interestingly, this simple branched FA has never been isolated from the nature. The amino-acid constituents of 1 and FA were then connected on the basis of COSY, CT-HMBC and NOESY correlations (Figure 4; Supplementary Figure S9).

Figure 3
figure 3

Structures of the FA moiety of tumescenamides A (1) and B (2).

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Figure 4
figure 4

Important COSY, CT-HMBC and NOESY correlations observed for tumescenamide A (1).

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The linkage of ABA to Tyr was established by CT-HMBC correlations from the NH amide proton of ABA (δH: 7.98) and the α-amino proton of Tyr (H-2, δH: 4.45) to the Tyr amide carbonyl carbon (C-1, δC: 169.5). The Z configuration of ABA was clarified by an NOE between the methyl and amide protons of ABA. The α-amino proton of Tyr (H-2, δH: 4.45) and the α-amino proton of Val (H-2, δH: 3.85) were correlated to the carbonyl carbon of Val (C-1, δC: 171.2) by the CT-HMBC spectrum. A NOESY cross-peak between the NH of Tyr (δH: 7.48) and the α-amino proton of Val (H-2) also supported the sequence of Tyr–Val. The connection between Val and Leu was determined through interpretation of CT-HMBC data, which showed correlations from the α-amino proton of Val (H-2, δH: 3.85) and the α-amino proton of Leu (H-2, δH: 4.29) to the Leu amide carbonyl carbon (C-1, δC: 176.6). The NOESY correlation between the amide NH proton of Val (δH: 8.02) and the α-amino proton of Leu (H-2) also confirmed this connectivity. The subsequent connection of Leu to Thr was achieved on the basis of CT-HMBC correlations from the NH of Leu (δH: 7.92) and the α-amino proton of Thr (H-2, δH: 4.92) to the Thr amide carbonyl (C-1, δC: 171.3). This connectivity was also supported by the presence of a NOESY correlation between the amide NH proton of Leu (δH: 7.92) and the α-amino proton of Thr (H-2, δH: 4.92). Further, CT-HMBC correlations from the FA proton (H-2, δH: 2.65) and the amide NH proton of Thr (δH: 7.04) to the FA carbonyl carbon (C-1, äC: 177.3) led to the assignment of Thr to FA linkage. A NOESY correlation was also observed between these protons. Finally, location of the ester linkage was established by the observation of CT-HMBC correlation from Thr (H-3, δH: 5.15) to ABA (C-1, δC: 165.3) completing the full planar structure of 1.

The relative stereochemistry of FA was determined by J-based configurational analysis using 3JH−H and 3JC−H (Figure 5).5, 6 For this experiment, we used J-resolved HMBC7 for efficient measurements (Supplementary Figure S10). Both the methyl carbon at C-2 and H-2 exhibited small three-bond couplings with Hb-3 (JCH3(2)−Ha3=3.3 Hz, JH2−H3b=5.4 Hz) and thus they were required to be gauche to Hb-3. Anti orientations between Ha-3 and H-2 and between Hb-3 and C-1 were determined from the large three-bond couplings (JHa3−H2=8.8 Hz, JHb3−C1=6.3 Hz). Both H-4 and C-5 exhibited small three-bond couplings with Ha-3 (JH4−Ha3 =5.9 Hz, JC5−Ha3=2.2 Hz) and were required to be gauche to Ha-3. Anti-orientations between Ha-3 and methyl at C-4 and between Hb-3 and H-4 were determined from the large three-bond couplings (JHa3−CH3 (4)=5.3 Hz, JHb3−H4=8.3 Hz). These relationships assigned the relative stereochemistry of FA to 2R*, 4R* as shown in Figure 5.

Figure 5
figure 5

J-based configurational analysis of FA at C-2 and C-4.

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Hydrolysis of 1 using 6 N HCl yielded a mixture of free amino acids. The hydrolysis product was derivatized using the Marfey reagent8 and analyzed by HPLC. Comparison with the retention times of authentic Marfey standards of L- and D-forms of Thr, Tyr, Leu and Val showed that the absolute configurations of these amino acids were L-Thr, D-Tyr, L-Leu and L-Val. The absolute stereochemistry of the FA remained to be determined.

Tumescenamide B (2) was obtained as a white powder, which analyzed for the molecular formula C40H63N5O8 by the HRFAB-MS ([M+H]+ m/z 742.4756, calcd. 742.4755). The 1H- and 13C-NMR spectral data of the amino-acid moiety of 2 were completely identical with those of 1 (see Experimental procedure) suggesting that the structural difference between them resided in the FA moiety (Supplementary Figures S11–S13). The molecular formula of 2 and its 13C-NMR spectral data indicated that 2 has additional carbons, CH3 (δC 21.0), CH2 (40.0) and CH (28.2). Its COSY and CT-HMBC spectra proved the structure of the FA moiety to be 2,4,6-trimethylnonanoic acid as shown in Figure 3 (Supplementary Figures S14–S16).

Biological activity

Tested so far, 1 showed weak activity only in the luciferase-reporter assay system, which enabled to observe the expression of insulin-degrading enzyme (IDE). This enzyme, a metalloprotease enzyme responsible for insulin degradation, had been shown to have a key role in β-amyloid (Aβ) peptide degradation both in vitro and in vivo, and was selective for Aβ monomer.9, 10, 11 Thus, substances which induce the upregulation of IDE enzyme are expected to be promising drugs for the treatment of Alzheimer’s disease. Compound 1 induced reporter gene expression under the control of the IDE promoter more than three times at a concentration of 100 μM.

Experimental procedure

General experimental procedures

Optical rotations were recorded on a Jasco DIP-140 polarimeter (Jasco, Tokyo, Japan). UV and IR spectra were recorded on a Hitachi U-3310 spectrophotometer (Hitachi, Ibaraki, Japan) and a Shimadzu 8300 FTIR spectrometer (Shimadzu, Kyoto, Japan), respectively. LC-NMR was carried out on a Varian Inova 500 equipped with a cryo probe (Varian, Palo Alto, CA, USA). The HPLC condition of LC-NMR was as follows: Senshu Pak Pegasil ODS column (4.6 × 250 mm, flow rate of 0.7 ml min−1; Senshu, Tokyo, Japan), linear gradients started with 70% CH3CN containing D2O and finished 100% CH3CN in 30 min, flow rate of 1 ml min−1).

Both one-dimensional 1H- and 13C-NMR spectra were obtained on a Jeol A-400 (Jeol, Akishima, Tokyo, Japan) or a Varian Inova 500 NMR spectrometer (Varian), and two-dimensional COSY, NOESY, heteronuclear single-quantum coherence, CT-HMBC and J-resolved HMBC spectra were recorded on a Varian Inova 500 NMR spectrometer. Samples were dissolved in acetone-d6 and the solvent peak was used as an internal standard (δH 2.16 and δC 30.8). High-resolution FAB mass spectra were obtained using an LCT-premier XE mass spectrometer (Waters, Milford, MA, USA).

HPLC purifications were carried out using a Senshu Pak Pegasil ODS column (20φ × 250 mm, at a flow rate of 14 ml min−1; Senshu) equipped with a Hitachi High Technologies L-2450 Diode array detector (Hitachi, Ibaraki, Japan). Merck silica gel 60 F254 plastic-backed sheets (Merck, Darmstadt, Germany) were used for TLC analysis. Preparative TLC was performed using Merck Si gel 60 F254 plastic-backed sheets.

Cultivation of S. tumescens YM23-260

S. tumescens YM23-260 was isolated from the sediments of Big drop-off of the Republic of Palau and is maintained at Marine Biotechnology Institute. It showed 99% homology to the type strain of S. tumescens. This strain was cultivated in 15 ml test tubes each containing 5 ml of a preliminary seed medium consisting of starch 1.0%, polypeptone 1.0%, molasses 1.0% and meat extract 1.0% (pH 7.2). The test tubes were shaken on a reciprocal shaker (200 r.p.m.) at 28 °C for 2 days. A total of 1 ml aliquot of the seed culture was inoculated into each of the 500 ml baffled Erlenmeyer flasks containing 100 ml of the production medium consisting of starch 2.5%, soybean meal 1.5%, yeast extract 0.2% and CaCO3 0.4% (pH 6.2). The fermentation was carried out at 28 °C with agitation at 170 r.p.m. After 7 days, the fermentation broth was separated into mycelial cake and filtrate by suction filtration. The mycelial cake was dipped in 60% acetone for 2 h and the mycelium residue was removed by filtration. The extract was evaporated in vacuo to remove the acetone and the aqueous residue was extracted with EtOAc. The organic layer was dried over anhydrous Na2SO4 and evaporated to afford a brown oil (ca. 3 g from 10 l of the fermentation broth).

Purification of tumescenamides A (1) and B (2)

The brown oil was subjected to Si gel column chromatography developed with CHCl3 and MeOH at 20:1 to give fractions containing 1 and 2. Both the fractions were combined and dried under reduced pressure. The dried material was finally purified by ODS HPLC. Detection of the compounds was carried out at UV 254 nm. The column was eluted with MeCN in H2O (70%) to give 1 and 2 in this order. These fractions were separately concentrated in vacuo to give pure 1 (white powder, 30.0 mg) and 2 (white powder, 2.5 mg).

Determination of the amino acids and FA stereochemistry

A sample of 1 (6.0 mg) was hydrolyzed in 6 N HCl at 140 °C for 4 h. After concentration to dryness, the residue was dissolved in 10 ml of EtOAc-H2O (7:3). An amino-acid mixture was recovered in the aqueous layer and FA in the organic layer.

Preparation and HPLC analysis of Marfey derivatives

The aqueous layer was dried in vacuo and was added with 5% NaHCO3 (500 μl) and FDAA (Marfey's reagent, 10 mg) in acetone (500 μl). The mixture was heated on a bath at 70 °C for 3 h. The reaction product was analyzed by HPLC as follows: Senshu Pak Pegasil ODS column (20φ × 250 mm, flow rate of 0.7 ml min−1), TFA mobile phase (0.05%) consisting of 1% methanol in both A and B with 5% MeCN in A and 60% MeCN in B (linear gradients started with 0% B and finished 100% B in 45 min, flow rate of 1 ml min−1). Retention times of the standard FDAA derivatives were as follows: L-Thr 23.8 min, D-Thr 27.8 min, L-Tyr 29.2 min, D-Tyr 30.9 min, L-Leu 31.4 min, D-Leu 40.7 min, L-Val 32.6 min and D-Val 36.9 min. The chromatograms of the hydrolysate derivatized with FDAA showed peaks corresponding to L-Thr, D-Tyr, L-Leu and L-Val.

Tumescenamide A (1)

White powder; [α]25D+30 (c 0.65, MeOH); UV (MeOH) λmax (ɛ) 277 (1850), 224 (18900), IR (KBr) 3300, 1720, 1650, 1520 cm−1; 1H-NMR (500 MHz, acetone-d6) and 13C-NMR (125 MHz, acetone-d6), see Table 1; HRFAB-MS m/z 700.4248 (calcd. for C37H58N5O8, [M+H]+ 700.4279).

Tumescenamide B (2)

White powder; HRFAB-MS m/z 742.4756 (calcd. for C40H63N5O8, [M+H]+ 742.4755). 1H-NMR (400 MHz, acetone-d6) δH 4.93 (d, 9.0, 3.2, Thr-2), 5.13, (dq, 3.2, 6.1, Thr-3), 1.34 (d, 6.1, Thr-4), 7.01 (d, 9.0, Thr NH), 6.59 (d, 7.0, ABA-3), 1.67 (d, 7.0, ABA-4), 8.01 (s, ABA NH), 4.43 (m, Tyr-2), 2.99 (dd, 13.7, 13.7, Tyr-3), 3.29 (dd, 3.4, 13.9, Tyr-3), 7.20 (d, 8.4, Tyr-5), 6.71 (d, 8.40, Tyr-6), 7.46 (d, 8.6, Tyr NH), 3.65 (dd, 3.9, 3.9, Val-2), 2.12 (m, Val-3), 0.86 (d, 7.1, Val-4), 0.58 (d, 7.1, Val-5), 8.02 (d, 3.9, Val NH), 4.30 (dd, 7.6, 7.6, Leu-2), 1.52 and 1.58 (m, Leu-3), 1.72 (m, Leu-4), 0.88 (d, 7.1, Leu-5), 0.98 (d, 6.6, Leu-6), 7.95 (br d, 2.0, Leu NH), 2.66 (m, FA-2), 0.97 and 1.72 (m, FA-3), 1.28 and 1.42 (m, FA-4), 0.78 and 1.12 (m, FA-5), 1.50 (CH, m, FA-6), 0.99 and 1.20 (CH2, m, FA-7), 1.22 and 1.31 (CH2, m, FA-8), 0.84 (CH3, d, 7.0, FA-9), 1.08 (CH3, d, 6.8, FA-2-Me), 0.78 (CH3, d, 6.3, FA-4-Me), 0.76 (CH3, d, 6.5, FA-6-Me). 13C NMR δc 171.3 (Thr-1), 55.5 (Thr-2), 75.1 (Thr-3), 16.2 (Thr-4), 165.2 (ABA-1), 128.3 (ABA-2), 134.8 (ABA-3), 14.7 (ABA-4), 169.3 (Tyr-1), 56.7 (Tyr-2), 36.6 (Tyr-3), 130.2 (Tyr-4), 130.9 (Tyr-5), 115.8 (Tyr-6), 156.8 (Tyr-7), 171.2 (Val-1), 61.7 (Val-2), 29.2 (Val-3), 19.2 (Val-4), 17.0 (Val-5), 176.6 (Leu-1), 54.4 (Leu-2), 40.7 (Leu-3), 25.2 (Leu-4), 22.3 (Leu-5), 22.9 (Leu-6), 177.1 (C, FA-1), 38.8 (CH, FA-2), 42.1 (CH2, FA-3), 28.2 (CH, FA-4), 45.6 (CH2, FA-5), 30.2 (CH, FA-6), 40.0 (CH2, FA-7), 20.5 (CH2, FA-8), 14.6 (CH3, FA-9), 19.2 (CH3, FA-2-Me), 21.0 (CH3, FA-4-Me) and 20.3 (CH3, FA-6-Me).

Cell culture and bioassay for luciferase activity

We used the reporter plasmid vector (pGL3 basic vector, Promega, Madison, WI, USA) in which the IDE promoter gene is inserted.12 COS7 cells were transfected with the IDE reporter gene and maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 50 μg per ml streptomycin and 5 units per ml penicillin. For screening, 1 μl of sample containing 4 × 104 cells was added into 96 multi-well plates and 100 μl of the medium into each well. After incubation for 18 h, the productivity of luciferase in each well was measured with Picagene BrillianStar-LT Luciferase assay system (Toyo B-Net, Tokyo, Japan) and a 2103 EnVision Multilabel Reader (Perkin-Elmer, Waltham, MA, USA).