Introduction

Microorganisms produce structurally unique metabolites with various biological activities.1, 2, 3 These metabolites have been used as agrochemicals, pharmaceutical leads and therapeutic agents.4 They are also important as bioprobes, the tools used to investigate biological functions in chemical biology studies.5 Numerous secondary metabolites have been isolated using various bioassay-guided separations. We have isolated new active metabolites such as reveromycins,6 epoxyquinols7 and azaspirene8 from actinomycetes and fungi using unique bioassay systems. However, it is difficult to discover active metabolites as they are less abundantly found, form complex mixtures in extract or are unsuitable for bioassay systems. To overcome this difficulty, we constructed a microbial metabolite fraction library comprising semipurified metabolites coupled with an in-house spectral database, Natural Products Plot (NPPlot),9, 10 that contains UV and mass spectral information on metabolites of each microbe for structure-oriented isolation. Using the NPPlot, we isolated various structurally unique metabolites, such as verticilactam,11 spirotoamides12 and pyrrolizilactone.13 In addition, we expanded the library by stocking broths prepared from microbes isolated from soils from various regions of Japan. Approximately 6000 extracts of the broth library were analyzed using diode-array detector LC/MS and subsequently screened for structurally interesting metabolites. During screening, a broth prepared from a fungus, Pyrenochaetopsis sp. RK10-F058, was found to produce interesting metabolites, one of which was possibly identical to phomasetin14 by UV and mass spectral data. The others were likely to be related to phomasetin with m/z of 400–500. Phomasetin is a bioactive metabolite isolated from the fungus, Phoma sp.; it comprises a tetramic acid and a decaline unit.14 We recently identified Diels–Alderase as a key enzyme that controls the stereoselective construction of the decaline skeleton of equisetin15 that had a similar skeleton to that of phomasetin.16 Interestingly, phomasetin has a completely opposite absolute structural configuration to that of equisetin, and this is interesting from the viewpoint of biosynthesis. Therefore, we focused on the decaline-related metabolites produced by Pyrenochaetopsis sp. RK10-F058, and isolated new metabolites designated as wakodecalines A (1) and B (2) along with phomasetin (3) (Figure 1). Here, we report the isolation and structures of compounds 1 and 2.

Figure 1
figure 1

Structures of wakodecalines A (1) and B (2) and phomasetin (3).

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Results and discussion

The fungal strain Pyrenochaetopsis sp. RK10-F058 collected in Wako, Saitama, Japan, was cultured in 4.8 l of culture medium for 13 days. The culture broth was partitioned thrice with half the volume of EtOAc and the resulting organic extract was evaporated in vacuo to obtain a light brown oil (1.30 g). The oil was separated into 8 fractions using SiO2 medium pressure liquid chromatography (MPLC) with a CHCl3/MeOH stepwise gradient. The fourth and fifth fractions were combined and subjected to C18-MPLC with an acetonitrile/0.05% aqueous formic acid gradient system to obtain seven subfractions. The fourth subfraction was separated using Sephadex LH-20 (GE Healthcare, Pittsburgh, PA, USA) to obtain four fractions. The third and second fractions were purified using C18-HPLC with acetonitrile/0.05% formic acid isocratic elution at 72:28 and 48:52 to obtain compounds 1 (77.7 mg) and 2 (26.8 mg), respectively, as colorless powders (Table 1 and Supplementary Figure S1). Compound 3 was isolated as a colorless powder (397.3 mg) from the first fraction of the SiO2 MPLC fractions using SiO2 MPLC (hexane/EtOAc) and Sephadex LH-20 separations and identified as phomasetin using the NMR and electronic CD (ECD) spectra.14

Table 1 Physicochemical properties of wakodecalines A (1) and B (2)
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The molecular formula of 1 was determined to be C25H37NO6 using high-resolution electrospray ionization time-of-flight MS (HRESITOFMS) (found m/z: 446.2545 [M–H]−, calcd for C25H36NO6 446.2543) (Supplementary Figure S2). The IR spectrum implied the presence of hydroxyl and carbonyl groups (3400–3500, 1730 and 1625 cm−1). The 1H and 13C NMR spectra of 1 in acetonitrile-d3 showed doubling and broadening signals (Supplementary Figures S3 and S4). These features were reported for those of phomasetin,14 equisetin16 and gabusectin17 that had a tetramic acid and a decaline unit in the molecules, suggesting that 1 was supposed to have a related substructure to phomasetin. The 1H NMR spectrum showed 5 methyl signals at δ 0.88 (d, J=6.3 Hz), 0.90 (s), 1.14 (d, J=6.3 Hz), 1.71 (s) and 3.09 (s) that was assigned as an N-methyl, and 3 olefin signals at δ 5.22 (brs), 5.53 (dd, J=15.5, 6.3 Hz) and 5.69 (dd, J=15.5, 9.2 Hz). It also contained an α-proton at δ 4.81 (dd, J=8.0, 5.1 Hz), indicating the presence of an amino acid. Most signals in the 13C NMR spectrum were observed as doublings with a signal strength of ∼2:1. The careful assignment of heteronuclear single quantum correlation (HSQC) spectrum through the 13C DEPT experiment led to the assignment of the 25 carbon signals as the main conformers (Supplementary Figures S5 and S6). It comprised 5 methyls, 4 methylenes including an oxygenated one at δ 61.0, 11 methines including an oxygenated one at δ 68.9, suggesting the presence of a hydroxyl group, and 5 quaternary signals that included three carbonyl signals at δ 171.4, 171.7 and 215.4. The carbon signals also contained 4 olefin signals at δ 128.3, 132.6, 134.3 and 138.2, and an α-carbon signal at δ 61.4 of an amino acid. These observations implied that 1 had a decaline skeleton and an N-methylated serine similar to that of 3. The detailed structure was confirmed by interpreting the double quantum filtered (DQF) COSY and heteronuclear multiple bond correlation (HMBC) spectra (Supplementary Figures S7 and S8). The DQF-COSY revealed the proton spin networks of a cyclohexane moiety from C-5 to C-11 including Me-19 attached at C-8, an olefin side chain from C-13, which also connected to C-3 and C-20, to C-17, and a primary alcohol at C-22 (Figure 2a). The decaline ring was confirmed by HMBC correlations from H-3 to C-2 and C-11, from H-5 to C-3 and from Me-18 to C-3, C-4 and C-5 (Figure 2a). Me-12 showed HMBC correlations to C-2, C-3 and C-11, suggesting that Me-12 was attached at the C-2. Me-12 also showed HMBC correlation to C-1 that was correlated from H-20, and these correlations led to the construction of a cyclopentanone-fused decaline skeleton. The N-methylated serine moiety was confirmed by HMBC correlations from H-22 and H-24 to C-23, from H-22 to C-21 and from N-Me-25 to C-21 and C-22 and considering the 13C NMR chemical shifts. It was confirmed to attach to C-20 by HMBC correlation from H-20 to C-21 and the planer structure was constructed.

Figure 2
figure 2

Key DQF-COSY and HMBC correlations (a) and NOESY correlations (b).

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The geometry at Δ14 was assigned as an E-configuration by the large coupling constant of 15.5 Hz and confirmed by NOESY correlation between H-13 and H-15. The relative stereochemistry of the decaline moiety was determined using the NOESY spectrum (Supplementary Figure S9). The decaline skeleton was confirmed as a trans-configuration by NOESY correlations between the H-6 and H-8, H-10ax and Me-12 and between H-11 and H-7ax and H-9ax (Figure 2b). These correlations also confirmed a chair form of the cyclohexane ring, α-configuration of Me-12 and β-configuration of Me-19. The cyclopentanone ring fused by a β-configuration to the decaline was assigned by NOESY correlations between the H-3 and Me-12 and between H-11 and H-13 that also confirmed an α-configuration of the olefin side chain at C-13. The N-methylated serine moiety was assigned as a β-configuration at C-20 by a NOESY correlation between H-3 and H-20. Therefore, the overall structure of 1, including the relative stereochemistry around the decaline moiety, was determined as (2R*, 3S*, 6R*, 8S*, 11S*, 13R* and 20S*).

The molecular formula of 2 was determined to be C25H35NO6 by HRESITOFMS (found m/z: 444.2392 [M-H]−, calcd for C25H34NO6 444.2386), which was lower than that of 1 by two hydrogen (Supplementary Figure S10). The 1H and 13C NMR spectra were almost identical to those of 1 showing doubling and broadening signals except for the signals assigned for the olefin side chain at C-13 (Supplementary Figures S11 and S12). The proton and carbon signals derived from the hydroxyl group at C-16 of 1 disappeared and the additional carbonyl signal was observed at δ 199.0. These observations suggested that 2 had a carbonyl group at C-16 that was oxidized from the hydroxyl group of 1. The planer structure was confirmed in the same manner as that of 1 based on the interpretation of NMR spectra including 13C DEPT, HSQC, DQF-COSY and HMBC (Supplementary Figures S13–S16). The olefin side chain was confirmed by the HMBC correlations from Me-17 to C-15 and C-16 (Supplementary Figure S17a). The relative stereochemistry, including the geometry of Δ14, was confirmed to be the same as that of 1 by the NOESY correlations (Supplementary Figures S17b and S18).

The absolute configurations of 1 and 2 were confirmed using a combination of chemical methods and the calculation of the ECD spectrum. The configuration at C-16 of 1 was determined by application of the modified Mosher’s method.18 The methyl ester 4 derived from 1 by treatment with TMSCHN2 was converted to the (R, R) and (S, S)-diMTPA esters (5 and 6) at C-16 and C-24 with (S)- and (R)-MTPA chloride, respectively (Scheme 1). They were used to calculate the differences of 1H NMR chemical shift values around C-16. The results suggested that the absolute configuration at C-16 was assigned as an S (Figure 3). The Marfey’s method19 was applied to determine the absolute configuration at C-22 on the N-methylated serine derived from 1 by hydrolysis using 6 N HCl for 18 h at 110 °C in a sealed ampule. The hydrolytic product, N-methylated serine, was reacted with 1-fluoro-2,4-dinitrophenyl-5- L-leucinamide (L-FDLA), and the resulting product, N-methylated serine-L-FDLA, was analyzed using LC/MS. At the same time, the standard L-FDLA derivatives of N-methylated L- and D-serines, which were derived from N-carbobenzoxy (cbz) L- and D-serines, were prepared and analyzed using LC/MS. The FDLA derivative of N-methylated serine from 1 eluted at 16.20 min, whereas the standard L- and D-derivatives eluted at 15.76 and 16.19 min, respectively (Supplementary Figure S19). The result showed that the N-methylated serine in 1 was the D-form and C-22 was assigned as an R-configuration. Compound 2 was confirmed to have the same absolute configurations as those of 1, except for the C-16, by direct comparison of the methyl ester 7, which was derived from 2 by treatment with TMSCHN2, with an oxidized derivative of the methyl ester 4 using the 1H NMR and ECD spectra (Scheme 1 and Supplementary Figure S20).

Figure 3
figure 3

ΔδS-R values obtained from 16,24-diMTPA esters 5 and 6.

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The absolute configuration of the decaline skeleton was deduced by comparing the experimental and calculated ECD spectra of 2.20 The conformers obtained by conformational analysis using Merck molecular force field, which gave 123 stable conformers, were optimized by Hartree–Fock STO-3G and further optimized by density functional theory at B3LYP/6-31G(d) level. The ECD spectra were simulated by time-dependent density functional theory at ωB97XD/6-31G(d) level and Boltzmann averaged. The resulting calculated ECD spectrum agreed with the experimental ECD spectrum (Figure 4). Based on the above results, the absolute configurations were determined to be 2R, 3S, 6R, 8S, 11S, 13R, 16S, 20S, 22R for 1 and 2R, 3S, 6R, 8S, 11S, 13R, 20S, 22R for 2, respectively, and found to be the same as those of phomasetin on the decaline moiety and N-methylated serine.

Figure 4
figure 4

Experimental and calculated electronic CD (ECD) spectra of wakodecaline B (2).

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We evaluated the cytotoxicities of compounds 1 and 2 as well as those of 3 against the human cervical epidermoid carcinoma cell line, HeLa, human promyelocytic leukemia cell line, HL-60, and rat kidney cells infected with ts25, a T-class mutant of Rous sarcoma virus Prague strain, srcts-NRK. Furthermore, their antimicrobial activities against Staphylococcus aureus 209, Escherichia coli HO141, Aspergillus fumigatus Af293, Pyricularia oryzae kita-1 and Candida albicans JCM1542, and antimalarial activity against P. falciparum 3D7 were evaluated. Compounds 1 and 2 did not show cytotoxicity or antimicrobial activity with IC50 of 30 μg ml−1, but they showed moderate antimalarial activity with IC50 values of 28 and 16 μg ml−1, respectively. Compound 3 showed moderate cytotoxicity against HeLa, HL-60 and srcts-NRK with IC50 values of 4.5, 1.4 and 4.4 μg ml−1, strong antimicrobial activities against S. aureus, E. coli and P. oryzae with IC50 values of 0.057, 0.53 and 0.018 μg ml−1 and strong antimalarial activity with an IC50 value of 0.74 μg ml−1. These results suggest that the tetramic acid unit might be important for the observed activities.

The broadening and doubling of signals in 1H and 13C NMR spectra of wakodecalines A and B suggested the existence of conformers. These features are reported for phomasetin14 and other tetramic acid containing decaline metabolites as mentioned before, and known to be caused by tautomerization.16, 17 Tetramic acid is reported to be a mixture of tautomers of a diketo form and enolized hydroxyl forms in the solution.21 Similar tautomerizations have been suggested for a ketone at C-1 position of phomasetin and equisetin.14, 16 From these reports, the diketo moiety of wakodecalines A and B is possibly associated with enolized hydroxyl-forms at C-1 or C-21.

Wakodecalines A and B have a cyclopentanone fused decaline skeleton that is rare in natural products. Only two fungal metabolites, fusarisetin A22, 23 and altercrasin A,24 have been reported as natural products with the same skeleton. However, the absolute stereochemistry of their decaline moiety was identical to that of equisetin, but opposite to those of the wakodecalines. D-Serine was assigned as the amino acid that is also the opposite of equisetin. From the abovementioned facts, we proposed that the wakodecalines might be biosynthesized from phomasetin, which has the same absolute stereochemistry as that of the wakodecalines, through a spiro intermediate similar to altercrasin A24 (Supplementary Scheme S1). We will continue to search related metabolites from this fungus to reveal the biosynthetic mechanism of decaline-type tetramic acid derivatives and examine their activities.

Experimental procedures

General experimental procedures

Analytical-grade solvent and reagents were purchased from commercial sources. UV and optical rotations were recorded on a JASCO V-630 BIO spectrophotometer (JASCO International, Tokyo, Japan) and a HORIBA SEPA-300 high sensitive polarimeter (HORIBA, Kyoto, Japan), respectively. ECD spectra were measured on a JASCO J-720 CD spectrometer. IR spectra were recorded on a HORIBA FT-720 with a DuraSampl IR II ATR instrument. NMR data were obtained at 500 MHz for 1H NMR and 125 MHz for 13C NMR on a JEOL JNM-ECA-500 spectrometer (JEOL, Tokyo, Japan). Chemical shifts (in ppm) were referenced against the residual undeuterated solvent. LC/MS analysis was performed on a Waters 2965 Alliance system with 2996PDA detector (Waters, Milford, MA, USA) connected to AB Sciex Qtrap by ESI probe (AB Sciex, Framingham, MA, USA) on a Waters Xterra C18 column (2.1 mm i.d. × 150 mm, 5 μm) with elution of acetonitrile/0.05% aqueous formic acid linear gradient system (acetonitrile: 5 to 100% in 30 min at 0.2 ml min−1), or Waters 600E pump system with 996 PDA detector connected to Waters ZQ by ESI probe on a Senshu Pak Pegasil ODS column (Senshu Scientific, Tokyo, Japan) (4.6 mm i.d. × 250 mm, 5 μm) with elution of acetonitrile/0.05% aqueous formic acid linear gradient system (acetonitrile: 20 to 100% in 20 min at 1.0 ml min−1). HRESITOFMS was obtained on a Waters Synapt G2. Teredyne ISCO CombiFlash Companion (Teredyne ISCO, Lincoln, NE, USA) was used for MPLC. Preparative HPLC was performed using a Waters 600E pump system with Senshu Pak Pegasil ODS column (20 mm i.d. × 250 mm or 10 mm i.d. × 250 mm, 5 μm).

Strain and culture condition

The fungal strain Pyrenochaetopsis sp. RK10-F058 was isolated from a soil sample collected in Wako, Saitama, Japan, in 2010, and identified using ITS-5.8s rDNA sequencing and morphological observation (Techno Suruga Laboratory, Shizuoka, Japan). It was deposited in Chemical Biology Research Group, RIKEN. The conidia freshly prepared was inoculated in 500 ml cylindrical flasks containing 100 ml of PDB medium with 0.1% agar at 28 °C for 6 days on a rotary shaker at 150 r.p.m. Then, 4 ml of the preculture was inoculated in 500 ml cylindrical flasks containing 100 ml of YMGS medium (0.5% yeast extract, 0.5% malt extract, 1% glucose and 1% soluble starch), and cultured at 28 °C for 13 days on the same rotary shaker.

Extraction and isolation

The 4.8 l of culture broth was partitioned with half volume of EtOAc three times to obtain an organic soluble material. It was evaporated in vacuo to afford 1.30 g of light brown oil. The brown oil was subjected to SiO2 MPLC with CHCl3/MeOH stepwise gradient system (MeOH: 0, 1, 2, 5, 10, 20, 50 and 100%) to afford 8 fractions. The first fraction was separated by SiO2 MPLC with hexane/EtOAc linear gradient and Sephadex LH-20 to afford 397.3 mg of compound 3 as colorless powder. The fourth and fifth fractions was combined and subjected to C18-MPLC with acetonitrile/0.05% aqueous formic acid linear gradient to obtain seven subfractions. The fourth subfraction was separated by Sephadex LH-20 to afford four fractions. The third and second fractions were purified by C18-HPLC with acetonitrile/0.05% aqueous formic acid isocratic elution of 72:28 and 48:52 to afford 77.7 and 26.8 mg of compounds 1 and 2 as colorless powder, respectively. Physicochemical properties of 1 and 2 are summarized in Table 1. 1H and 13C NMR chemical shifts in acetonitrile-d3 of 1 and 2 are summarized in Table 2.

Table 2 1H and 13C NMR chemical shifts of wakodecalines A (1) and B (2) in acetonitrile-d3
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3: colorless powder; (c 0.1, MeOH) +558; ECD (MeOH) λmax (Δɛ) 234 (+18.2), 260 (+6.46), 293 (+15.1), 360 (0); 1H NMR (CDCl3) δ 0.87 (m), 0.89 (d, J=6.3 Hz, 3H), 1.01 (m), 1.08 (m), 1.36 (m), 1.49 (m), 1.63 (m), 1.66 (d, J=6.3 Hz, 3H), 1.72 (m), 1.77 (m), 1.80 (m), 1.94 (m), 3.01 (s, 3H), 3.12 (brd, J=8.6 Hz), 3.60 (brs), 3.86 (m), 3.99 (m), 5.16 (m), 5.21 (m), 5.48 (m), 5.77 (m), 5.87 (m); 13C NMR (CDCl3) δ 14.1, 18.2, 22.4, 22.7, 27.5, 28.4, 33.7, 35.9, 39.4, 39.8, 42.7, 49.4, 49.6, 60.6, 66.9, 100.1, 126.3, 128.1, 130.4, 131.6, 131.7, 132.6, 177.3, 190.8, 199.2.

Preparation of methyl ester 4

To a stirred solution of 1 (4.8 mg, 10.7 μM) in benzene-MeOH (0.6 ml, 4:1) was added 2.0 M solution of TMSCHN2 in diethyl ether (10.8 μl; 21.6 μmol) at room temperature. The reaction mixture was stirred for 10 min and then concentrated to provide methyl ester 4 (4.9 mg, 99%).

4: 1H NMR (CDCl3) δ 0.87 (d, J=6.5 Hz, 3H), 0.95 (s, 3H), 1.25 (d, J=6.5 Hz, 3H), 1.71 (brs, 3H), 2.01 (d, J=11.0 Hz, 1H), 3.18 (s, 3H), 3.28 (m, 1H), 3.48 (d, J=10.5 Hz, 1H), 3.71 (s, 3H), 3.96 (m, 1H), 4.06 (m, 1H), 4.27 (m, 1H), 4.96 (dd, J=7.5, 5.5 Hz, 1H), 5.21 (brs, 1H), 5.65 (dd, J=15.0, 5.0 Hz, 1H), 5.67 (dd, J=15.0, 7.5 Hz, 1H).

Preparation of 16,24-di(R)-MTPA ester 5

To a stirred solution of crude 4 (1.6 mg, 3.5 μmol) in CH2Cl2 (0.5 ml) were added pyridine (1.8 μl, 20.8 μmol), DMAP (0.1 mg) and (S)-MTPA chloride (2.6 μl, 13.8 μmol) at room temperature. The reaction mixture was heated under reflux for 12 h. The reaction mixture was evaporated to afford a yellow oil that was purified by preparative TLC (n-hexane: EtOAc=2:1) to provide 16,24-di(R)-MTPA ester 5 (0.3 mg, 10%).

5: 1H NMR (CDCl3) δ 0.88 (d, J=6.5 Hz, 3H), 0.93 (s, 3H), 1.39 (d, J=6.5 Hz, 3H), 1.64 (brs, 3H), 2.94 (s, 3H), 3.30 (m, 2H), 3.51 (s, 3H), 3.53 (s, 3H), 3.57 (s, 3H), 4.61 (dd, J=12.0, 3.0 Hz, 1H), 4.81 (dd, J=12.0, 6.0 Hz, 1H), 4.98 (dd, J=6.0, 3.0 Hz, 1H), 5.19 (brs, 1H), 5.56 (m, 1H), 5.57 (dd, J=15.5, 6.5 Hz, 1H), 5.63 (dd, J=15.5, 5.5 Hz, 1H).

Preparation of 16,24-di(S)-MTPA ester 6

This compound was prepared from 4 with (R)-MTPA chloride in 10% yield using the same procedure for preparation of 5.

6: 1H NMR (CDCl3) δ 0.88 (d, J=6.5 Hz, 3H), 0.92 (s, 3H), 1.31 (d, J=7.0 Hz, 3H), 1.67 (brs, 3H), 3.00 (s, 3H), 3.37 (m, 2H), 3.50 (s, 6H), 3.60 (s, 3H), 4.67 (dd, J=12.5, 4.0 Hz, 1H), 4.75 (dd, J=12.5, 6.0 Hz, 1H), 5.09 (m, 1H), 5.21 (brs, 1H), 5.55 (m, 1H), 5.70 (dd, J=15.0, 5.0 Hz, 1H), 5.74 (dd, J=15.0, 7.0 Hz, 1H).

Oxidation of methyl ester 4

To a stirred solution of methyl ester 4 (0.8 mg, 1.7 μM) in CH2Cl2 (0.2 ml) was added active MnO2 (1.2 mg, 13.8 μmol) at room temperature. After stirring for 1 h, the mixture was purified by SiO2 column chromatography (n-hexane: EtOAc=1:3) to provide colorless solid 7 (0.6 mg, 75%).

7: 1H NMR (CDCl3) δ 0.88 (d, J=6.5 Hz, 3H), 0.98 (s, 3H), 1.69 (brs, 3H), 2.12 (d, J=9.5 Hz, 1H), 2.25 (s, 3H), 2.48 (brt, J=3.0 Hz, 1H), 3.18 (s, 3H), 3.54 (m, 2H), 3.70 (s, 3H), 3.98 (m, 1H), 4.07 (m, 1H), 4.89 (dd, J=6.5, 6.0 Hz, 1H), 5.25 (brs, 1H), 6.29 (d, J=15.5 Hz, 1H), 6.84 (ddd, J=15.5, 8.0, 1.0 Hz, 1H); ECD (MeOH) λmax nm (Δɛ) 230 (+9.03), 297 (−2.51), 340 (0) (Supplementary Figure S20).

Esterification of 2

To a stirred solution of 2 (1.0 mg, 2.3 μmol) in benzene-MeOH (0.2 ml, 4:1) was added 2.0 M solution of TMSCHN2 in diethyl ether (2.3 μl, 4.6 μmol) at room temperature. The reaction mixture was stirred for 10 min and then concentrated to provide methyl ester 7 (1.0 mg, 95%).

7: ECD (MeOH) λmax nm (Δɛ) 231 (+8.06), 296 (−2.10), 340 (0) (Supplementary Figure S20).

Acid hydrolysis of 1 and preparation of FDLA derivative

A mixture of 1 (2.4 mg, 5.2 μmol) and 6 N HCl (100 μl) was heated at 110 °C for 18 h in a sealed ampule. After cooling to room temperature, lipophilic materials were removed by extraction with CHCl3. The aqueous layer was dried up and dissolved in 100 μl of H2O. The resulting solution (50 μl) was treated with 50 μl of 1% L-FDLA in acetone and 50 μl of 1 M NaHCO3. The mixture was heated at 40 °C for 1.5 h. After cooling to room temperature, the reaction mixture was neutralized with 50 μl of 1 N HCl, and the resulting mixture was added to 300 μl of MeOH to total volume of 500 μl. From the solution, 200 μl aliquot was withdrawn and dried up, and then redissolved in 200 μl of MeOH. The solution (10 μl) was analyzed by LC/MS on a Waters 600E pump system connected to Waters ZQ.

Preparation of FDLA derivatives of N-methyl-L-serine and N-methyl-D-serine

Authentic samples of N-methyl-L-serine and N-methyl-D-serine were prepared from N-cbz-L-serine and N-cbz-D-serine, respectively, according to the procedure reported by Hughes and colleagues.25 N-methyl-D-serine or N-methyl-L-serine was dissolved in H2O by 1% (w/v), and each of 50 μl aliquot was treated for LC/MS as the same manner as that of hydrolysate of 1.

Computer calculation of ECD spectrum of compound 2

The conformational analysis and calculation of ECD spectra were performed with Spartan '16 (Wave function, Irvine, CA, USA) on Mac Pro Apple (Early 2008) and Gaussian 09 revision E.01 (Gaussian, Wallingford, CT, USA)26 on HOKUSAI GreatWave in RIKEN (Wako, Japan). Wakodecaline B (2) was submitted to a conformational search employing Merck molecular force field to afford 123 stable conformers. Each conformer was optimized by Hartree–Fock/STO-3G with Spartan '16 and further optimized by density functional theory at B3LYP/6-31G (d) level with Gaussian 09. The optimized conformers were subjected to a time-dependent density functional theory at ωB97XD/6-31G (d) level with Gaussian 09 to obtain calculated ECD spectra. They were averaged based on the Boltzmann distribution of the 5 most stable conformers that were selected with >1.0% of Boltzmann distribution and covered 92.0% of population (see Supplementary Information).

Cytotoxicity, antimicrobial activity and antimalarial tests

The in vitro cytotoxicity assay methods against HeLa, HL60 and srcts-NRK cells have been described in the previous report.26 The microdilution assay against S. aureus 209, E. coli HO141, A. fumigatus Af293, P. oryzae kita-1 and C. albicans JCM1542 have been in the previous report.27 The antimalarial test against P. falciparum 3D7 has been described in the previous report.28

Dedication

This article is dedicated to the special issue for Professor Hamao Umezawa.

scheme 1

Preparation of 16,24-di(R)-MTPA ester 5, 16,24-di(S)-MTPA ester 6 and methyl ester 7.