Yakushinamides, Polyoxygenated Fatty Acid Amides That Inhibit

Aug 22, 2016 - Abstract Image. Yakushinamides A (1) and B (2), prolyl amides of polyoxygenated fatty acids, have been isolated from the marine sponge ...
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Yakushinamides, Polyoxygenated Fatty Acid Amides That Inhibit HDACs and SIRTs, from the Marine Sponge Theonella swinhoei Kentaro Takada,*,†,‡ Yasufumi Imae,†,‡ Yuji Ise,§ Susumu Ohtsuka,⊥ Akihiro Ito,∥,# Shigeru Okada,† Minoru Yoshida,∥,# and Shigeki Matsunaga*,† †

Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan § Sugashima Marine Biological Laboratory, Nagoya University, Toba, Mie 517-0004, Japan ⊥ Takehara Marine Station, Hiroshima University, Takehara, Hiroshima 725-0024, Japan ∥ Chemical Genomics Reserach Group, RIKEN Center for Sustainable Resource Science, Wako, Saitama 351-0198, Japan # Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan S Supporting Information *

ABSTRACT: Yakushinamides A (1) and B (2), prolyl amides of polyoxygenated fatty acids, have been isolated from the marine sponge Theonella swinhoei as inhibitors of HDACs and SIRTs. Their planar structures were determined by interpretation of the NMR data of the intact molecules and tandem FABMS data of the methanolysis products. For the assignment of the relative configurations of the three contiguous oxymethine carbons in 1 and 2, Kishi’s universal NMR database was applied to the methanolysis products. During the assignments of relative configurations of the isolated 1-hydroxy-3-methyl moiety in 1 and the isolated 1-hydroxy-2-methyl moiety in 2, we found diagnostic NMR features to distinguish each pair of diastereomers. The absolute configurations of 1 and 2 were determined by a combination of the modified Mosher’s method and Marfey’s method. Although the modified Mosher’s method was successfully applied to the methanolysis product of 1, this method gave an ambiguous result at C-20 when applied to the methanolysis product of 2, even after oxidative cleavage of the C-14 and C-15 bond.

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we report the isolation, structure elucidation, and HDAC inhibitory activities of 1 and 2.

cetylation and deacetylation of histone proteins are regulated by histone acetyltransferases and histone deacetylases (HDACs), respectively. HDACs remove the acetyl groups to consolidate interactions between negatively charged DNA and the histone proteins, resulting in the repression of transcription. Eighteen HDACs have been identified in the human genome and are grouped into four classes based on the functions and structures: Zn2+-dependency (class I, II, and IV) and NAD+-dependency (class III);1 the latter are called sirtuins (SIRTs).2 HDAC1 is a member of the class I HDAC family.3 The overexpression of HDAC1 has been correlated with abnormal proliferation of tumor cells,4−6 dysfunction of the apoptosis pathways,7 and acquired resistance to chemotherapy.8 It has also been documented that HDAC1 knockdown results in cell cycle arrest,9 selective apoptosis of tumor cells,7,9 and inhibition of tumor cell proliferation.6,9,10 These observations indicate that HDAC inhibitors are potential candidates as anticancer agents.11 In fact, a couple of HDAC inhibitors are used as cancer chemotherapeutics.12 In the course of our continuing search for biologically active molecules from marine sponges, we found that the extract of the sponge Theonella swinhoei inhibited HDAC1. Bioassayguided fractionation afforded two active compounds, termed yakushinamide A (1) and yakushinamide B (2). In this paper, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The MeOH and EtOH extracts of the sponge (340 g, wet weight) were combined and subjected to solvent partitioning between H2O and CHCl3. The organic layer was separated by ODS flash chromatography, gel permeation chromatography, and silica gel column chromatography followed by ODS HPLC. Because the HPLC peaks were broad under neutral conditions, we added AcOH in the mobile phase. Although the peaks became sharper, the isolated compounds decomposed after evaporating the solvent.13 Therefore, HPLC was performed using a phosphate buffer at neutral pH to afford yakushinamides A (1, 5.7 mg) and B (2, 5.8 mg). Yakushinamide A (1) had a molecular formula of C45H80NNaO14S. The presence of a prominent fragment ion due to a loss of SO3Na (m/z 102) from the sodium adduct ion in the FABMS spectrum indicated the presence of a sulfate group. Interpretation of the 1H NMR data in conjunction with the HSQC data showed the presence of five oxygenated Received: June 27, 2016

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DOI: 10.1021/acs.jnatprod.6b00588 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

that the triacetoxy moiety in 1 was in the anti−anti relative configuration (Figure 3). It was not possible to determine the 1H−1H coupling constants between H-28 and H2-29 and between H2-29 and H30 due to the heavily overlapped 1H NMR signals, precluding the assignment of the relative configuration between C-28 and C-30. To the best of our knowledge, no convenient method to assign the relative configuration of the 1-hydroxy-3-methyl moiety is available. In our previous study on shulzeines,15 we noticed that the 1H NMR signals of the central geminal methylene protons in the 1-hydroxy-3-methyl moiety were highly diastereotopic. However, the corresponding methylene protons in 1 were less diastereotopic. The difference in the extent of diastereotopicity was emphasized in the MTPA esters.15 From these observations, we considered it possible to distinguish the relative configuration of the 1-hydroxy 3-methyl moiety based on the degree of diastereotopicity of the central methylene proton signals.16 In order to confirm this idea, we elected to prepare diastereomers of 3-methyl-5-decanol and analyze their NMR data. (S)-2-Methyl-1-butanol was converted to (S)-2-methylbutyl bromide,19 which was coupled with hexanal through a Grignard reaction to afford a diastereomeric mixture of (3S,5R)- and (3S,5S)-3-methyl-5-decanol (4 and 5, respectively). By contemplating that the absolute configuration of the newly generated secondary alcohol at C-5 can be determined by the Mosher’s method, we converted the mixture of 4 and 5 to the mixture of the (S)-MTPA esters (6a and 7a) and the mixture of the (R)-MTPA esters (6b and 7b). We used CDCl3−C5D5N (1:1) as the solvent for NMR measurements, in order to distinguish the oxymethine protons in the MTPA esters. Because one isomer was twice as abundant as the other, we were able to assign the 1H and 13C signals of each isomer separately. The distribution of the Δδ values suggested that the (3S,5R)-isomer was the major product and the (3S,5S)-isomer the minor. The H2-4 signals of the (3S,5R)-isomers (6a and 6b) converged, whereas those of the (3S,5S)-isomers (7a and 7b) were highly diastereotopic (Figure 4a). Then, 3 was converted to the tetra-(S)-MTPA ester (8a) and the tetra-(R)MTPA ester (8b), in both of which the MTPA esters were introduced at C-13, C-15, C-20, and C-28. The H2-29 signals in 8a and 8b were less diastereotopic (Figure 4b), and the distribution of the Δδ values suggested the 28S-configuration. Therefore, the configuration at C-30 was assigned as R. The distribution of the Δδ values around the C-13 to C-15 portion suggested the 13S and 15R configurations. By taking into account the relative configuration of the triol portion, the configuration at C-14 was assigned as S. The absolute configuration of the proline residue was determined to be L by Marfey’s method (Figure S1).

methines, one O-methyl, one each of nitrogenous methine and nitrogenous methylene, three acetyl methyls, one doublet methyl, one terminal methyl, and methylenes in saturated alkyl chains. Extensive analysis of the 2D NMR data revealed the presence of five partial structures (Figure 1): an acylated proline methyl

Figure 1. Partial structures in 1 and 2.

ester (partial structure a), which appeared as a 1:4 mixture due to the geometrical isomerism of the amide bond; three contiguous oxygenated methines (partial structure b); an isolated oxygenated methine (partial structure c); a 1hydroxy-3-methyl moiety (partial structure d); and a terminal portion of the alkyl chain (partial structure e). HMBC data indicated that the oxygen atoms in partial structure b were all acetylated. The 1H and 13C chemical shift values of the oxymethines (δH 4.32/δC 80.8 in partial structure c and δH 3.61/δC 70.5 in partial structure d) indicated that the oxygen atom in the former was sulfonated and the latter was free (Table 1). Because partial structures b−d were separately placed in a long methylene chain, it was not possible to determine their locations on the basis of the NMR data. Therefore, we conducted tandem FABMS analysis of the pentaol 3 derived from 1 by methanolysis. Charge-remote fragmentations observed with the lithiated proline moiety gave diagnostic product ions separated by 30 and 28 mass units, permitting us to locate the five hydroxy groups at C-13, 14, 15, 20, and 28 and the methyl group at C-30 (Figure 2). Because of the overlapped H-13 and H-15 signals and the medium-sized coupling constants between H-14 and both H-13 and H-15, it was not possible to assign the relative configuration of this portion in 1 and 3. Therefore, we employed Kishi’s universal NMR database, in which NMR data of all possible diastereomers of contiguous triols are reported.14 The data of 3 matched with those of (5R,6S,7S)-decane-1,5,6,7tetraol but not with those of other diastereomers,14 suggesting B

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Table 1. 1H and 13C NMR Data of Yakushinamides A (1) and B (2) in CD3OD (1H 600 MHz, 13C 150 MHz) yakushinamide A (1) position

174.5a [174.2],b C 60.2 [60.9], CH 30.3 [32.1], CH2 25.7 [23.5], CH2 48.5 [47.5], CH2 174.6a [174.8], C 35.1 [35.0], CH2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 29-Me 30 30-Me 31 32 33 34 35 36 37 1-OMe 13-OAc 14-OAc 15-OAc a,c−e

δC, type

25.7 [25.8], CH2 30.0 [29.9], CH2 30.0, CH2 26.0, CH2 30.6, CH2 73.1, CH 74.8, CH 73.1, CH 30.6, CH2 26.0, CH2 25.7, CH2 35.1, CH2 80.6, CH 35.4, CH2 26.0, CH2 30.8,d CH2 30.7,d CH2 30.8,d CH2 26.7, CH2 38.7, CH2 70.4, CH 46.1, CH2 30.6, CH 21.0, CH3 37.7, CH2 27.9, CH2 31.1, CH2 30.5, CH2 33.1, CH2 23.7, CH2 14.5, CH3 52.7 [53.2], CH3 172.2,C; 20.9, CH3 171.8,C; 20.7, CH3 172.2, C;20.9, CH3

yakushinamide B (2)

δH (J in Hz) 4.41, 2.23, 2.01, 3.64,

δC, type

dd (8.7, 4.3) [4.62 dd (8.7, 2.5)] m; 1.95, m [2.31, m, 2.16, m] m [1.92, m, 1.86, m] m; 3.60, m [3.55, m, 3.48, m]

2.38, dd (15.3, 7.6); 2.36, dd (15.3, 7.6) [2.24, m, 2.11, m] 1.60, m [1.61, m, 1.58, m] 1.35, m [1.30, m] 1.30, m 1.36, m; 1.27, m 1.66, m; 1.59, m 5.00, m 5.09, t (5.0) 5.02, m 1.67, m; 1.61, m 1.33, m 1.40, m 1.61, m 4.31, quint (5.9) 1.64, m; 1.59, m 1.40, m 1.32, m 1.32, m 1.32, m 1.44, m; 1.33, m 1.43, m; 1.34, m 3.60, m 1.34, m; 1.27, m 1.60, 0.89, 1.36, 1.35, 1.28, 1.29, 1.28, 1.30, 0.90, 3.70, 2.03, 2.06, 2.03,

m d (6.8) m; 1.08, m m; 1.27, m m m m m t (7.0) s [3.76 s] s s s

174.5c [174.2], C 60.2 [60.9], CH 30.3 [32.1], CH2 25.7 [23.5], CH2 48.5 [47.5], CH2 174.6b [174.8], C 35.1 [35.0], CH2

δH (J in Hz) 4.41, 2.23, 2.01, 3.64,

dd (8.7, 4.3) [4.62, dd (8.7, 2.5)] m; 1.95, m [2.31, m, 2.16, m] m [1.92, m, 1.86, m] m; 3.60, m [3.55, m, 3.48, m]

25.7 [25.8], CH2 30.0 [29.9], CH2 30.0, CH2 26.1, CH2 30.6, CH2 73.1, CH 74.8, CH 73.1, CH 30.6, CH2 26.3, CH2 26.4, CH2 38.1, CH2 72.3, CH 38.5, CH2 26.8, CH2 30.7,e CH2 30.8,d CH2 30.8,e CH2 26.6, CH2 31.8, CH2 84.4, CH 36.9, CH 15.1, CH3 33.3, CH2

2.38, dd (15.3, 7.6); 2.36, dd (15.3, 7.6) [2.24, m, 2.11, m] 1.60, m [1.61, m, 1.58, m] 1.35, m [1.30, m] 1.30, m 1.35, m; 1.28, m 1.66, m; 1.55, m 5.01, ddd (9.3, 4.9, 3.2) 5.11, t (5.0) 5.01, ddd (9.3, 4.9, 3.2) 1.67, m; 1.61, m 1.27, m 1.44, m; 1.36, m 1.44, m; 1.38, m 3.49, m 1.43, m 1.42, m; 1.31, m 1.32, m 1.32, m 1.32, m 1.44, m; 1.35, m 1.68, m; 1.57, m 4.22, ddd (6.8, 5.7, 3.8) 1.83, m 0.89, d (6.8) 1.58, m; 1.12, m

28.6, CH2 30.7,d CH2 33.1, CH2 23.8, CH2 14.5, CH3

1.33, 1.32, 1.29, 1.31, 0.89,

m; 1.29, m m m m t (6.8)

3.70, 2.03, 2.06, 2.03,

s [3.76 s] s s s

52.7 [53.2], CH3 172.2, C; 20.9, CH3 171.8, C; 20.7, CH3 172.2, C; 20.9, CH3

Assignments may be interchanged. bSignals for the minor isomer are in brackets.

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Figure 2. Intense product ions in the tandem FABMS of 3.

moiety is not considered in Kishi’s universal NMR database. Therefore, we sought in the literature the diastereomers of an isolated 1-hydroxy-2-methyl moiety whose 13C NMR data were reported. We found that diasteromers of cruentol (5methyloctan-4-ol) and ferrugineol (4-methylnonan-5-ol) were suitable for comparison of the NMR data with those of 9.21−24 Although the 13C NMR data of these compounds were reported in CDCl3 without assignments, it was possible to single out the chemical shift values of the branched methyl group. The chemical shifts of the relevant carbon were not identical for the (R*,R*)- (or threo-) and (S*,R*)- (or erythro-) isomers in both compounds (δC 13.6 for the threo-isomer and δC 15.2 for the erythro-isomers), implying that the branched methyl in the threo-isomers is more shielded than that in the erythro-isomers. Then we measured the NMR spectra and assigned the chemical shifts of the commercially available 2:1 diastereomeric mixture of 4-methylnonan-5-ol (10 and 11) (Figure S49). The branched methyls in CD3OD resonated at 14.3 and 15.7 ppm in the threo-isomer (10) and the erythroisomer (11), respectively (Figure S50). The chemical shift of 14.5 ppm for the branched-methyl carbon in 9 suggested the threo-configuration for C-28 and C-29.25 The absolute configuration of yakushinamide B was studied by the modified Mosher’s analysis applied to the tetra-MTPA esters 12a and 12b, which were derived from 9. The absolute configurations of C-13, C-14, and C-15 were identical with those in 1, whereas the 28R configuration was assigned by this analysis. By considering the threo-relative configuration, the 29R configuration was deduced. Unexpectedly we were not able to assign the absolute configuration at C-20 by the modified

Figure 3. 1H and 13C chemical shift values (CD3OD) of the contiguous triol portion in 3 and that in (5R,6S,7S)-decane-1,5,6,7tetraol.

The molecular formula of yakushinamide B (2) was determined to be C43H76NNaO14S by HRESIMS. Five partial structures (partial structures a, b, c, e, and f) were deduced from the 2D NMR data. The NMR data of partial structure a and partial structure b in 1 were identical with those in 2. Partial structure c was assigned as an isolated secondary alcohol on the basis of the NMR data of the oxymethine (δH 3.49 and δC 72.3). In partial structure f an oxymethine and a branched methyl were placed side by side, in which the oxymethine was suggested to be sulfonated on the basis of its NMR data (δH 4.24 and δC 84.4). In order to determine the location and orientation of these partial structures, yakushinamide B (2) was converted to the pentaol 9 and subjected to the tandem FABMS analysis, which permitted us to assign the locations and orientation of substituents in the aliphatic chain. The coupling constant of 3.7 Hz between H-28 and H-29 was not instrumental in assigning the relative configuration between C-28 and C-29,20 and an isolated 1-hydroxy-2-methyl

Figure 4. HSQC spectra for the geminal methylene proton region of (a) the mixture of 6a and 7a, (b) the mixture of 6b and 7b, (c) 8a, and (d) 8b. D

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between H2O and CHCl3. The CHCl3 layer was concentrated and partitioned between 90% MeOH and n-hexane. The 90% MeOH layer was diluted with H2O to give a 60% MeOH solution, which was extracted with CHCl3. The latter CHCl3 layer was separated with ODS flash chromatography using a stepwise gradient elution (20−70% MeOH, 70−90% MeCN, and MeOH). The 70% MeCN fraction was separated by gel filtration on a Sephadex LH-20 column with CHCl3− MeOH (1:1). The active fractions were combined and chromatographed on silica gel using a stepwise gradient elution (CHCl3− MeOH−H2O, 98:2:0, 9:1:0, 8:2:0.1, 7:3:0.5, 6:4:1, and 5:5:1). The fractions eluted with 9:1:0 and 8:2:0.1 CHCl3−MeOH−H2O were combined and separated by RP-HPLC using two columns connected in tandem (Cosmosil AR-II; 20 × 250 mm, 8.0 mL/min) with eluent consisting of 8:2 MeOH−phosphate buffer I (28 mM Na2HPO4 and 12 mM KH2PO4) to provide four active fractions. Fraction 3 was purified by RP-HPLC (Cosmosil AR-II; 20 × 250 mm) with 5:5 MeCN−phosphate buffer II (14 mM Na2HPO4 and 8 mM KH2PO4) to give yakushinamide A (1, 5.7 mg). Fraction 4 was purified by RPHPLC (Cosmosil AR-II; 20 × 250 mm) with MeCN−phosphate buffer II (65:35) to provide yakushinamide B (2, 5.8 mg). All the fractions were desalted using a short ODS column. Yakushinamide A (1): clear oil; [α]24D −26 (c 0.25, MeOH); 1H and 13C NMR data, Table 1; HRESIMS m/z 936.5058 [M + Na]+ (calcd for C45H80NNa2O14S, 936.5095). Yakushinamide B (2): clear oil; [α]23D −26 (c 0.25, MeOH); 1H and 13C NMR data, Table 1; HRESIMS m/z 908.4767 [M + Na]+ (calcd for C43H76NNa2O14S, 908.4782). Methanolysis of 1 and 2. Yakushinamide A (1, 0.5 mg) was dissolved in 10% HCl−MeOH (1 mL) and heated at 100 °C for 1 h. The solution was neutralized with 1 M NaHCO3 and desalted with InertSep RP-1 (GL Science Inc.) to give 3. The methanolysis product 9 was prepared from 2 in the same manner. 3: 1H and 13C NMR data, Table S1; tandem FABMS spectrum, Figure S50. 9: 1H and 13C NMR data, Table S1; tandem FABMS spectrum, Figure S51. Marfey’s Analysis. Compound 1 was dissolved in 6 N HCl (150 μL) and heated at 110 °C for 2 h. The solution was concentrated and redissolved in 50 μL of H2O. 1% L-FDAA in acetone (100 μL) and 1 M NaHCO3 (20 μL) were added to the solution. The mixture was heated at 40 °C for 1 h. After cooling to room temperature (rt), the reaction mixtures were quenched with 5 N HCl (4 μL), concentrated, and redissolved in DMSO. DL- and L-Proline standards were treated with L-FDAA in the same manner. The L-FDAA derivatives were analyzed by LC-MS (Cosmosil 2.5C18-MS-II (2.0 × 100 mm); flow rate, 0.5 mL/min; solvent, 10−50% MeCN containing 0.5% AcOH (22 min)). The retention time (tR) of each amino acid was as follows: L-proline (10.5 min), DL-proline (10.4 and 11.3 min). The absolute configuration of the proline residue in 1 was determined as L (tR 10.5 min). Preparation of MTPA Derivatives. To a half-solution of 3 in dry pyridine−CH2Cl2 (1:1, 50 μL) was added (R)-(−)-MTPACl (5 μL). The solution was left at room temperature for 1 h. The reaction mixture was diluted with H2O and extracted with CHCl3. The organic layer was purified by RP-HPLC to afford tetra-(S)-(−)-MTPA ester (8a). Tetra (R)-(+)-MTPA ester (8b) was prepared in a similar way using (S)-(+)-MTPACl. 8a: 1H and 13C NMR data, Table S2; ESIMS m/z 1573. 8b: 1H and 13C NMR data, Table S2; ESIMS m/z 1573. Similarly, 9 was converted to (S)-(−)-MTPA ester (12a) and (R)(+)-MTPA ester (12b). 12a: 1H and 13C NMR data, Table S3; ESIMS m/z 1545. 12b: 1H and 13C NMR data, Table S3; ESIMS m/z 1545. Preparation of MTPA Esters of (S)-3-Methyl-5-decanol. PBr3 (25.7 g) was added dropwise to a mixture of (S)-2-methylbutanol (20.0 g) and pyridine (6.5 g) at 0 °C for 2 h. The reaction mixture was distilled to afford (S)-2-methylbutyl bromide. The mixture of a portion of (S)-2-methylbutyl bromide (1.8 g) and magnesium (0.29 g) in dried Et2O (5 mL) was treated with hexanal (1.2 g) at room temperature to afford a mixture of (3S,5R)-3-methyl-5-decanol (4) and (3S,5S)-3-

Figure 5. 13C chemical shift values of the 1-hydroxy-2-methyl portion of 9−11.

Mosher’s analysis, because the signs of the Δδ values around C20 were all small and positive. Larger Δδ values were observed for the protons closer to C-15, indicating that these positive Δδ values arose from the influence of the MTPA esters at C-13 and C-15. Therefore, we prepared derivatives that lack these MTPA esters. Selective removal of the C-13 and C-15 MTPA esters from either 12a or 12b was realized by mild base treatment. The product was oxidized with NaIO4 followed by oxidation with a mixture of HCO2H and H2O2 and methylation with TMS diazomethane to afford 13a and 13b. The Δδ values around C-6 (corresponds to C-20 in 2) were still small. We speculate that this anomaly arose from the interaction between the two MTPA ester groups.

Yakushinamides exhibit moderate inhibitory activity against histone deacetylases. The inhibitory activities of yakushinamide A against HDAC1, HDAC4, HDAC6, SIRT1, SIRT2, and SIRT3 were 26, >200, >200, 16, >200, and 79 μM, respectively, whereas the inhibitory activities of yakushinamide B against the corresponding enzymes were 29, 75, 52, 34, 150, and 78 μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Methods. Optical rotations were measured on a JASCO DIP-1000 digital polarimeter in MeOH. NMR spectra were recorded on a JEOL alpha 600 MHz NMR spectrometer at 300 K. Chemical shifts were referenced to solvent peaks: δH 7.24 and δC 77.0 for CDCl3; δH 3.30 and δC 49.0 for CD3OD. ESI mass spectra were measured on a JEOL JMS-T100LC. FABMS and tandem FABMS were measured on a JEOL JMS-700T. LC-MS was conducted on an Amazon SL mass spectrometer with UFLC performed on a Shimadzu LC 20AT with a SPD-M20A detector. HPLC was carried out on a Shimadzu LC 20AT with a SCL10Avp controller and a SPD-10Avp detector. . Animal Material. The sponge Theonella swinhoei was collected by dredging at a depth of 130 m at the seamount “Yaku-Shinsone” (29°47.22 N, 130°19.88 E) near Yakushima Island, southern Japan, during a cruise of T/S Toyoshio-maru, on May 23, 2007. Spicule characteristics of our specimen are very similar to those of the type specimen of Theonella swinhoei Gray, 1868 redescribed by Pisera and Lévi (2002), and here we identified our specimen as T. swinhoei.31 The specimen used for identification (NSMT-Po-2486) was deposited at the National Museum of Nature and Science, Tokyo. Isolation of Yakushinamides A and B. The sponge (340 g, wet weight) was homogenized and extracted with MeOH (900 mL × 2) and EtOH (900 mL). The extracts were combined and partitioned E

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HDAC Assay. Measurements of HDAC activities of human sirtuins including SIRT1, -2, and -3 were carried out using fluorogenic peptide substrates. Recombinant sirtuin proteins were incubated with a fluorescent peptide (100 μM Ac-RHKK(Ac)-MCA for SIRT1 and SIRT2; 100 μM Ac-VSTPVK(Ac)-MCA for SIRT3) and NAD (final concentration 1 mM) in 20 μL of assay buffer (50 mM Tris-HCl [pH 9.0], 4 mM MgCl2, 0.2 mM DTT). After 60 min at 37 °C, trypsin (final concentration 20 mg/mL) was added and samples were incubated for an additional 15 min at 37 °C. The fluorescence of released amino methyl coumarin (λex: 370 nm and λem: 460 nm) was measured using a fluorescence plate reader (Molecular Devices). Enzymatic activity assays for HDAC1, -4, and -6 were performed as described previously.34

methyl-5-decanol (5) (a combined weight of 1.5 g). A portion of this mixture (30 mg) was converted to the mixture of (S)-(−)-MTPA ester (6a and 7a) with (R)-(−)-MTPACl (50 μL) in pyridine−CH2Cl2 (1:1, 500 μL). After the addition of H2O, the mixture was extracted with CHCl3, and the organic layer was purified by RP-HPLC. The 2D NMR data showed that the product was a 2:1 mixture of 6a and 7a. The corresponding (R)-(+)-MTPA esters (6b and 7b) were prepared in the same manner. (S)-MTPA ester of 6a: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz) δH 7.71, 7.40, 5.31 (1H, m, H-5), 1.67 (2H, m, H-6), 1.53 (1H, m, H4), 1.48 (1H, m, H-4), 1.42 (1H, m, H-2), 1.38 (2H, m, H-7), 1.33 (1H, m, H-3), 1.33 (2H, m, H-8), 1.09 (1H, m, H-2), 0.93 (3H, t, H10), 0.87 (3H, t, H-1), 0.85 (3H, d, 3-Me); 13C NMR (CDCl3− C5D5N) δC 76.0 (CH, C-5), 40.7 (CH2, C-4), 34.3 (CH2, C-6), 31.7 (CH2, C-9), 30.8 (CH, C-3), 28.7 (CH2, C-2), 25.0 (CH2, C-7), 22.6 (CH2, C-8), 19.0 (CH3, 3-Me), 14.1 (CH3, C-10), 11.2 (CH3, C-1); ESIMS m/z 411 [M + Na]+. (S)-MTPA ester of 7a: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz) δH 7.71, 7.40, 5.31 (1H, m, H-5), 1.78 (1H, m, H-4), 1.60 (2H, m, H6), 1.46 (1H, m, H-3), 1.32 (1H, m, H-2), 1.31 (1H, m, H-4), 1.26 (2H, m, H-8), 1.23 (2H, m, H-7), 1.22 (1H, m, H-2), 0.95 (3H, d, 3Me), 0.91 (3H, t, H-1), 0.90 (3H, t, H-10); 13C NMR (CDCl3− C5D5N) δC 76.0 (CH, C-5), 41.0 (CH2, C-4), 34.5 (CH2, C-6), 32.1 (CH2, C-9), 31.0 (CH, C-3), 30.0 (CH2, C-2), 24.6 (CH2, C-7), 22.6 (CH2, C-8), 19.3 (CH3, 3-Me), 14.1 (CH3, C-10), 11.2 (CH3, C-1) ; ESIMS m/z 411 [M + Na]+. (R)-MTPA ester of 6b: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz) δH7.71, 7.40, 5.31 (1H, m, H-5), 1.59 (2H, m, H-6), 1.58 (2H, m, H4), 1.46 (1H, m, H-2), 1.46 (1H, m, H-3), 1.25 (2H, m, H-8), 1.21 (2H, m, H-7), 1.16 (1H, m, H-2), 0.93 (3H, d, 3-Me), 0.89 (3H, t, H1) 0.87 (3H, t, H-10); 13C NMR (CDCl3−C5D5N) δC 76.0 (CH, C5), 41.0 (CH2, C-4), 33.9 (CH2, C-6), 31.7 (CH2, C-9), 31.1 (CH, C3), 29.2 (CH2, C-2), 24.6 (CH2, C-7), 22.7 (CH2, C-8), 19.5 (CH3, 3Me), 14.1 (CH3, C-10), 11.2 (CH3, C-1) ; ESIMS m/z 411[M + Na]+. (R)-MTPA ester of 7b: 1H NMR (CDCl3−C5D5N (1:1), 600 MHz) δH 7.71, 7.40, 5.31 (1H, m, H-5), 1.69 (1H, m, H-4), 1.71 (1H, m, H6), 1.60 (1H, m, H-6), 1.37 (2H, m, H-7), 1.32 (2H, m, H-8), 1.24 (1H, m, H-4), 1.23 (1H, m, H-3), 1.21 (1H, m, H-2), 1.12 (1H, m, H2), 0.91 (3H, t, H-10), 0.88 (3H, d, 3-Me), 0.79 (3H, t, H-1); 13C NMR (CDCl3−C5D5N) δC 76.0 (CH, C-5), 40.9 (CH2, C-4), 34.9 (CH2, C-6), 31.7 (CH2, C-9), 31.1 (CH, C-3), 30.1 (CH2, C-2), 25.1 (CH2, C-7), 22.7 (CH2, C-8), 18.9 (CH3, 3-Me), 14.1 (CH3, C-10), 11.4 (CH3, C-1); ESIMS m/z 411 [M + Na]+. Preparation of 13a and 13b. Compound 12a was dissolved in 10 M NaOH−MeOH (1:9, 500 μL) and left at rt for 1 h to afford the 20,28-bis-MTPA ester as detected by LCMS. After neutralization with 1 N HCl the solution was extracted with CHCl3. The organic layer was dried and the residue was dissolved in MeOH. To this solution was added NaIO4, and the solution stirred at rt for 2 h. The resulting aldehyde as detected by LCMS was dissolved in 90% HCOOH−35% H2O2 (2:1, 500 μL) and left at rt. The product was dried and redissolved in MeOH. To this solution was added TMS diazomethane in n-hexane, and the reaction mixture was dried and purified by RPHPLC (80−100% MeCN) to afford the bis-MTPA ester 13a. BisMTPA ester 13b was prepared in the same manner. 13a: 1H and 13C NMR, Table S4. 13b: 1H and 13C NMR, Table S4. Preparation of Recombinant Human HDAC Proteins. Purification of recombinant GST-fused SIRT2 catalytic domain was performed as described previously.32 Purification of the recombinant His-tagged SIRT3 protein was performed as described in the literature.33 For preparation of the SIRT1 protein, pCold TF DNASIRT1 was introduced into Escherichia coli BL21 (DE3). The expression of recombinant proteins was induced with 0.3 mM isopropyl-β-D-galactopyranoside (IPTG) at 16 °C for 24 h. Purification of the (His)6-fused proteins was carried out using the nickel affinity (GE Healthcare) interaction, followed by anionexchange chromatography (GE Healthcare). Purifications of the zinc-dependent HDACs including HDAC1, -4, and -6 were performed as described previously.34

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00588. NMR data for 1, 2, 3, 8a, 8b, 9, 10, 11, 12a, 12b, 13a, 13b, a mixture of 6a and 6b, and a mixture of 7a and 7b, and tandem FABMS data for 3 and 9 (PDF) Spectra (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (K. Takada): [email protected]. *Tel (S. Matsunaga): 81-3-5841-5297. Fax: 81-3-5841-8166. Email: [email protected]. Author Contributions ‡

K. Takada and Y. Imae contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemical Biology of Natural Products” (23102007) and JSPS KAKENHI Grant Numbers 25252037, 25712024, 25660163, 15K14800, and 15K14799 from The Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Professor T. Kusumi, Professor Emeritus, Tokushima University, for valuable discussion and Ms. A. Nakata, RIKEN, for conducting the enzyme inhibitory assay.

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REFERENCES

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