Cladosins A–E, Hybrid Polyketides from a Deep ... - ACS Publications

Feb 5, 2014 - derived fungus Cladosporium sphaerospermum 2005-01-E3. Their structures. (1−5) were elucidated through a combination of spectroscopic ...
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Cladosins A−E, Hybrid Polyketides from a Deep-Sea-Derived Fungus, Cladosporium sphaerospermum Guangwei Wu, Xinhua Sun, Guihong Yu, Wei Wang, Tianjiao Zhu, Qianqun Gu, and Dehai Li* Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *

ABSTRACT: Five new fungal hybrid polyketides, cladosins A−D (1−4), that contain a novel linear 6(3)-enamino-8,10-dihydroxy-tetraketide (1 and 2) or 6-enamino-7(8)-en-10-ol (3 and 4) moiety, as well as the biogenetically related cladosin E (5), were isolated from the deep-seaderived fungus Cladosporium sphaerospermum 2005-01-E3. Their structures (1−5) were elucidated through a combination of spectroscopic data, chemical conversion, and both Mosher’s and Marfey’s methods for stereochemical assignment. A plausible biogenetic pathway to 1−5 is proposed. Cladosin C (3) possesses mild anti-influenza A H1N1 virus activity.

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atural products containing the 2,4-pyrrolidinedione (tetramic acid) core are well known as hybrids generally constructed by fusion of amino acid and polyketide units.1 This family of structures has attracted particular attention because of the structurally diverse scaffolds,2 such as pyrrospirones A and B,3 diaporthichalasin,3 cylindramide,4 cyclopiazonic acid,5 and the penicillenols,6 as well as a range of biological effects including antibiotic, antiviral, antitumor, anti-HIV, herbicidal, and RNA polymerase inhibitory activities.7 This has led to several studies of their total synthesis and biogenesis in recent years.1,3,8 Although new members of this large family are being discovered, there has been no report of congeners with a polyketide component containing 1,3-diol, polyol, or enamino moieties to date. As part of our ongoing work exploring the bioactive secondary metabolites from deep-sea-derived fungi,9 the strain Cladosporium sphaerospermum 2005-01-E3, isolated from sediments collected in the Pacific Ocean, was selected for further development due to its interesting HPLC-UV profiles and bioactivity. Chemical investigation of this fungal extract finally led to the isolation of four novel tetramic acid derivatives containing either 6(3)-enamino-8,10-dihydroxy or 6(3)-enamino-7(8)-en-10-ol side chains, named cladosins A−D (1−4). Each compound exists as two tautomeric forms differing in configuration of the enamine. We also isolated a new biosynthetically related compound, cladosin E (5), from the same culture. Details of the structure elucidation, bioactivity screening, and a hypothetical biogenesis of 1−5 are reported herein.

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the HRESIMS adduct ion detected at m/z 305.1475 [M + Na]+, the mixture (1a/1b) had the same molecular formula of C14H22N2O4, requiring five degrees of unsaturation. The major 1D NMR resonances (1a) were categorized into four sp3 methyls (including a methoxy), two sp3 methylenes, two sp3

RESULTS AND DISCUSSION

Cladosin A (1) was isolated as an inseparable mixture of two geometric isomers (1a:1b) present in a ratio of 5:3. Based on © 2014 American Chemical Society and American Society of Pharmacognosy

Received: October 7, 2013 Published: February 5, 2014 270

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Figure 1. Key 2D NMR correlations of 1−6.

Table 1. 13C NMR (150 MHz) and 15N (90 MHz) Data of 1−4 in DMSO-d6 (δ ppm) 1

a

2

3

4

no.

a

b

a

b

a

b

a

b

2 3 4 5 6 7 8 9 10 11 12 13 14 15 NHb NH2b

168.6 97.2 186.5 130.3 169.6 36.4 77.8 43.6 63.7a 23.9 118.4 21.3 18.4a 56.7a 254.2 257.5

171.4 96.0 183.5 131.0 169.6 36.7 77.4 43.5 63.7a 24.0 118.6 21.4 18.4a 56.7a 254.9 258.3

169.0 97.3 186.4 130.3 170.0 39.5 68.2 47.0 64.7 23.9 118.4 21.3 18.4a

171.5 96.0 183.7 131.0 170.2 39.8 68.0 46.8 64.8 24.0 118.5 21.4 18.4a

168.3 96.9 187.3 130.3 161.2 123.6 142.1a 43.4 66.1 23.8a 118.5 21.3 18.5

169.7 95.6 183.8 130.9 161.5 123.3 142.1a 43.4 66.1 23.8a 118.7 21.4 18.4

166.5 103.8 183.8 129.8 166.9 120.4a 144.1 31.0a 74.0a 20.7a 119.0 21.4 18.5

168.0 103.7 186.4 130.6 165.0 120.4a 144.7 31.0a 74.0a 20.7a 119.4 21.5 18.8

N.D.c N.D.c

N.D.c N.D.c

N.D.c N.D.c

N.D.c N.D.c

N.D.c N.D.c

N.D.c N.D.c

Tautomers exhibited the same shifts for these carbons. bChemical shifts were recorded in the 1H−15N HSQC spectra. cN.D.: not detected.

oxygenated methines, an amide carbonyl, an α/β-unsaturated ketone carbon, and two tetrasubstituted double bonds, indicating one additional ring existed in 1a to match the degrees of unsaturation. The gross structure of 1a was established by comprehensive 2D NMR analysis (Figure 1). The sequential COSY correlations of H2-7/H-8/H2-9/H10(10-OH)/H3-11 unambiguously established the spin system from C-7 to C-11, with the methyl ether (δC 56.7) and hydroxy groups attached to C-8 and C-10, respectively, deduced by the HMBC correlations between H3-15 (δH 3.24) and C-8 (δC 77.8), and the exchangeable proton (δH 4.51) with C-10. This spin system was extended to include an enamine double bond (δC 97.2, C-3; 169.6, C-6) by the corresponding HMBC correlations from H-7 to C-3, with the amino group attached to C-6 supported by the long-range 1H−15N HMBC from H-7 to N-6 (Figure 1). The HMBC correlations from H3-13 and H3-14 to C-5 (δC 130.3) and C-12 enabled identification of an isobutenyl moiety. Finally, a highly substituted 2,4-pyrrolidinedione (tetramic acid) scaffold10 was established based on the HMBC correlations from the exchangeable amide proton NH-1 (δH 9.13) to alkene carbon C-3, α/β-unsaturated ketone carbon

C-4 (δC 186.5), isobutenyl carbon C-5, and the remaining upfield carbonyl C-2 (δC 168.6). To confirm the 2,4pyrrolidinedione core, compound 1 was hydrogenated to form the reduced diastereomers 6a/6b, which displayed the relevant COSY correlation between NH-1/H-5 and the HMBC correlation from the additional H-5 to C-3 (Figure 1 and Table S1, Supporting Information). Thus, the planar structure of 1a was assigned as a tetramic acid derivative with the new 6(3)enamino-8,10-dihydroxy system. Comparison of the NMR data of 1a and 1b indicated that they had a similar planar core structure. The only significant differences between 1a and 1b were the chemical shifts of carbonyls C-2 and C-4 (Table 1), indicating 1 likely existed as a pair of interconverting geometric isomers in solution.11 The only reported example of an enaminotetramic acid core is cyclopiazonic acid imine, which existed as a pair of tautomers and was explained probably by the interconversion of ketoenamine and enol-imine forms.12 However, the cladosin A isomers 1a/1b were only found in the keto-enamine form, in obvious difference from cyclopiazonic acid imine. The isomeric behavior of 1 was readily explained through conversion via 271

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tautomerism of the 3-acyltetramic acids,11 with 1a (major) and 1b (minor) assigned separately as the exo-form A (Δ3(6): E) and exo-form B (Δ3(6): Z). The forms of 1a and 1b were assigned as exo-form A and exo-form B, respectively, based on similar NMR evidence between 1 and 3-acyltetramic acids, where the hydrogen-bonded carbonyl is shifted downfield when compared to that of the corresponding free carbonyl.11,13 The molecular formula of tautomeric cladosin B (2) was established as C13H20N2O4 from an adduct ion [M + Na]+ at m/z 291.1316. The UV spectrum of 2 was almost identical to that of 1, indicating a 6(3)-enaminotetramic acid system. The 1 H and 13C NMR data of 2 showed similar resonances to those of 1, except that the methyl ether of 1 was replaced by an alcohol (δH 4.96) in 2. Similar to 1, 2 also existed as exo-A (major) and exo-B (minor) geometric isomers in the same ratio of 5:3. The relative configuration of the 1,3-diol unit in 2 was determined from the 13C NMR analysis after derivatization to acetonide 7 (Figure 2). The signals of the methyl carbons of

for the geminal protons H2-9 between the two compounds (Table 2). The 10S configuration in 1 was assigned by the modified Mosher’s method (Figure 3). Accordingly, the absolute configuration of 1 was determined to be 8R, 10S, in complete agreement with congener 2. To assess whether 1 was an artifact of isolation, compound 2 was dissolved in MeOH and then stirred at room temperature for a week. Monitoring by HPLC indicated no conversion of 1 to 2. It is also notable that when 2 was treated under harsher conditions, such as elevated temperature (40 °C), the compound degraded quickly. Cladosins C and D (3 and 4) were assigned the same molecular formula C13H18N2O3 from their adduct ions detected under HRESIMS conditions, being 18 mass units less than that of 2. Their UV absorption spectra suggested an extended 6(3)enaminotetramic acid chromophore. Analysis of the NMR data of 3 revealed that the side chain differed from the 1,3-diol units in 1 and 2, with COSY correlations between H-7/H-8/H2-9/H10/H3-11 establishing a homoallylic alcohol subunit, which was further supported by relevant HMBC cross-peaks as shown in Figure 1. The Δ7 moiety was unambiguously connected to C-6 based on the correlations from both H-7 and H-8 to enamine C-6. The E geometry of Δ7 was deduced from the large vicinal coupling constant 3JH‑7,H‑8 = 16.5 Hz. Moreover, the 10Sconfiguration was assigned by the modified Mosher’s method. The 1H and 13C NMR spectra of 3 and 4 were almost identical, and analysis of 2D NMR spectra of 4 established the same planar structure as 3. The only difference between 3 and 4 was the Z geometry of Δ7 deduced from the smaller vicinal coupling constant (3JH‑7,H‑8 = 9.9 Hz) in 4. Due to lack of material and slow decomposition under storage, the absolute configuration at C-10 in 4 could not be established. However, considering their common biogenetic origin, compound 4 is a Z/E geometric isomer of compound 3 and is likely to have the 10S configuration. Unlike compounds 1−3, exo-form B of 4 dominated exo-form A in a ratio of 5:3. This suggests that changes in the side chain of the 6(3)-enaminotetramic acid affect the tautomeric equilibrium. Cladosin E (5) was isolated as a white, amorphous powder and had the molecular formula C13H17NO4 by HRESIMS. The NMR resonances of 5 were attributed to a trisubstituted benzene ring, two upfield carbonyl carbons, two sp3 methines, two geminal methyls, an aryl methyl, a hydrogen-bonded proton, and an exchangeable amide proton. The assembly of a 6-methylsalicylic acid moiety, which is one of the oldest fungal polyketide skeletons known,17,18 was readily established from the 1D NMR data. This was further confirmed from key COSY and HMBC correlations in its 2D NMR spectra and in particular identifying a valine residue, which accounted for the remaining atoms required by the molecular formula (Figure 1). The 6-methylsalicylic acid moiety and valine residue were linked via an amide bond that was confirmed by the key HMBC correlation from H-2′ to C-1 and from the presence of an amide proton (δH 7.46). The absolute configuration of the valine residue was established as L by application of Marfey’s method.19 Biosynthetically, all of the isolated compounds (cladosins A− E) are composed of a tetraketide unit and a valine residue, involving both PKS and amino acid pathways (Figure 4). In the putative biogenesis of cladosins A−D, the linear tetraketide initially condenses with an activated valine to form the intermediate ii, a 1,3-dione-5,7-diol conjugate.1,20 The condensed hybrid molecule ii is released as a tetramic acid core

Figure 2. NMR data of acetonide 7 and the consequential syn-1,3-diol form.

acetonide 7 were distinct at δC 19.5 and 29.0 ppm, requiring a syn 1,3-diol system in 2.14 Furthermore, the coupling pattern for the C-9 methylene protons of 7 provided further evidence for a syn 1,3-diol.15 The modified Mosher’s method has been applied to ascertain the absolute configuration of the simple acyclic syn 1,3-diols from their di-MTPA esters.16 The systematically distributed Δδ values of positive (ΔδL), negative (ΔδC), and negative (ΔδR) corroborated the 8R,10S configurations in 2 (Figure 3). Biogenetically, compound 1 may be the methylation product of 2, which suggested the same syn 1,3diol unit as 2 and was supported by the similar splitting pattern

Figure 3. ΔδS−R values for the Mosher’s ester derivatives of 1−3. 272

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Table 2. 1H NMR (600 MHz) Data of 1−4 in DMSO-d6 (δ ppm, J in Hz) 1 no. NH-1 7 8 9 10 11 13 14 15 8-OH 10-OH 6-NH2

2

a 9.13, 2.96, 3.01, 3.64, 1.71, 1.36, 3.77, 1.04, 1.75, 2.16, 3.24,

s m; m m m; m m d (6.0) s s s

4.51, d (4.4) 10.06, brs; 8.85, brs

b 9.31, 2.96, 3.01, 3.62, 1.71, 1.36, 3.77, 1.04, 1.74, 2.13, 3.26,

s m; m m m; m m d (6.0) s s s

4.49, d (4.4) 9.46, brs; 8.73, brs

3

a 9.10, 3.08, 2.65, 3.93, 1.56, 1.39, 3.80, 1.03, 1.73, 2.14,

s m; m brs m; m brs d (5.5) s s

4.96, s 4.59, s 10.05, brs; 8.77, brs

b 9.25, 3.08, 2.65, 3.93, 1.56, 1.39, 3.80, 1.03, 1.72, 2.11,

s m; m brs m; m brs d (5.5) s s

4.94, 4.59, 9.45, 8.65,

s s brs; brs

4

a

b

a

b

9.22, s 7.43, d (16.5)

9.40, s 7.43, d (16.5)

6.92, m 2.34, m

6.92, m 2.34, m

3.77, 1.11, 1.75, 2.16,

3.77, 1.11, 1.74, 2.13,

9.80, s 7.72, dd (9.9, 1.6) 6.94, m 2.38, m; 2.57, m 4.42, m 1.40, d (6.6) 1.75, s 2.11, s

9.65, s 7.68, dd (9.9, 1.6) 6.97, m 2.35, m; 2.60, m 4.42, m 1.40, d (6.6) 1.75, s 2.12, s

m d (6.4) s s

4.71, d (5.0) 9.89, brs; 8.68, brs

m d (6.4) s s

4.71, d (5.0) 9.26, brs; 8.58, brs

Figure 4. Plausible biogenetic origin of 1−5.

acid,23 dysidin,24 and L-tenuazonic acid25 incorporate a valine residue within a 2,4-pyrrolidinedione core. In addition, all the isolated compounds were evaluated for antiviral activity against the influenza A H1N1 virus, as well as antitumor, antitubercular, and NF-κB inhibitory activities. Only compound 3 exhibited activity with an IC50 = 276 μM against the influenza A H1N1 virus (ribavirin as positive control, IC50 131 μM).

through a catalyzed Dieckmann cyclization and a series of subsequent transformations, including hydroxylation, dehydration, transamination,21 and O-methylation that lead to the production of cladosins A−D.1,20 However, the linear intermediate i could undergo cyclization, dehydration, and aromatization to produce a 6-methylsalicylic acid core and finally release cladosin E (5). Cladosins A−D (1−4) represent a novel class of tetramic acid congeners characterized by previously unknown 6(3)enamino-8,10-dihydroxy- or 6(3)-enamino-7(8)-en-10-ol side chains. To date, among the numerous known natural products containing the tetramic acid core, only xyrrolin22 and preaspyridone20 contain a C8 polyketide unit. Due to the different side chains in 1−4 in relation to those previously reported in other tetramic acids, the formation of the C8 polyketide chains in cladosins A−D may be encoded through a PKS gene with a rare aminotransferase domain. An extensive literature survey has also indicated that only cyclopiazonic acid imine contains a similar enamine motif in the polyketide chain,12b and only three examples, 3-acetyl-5-isopropytetramic

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. NMR spectra were recorded on a JEOL JNM-ECP 600 spectrometer using TMS as internal standard. 1H−15N HSQC spectra were recorded on an Agilent 500 MHz DD2 spectrometer. ESIMS spectra were acquired on a Thermo Scientific LTQ Orbitrap XL mass spectrometer or a Micromass Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. Semipreparative HPLC was performed using an ODS column [HPLC (YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/ min)]. Medium-pressure preparation liquid chromatography (MPLC) 273

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was performed on a Bona-Agela CHEETAH HP100 (Beijing Agela Technologies Co., Ltd.). Column chromatography (CC) was performed with silica gel (200−300 mesh, Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Amersham Biosciences), respectively. Fungal Material. The fungal strain C. sphaerospermum 2005-01-E3 was isolated from deep-sea sludge in the Pacific Ocean (W 102°22′0, N 00°15′9; depth over 1000 m). The isolate was identified as C. sphaerospermum 2005-01-E3 according to morphological traits and ITS rDNA sequence analysis. The sequence data have been submitted to GenBank, accession number JQ670942.1. The strain was deposited in the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, PR China. Working stocks were prepared on potato dextrose agar slants stored at 4 °C. Fermentation and Extraction. The fungus C. sphaerospermum 2005-01-E3 was cultured under static conditions at 28 °C in 1 L Erlenmeyer flasks containing rice media (rice 80 g, artificial seawater 120 mL). After 30 days of cultivation, the fermentation from 50 flasks was extracted with MeOH. The MeOH extract was evaporated under reduced pressure to afford an aqueous solution that was extracted with EtOAc. The EtOAc extract was redissolved in MeOH containing 20% H2O and was extracted by hexane (three times). The MeOH layer was concentrated under reduced pressure to give a defatted extract (5.0 g). Isolation. The extract (5.0 g) was applied on a C-18 ODS column using a stepped gradient elution of MeOH−H2O, yielding six subfractions (A1−A6). Fraction A2, that eluted with 30:70 MeOH− H2O, was separated by MPLC (C-18 ODS) using a stepped gradient elution of MeOH−H2O (5:95 to 50:50) to furnish five subfractions (A2.1−A2.5). A2.3 was chromatographed on SephadexLH-20 (MeOH) and further purified by semipreparative HPLC (20:80 MeCN−H2O, 4 mL/min) to afford compounds 1 (30.0 mg) and 2 (12.0 mg). A3 was fractionated on SephadexLH-20 (MeOH) and further purified by semipreparative HPLC (60:40 MeOH−H2O, 4 mL/min) to afford compounds 3 (5.0 mg) and 4 (1.3 mg). A1 was applied to a SephadexLH-20 column (MeOH) and further purified by semipreparative HPLC (30:70 MeOH−H2O) to afford 5 (25.0 mg). Cladosin A (1): pale yellow oil; [α]24D +17.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 278 (4.07) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS m/z 305.1475 [M + Na]+ (calcd for C14H22N2O4Na, 305.1472). CladosinB (2): pale yellow oil; [α]24D +20.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 279 (3.95) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS m/z 291.1316 [M + Na]+ (calcd for C13H20N2O4Na, 291.1315). Cladosin C (3): pale yellow oil; [α]24D +10.5 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 278 (3.40), 322 (3.98) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS m/z 273.1213 [M + Na]+ (calcd for C13H18N2O3Na, 273.1210). Cladosin D (4): pale yellow oil; [α]24D +8.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 278 (3.45), 322 (4.05) nm; 1H and 13C NMR data, see Tables 1 and 2, respectively; HRESIMS m/z 251.1389 [M + H]+ (calcd for C13H19N2O3, 251.1390). Cladosin E (5): colorless powder; [α]24D +15.2 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 225 (3.76), 268 (1.07); 1H NMR (DMSOd6 600 MHz) δ 6.60 (1H, d, J = 8.0 Hz, H-4); 7.02 (1H, t, J = 8.0, 7.7 Hz, H-5); 6.56 (1H, d, J = 7.7 Hz, H-6); 2.19 (3H, s, H-8); 4.02 (1H, d, J = 4.4 Hz, H-2′); 2.23 (1H, m, H-3′); 0.81 (3H, d, J = 7.1 Hz, H4′); 0.91 (3H, d, J = 6.6 Hz, H-5′); 7.46 (1H, brs, −NH); 11.99 (1H, brs, 3-OH); 13C NMR data (DMSO-d6 150 MHz) δ 168.8(C, C-1), 126.2 (C, C-2), 156.4 (C, C-3), 114.8 (CH, C-4), 129.6 (CH, C-5), 119.9 (C, C-6), 136.0 (C, C-7), 19.8 (CH3, C-8), 174.6 (C, C-1′), 60.9 (CH, C-2′), 29.7 (CH, C-3′), 18.5 (CH3, C-4′), 20.7 (CH3, C-5′); HRESIMS m/z 274.1050 [M + Na]+ (calcd for C13H17NO4Na, 274.1050); 252.1228 [M + H]+ (calcd for C13H18NO4, 252.1230). Hydrogenation of Compound 1. To a solution of 1 (4 mg) in MeOH (1 mL) was added 10% palladium on activated charcoal (5 mg). The flask was degassed and filled with hydrogen. After stirring for 12 h at room temperature (rt), the reaction mixture was filtered through a microporous filter (0.22 μm) and the filtrate was evaporated to dryness. The residue was purified by a semipreparative RP-18

HPLC column to give an isomeric mixture of 6 (2.2 mg). For 1D and 2D NMR data, see Table S1 (Supporting Information); HRESIMS m/ z 285.1807 [M + H]+ (calcd for C14H25N2O4, 285.1809); 307.1626 [M + Na]+ (calcd for C14H24N2O4Na, 307.1628) Preparation of Acetonide 7. To a solution of 2 (4 mg), in dry MeOH (1 mL) was added 2,2-dimethoxypropane (1 mL) and pTsOH (5 mg) with stirring at rt under a nitrogen atmosphere for 12 h. The reaction mixture was poured into cold saturated aqueous NaHCO3 and subsequently extracted with EtOAc. The organic layer was washed with H2O and evaporated to dryness, finally yielding a mixture of 7 and 2. 1H NMR (CD3OD 600 MHz) exo-form A [exoform B] δ 4.29 (1H, m, H-10) [4.29 (1H, m, H-10)]; 4.03 (1H, m, H8) [4.10 (1H, m, H-8)]; 3.25 (1H, m, H-7) [3.25 (1H, m, H-7)]; 2.71 (1H, m, H-7) [2.71 (1H, m, H-7)]; 2.22 (3H, s, H-14) [2.21 (3H, s, H-14)]; 1.82 (3H, s, H-13) [1.82 (3H, s, H-13)]; 1.65 (1H, m, H-9) [1.65 (1H, m, H-9)]; 1.40 (3H, s, (O)2C(CH3)2) [1.40 (3H, s, (O)2C(CH3)2]; 1.34 (3H, s, (O)2C(CH3)2) [1.34 (3H, s, (O)2C(CH3)2]; 1.28 (3H, s, H-11) [1.28 (3H, s, H-11)]; 1.15 (1H, m, H-9) [1.15 (1H, m, H-9)]; 13C NMR data of 7 (CD3OD 150 MHz) exoform A (exo-form B) δ 186.5 (184.2), 171.6 (170.6), 169.0 (169.5), 129.6 (129.6), 120.6 (120.9), 98.7 (98.7), 96.8 (97.0), 67.9 (67.6), 65.0 (65.6), 38.0 (38.0), 37.8 (38.3), 29.0 (29.3), 22.0 (22.8), 20.9 (22.3), 19.5 (19.7), 17.1 (17.2); ESIMS m/z 309.2 [M + H]+. Preparation of MTPA Esters of 1−3. To a mixture of 1 (1 mg) and DMAP (cat.) in pyridine (800 μL) was added (R)-MTPACl (10 μL) at rt under a nitrogen atmosphere for 2 h. The organic phase was evaporated to dryness and separated by semipreparative HPLC to afford the (S)-MTPA ester 1. In a similar manner, the (R)-MTPA ester 1 was prepared from 1 using (S)-MTPACl and DMAP. The same procedure was used for the preparation of the (R)- or (S)-MTPA diesters 2 and (R)- or (S)-MTPA esters 3 obtained from 2 and 3, respectively. (S)-MTPA ester 1: 1H NMR (DMSO-d6) exo-form A (exo-form B) δ 10.00 (9.40), 9.11 (9.32), 8.87 (8.79), 7.57−7.30 (7.57−7.30), 5.22 (5.22), 3.67 (3.61), 3.46 (3.45), 3.24 (3.23), 3.09 (3.04), 2.93 (2.91), 2.15 (2.10), 1.93 (1.93), 1.75 (1.72), 1.65 (1.65), 1.21(1.21); HRESIMS m/z 499.2048 [M + H]+ (calcd for C24H30F3N2O6, 499.2050), 521.1862 [M + Na]+ (calcd for C24H29F3N2O6Na, 521.1870). (R)-MTPA ester 1: 1H NMR (DMSO-d6) exo-form A (exo-form B) δ 9.97 (9.36), 9.14 (9.36), 8.80 (8.73), 7.49−7.36 (7.49−7.36), 5.20 (5.26), 3.51 (3.49), overlap with H2O (3.38), 3.08 (3.01), 2.96 (2.95), 2.93 (2.84), 2.16 (2.13), 1.87 (1.87), 1.76 (1.74), 1.57 (1.57), 1.30 (1.31); HRESIMS m/z 499.2042 [M + H]+ (calcd for C24H30F3N2O6, 499.2050). (S)-MTPA diesters 2: 1H NMR (DMSO-d6) exo-form A (exo-form B) δ 9.78 (9.19), 9.13 (9.34), 8.81 (8.68), 7.51−7.32 (7.51−7.32), 5.70 (5.60), 5.16 (5.21), 3.49 (3.51), 3.42 (overlap with H2O), 3.10 (3.10), 3.03 (2.95), 2.17 (2.04), 2.09 (2.09), 1.94 (1.94), 1.75 (1.72), 1.41 (1.42); HRESIMS m/z 701.2292 [M + H]+ (calcd for C33H35F6N2O8, 701.2292). (R)-MTPA diesters 2: 1H NMR (DMSO-d6) exo-form A (exo-form B) δ 9.98 (9.40), 9.18 (9.37), 9.05 (8.96), 7.53−7.37 (7.53−7.37), 5.71 (5.64), 5.03 (5.12), 3.24 (overlap with H2O), 3.15 (2.98), 2.17 (2.08), 2.01 (2.01), 1.91 (1.91), 1.75 (1.72), 1.24 (1.23); HRESIMS m/z 701.2291 [M + H]+ (calcd for C33H35F6N2O8, 701.2292). (S)-MTPA ester 3: 1H NMR (DMSO-d6) exo-form A (exo-form B) δ 6.89 (6.89), 5.27 (5.27), 2.67 (2.67), 2.62 (2.62), 2.16 (2.12), 1.76 (1.74), 1.29 (1.29); HRESIMS m/z 467.1782 [M + H]+ (calcd for C23H26F3N2O5, 467.1788). (R)-MTPA ester 3: 1H NMR (DMSO-d6) exo-form A (exo-form B) δ 6.79 (6.79), 5.28 (5.43), 2.62 (2.54), 2.17 (2.11), 1.75 (1.74), 1.38 (1.38); HRESIMS m/z 467.1781 [M + H]+ (calcd for C23H26F3N2O5, 467.1788). Absolute Configuration of Valine. Compound 5 (1 mg) was dissolved in 0.5 mL of 6 N HCl and heated at 95 °C for 24 h. After removing the solvent under reduced pressure, the reaction mixture (0.25 μmol) was redissolved in 50 μL of H2O, and then 0.25 μmol of L-FDAA in 100 μL of acetone was added, followed by 25 μL of 1 N NaHCO3. The mixture was heated for 1 h at 43 °C. After cooling to rt, 274

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Journal of Natural Products

Article

the reaction was quenched by the addition of 25 μL of 2 N HCl. Finally the resulting solution was filtered through a small 4.5 μm filter and stored in a freezer until HPLC analysis. The standard L- and Dvaline were also derivatized with FDAA in the above-described manner. The resulting mixture containing the L-FDAA derivative was analyzed by reversed-phase HPLC under the following conditions: a 5 mm × 250 mm YMC C18 column, 5 μm, with a linear gradient of (A) CH3CN and (B) 0.05% aqueous TFA from 10% to 50% (A) over 60 min at a flow rate of 1 mL/min, UV detection at 320 nm. The chromatographic peaks were identified by comparing the retention times for the L-FDAA derivatives of the L- and D-valine and FDAA standards. The standards gave the following retention times: 32.70 min for FDAA; 35.91 min for L-Val; 39.52 min for D-Val. The L-FDAAderivatized hydrolysate mixture showed a peak at 35.91 min. The result demonstrated the valine in compound 5 was the L-form. Biological Assay. Biological evaluation was carried out as previously reported.9a,b,26

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ASSOCIATED CONTENT

S Supporting Information *

The MS and NMR spectra of 1−5 and Marfey’s experimental data of 5. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Fundation of China (Nos. 41176120 and 21372208), the Promotive Research Fund for Excellent Young and Middleaged Scientists of Shandong Province (No. BS2010HZ027), the National High Technology Research and Development Program of China (No. 2013AA092901), the Program for New Century Excellent Talents in University (No. NCET-120499), the Public Projects of State Oceanic Administration (No. 2010418022-3), and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0944). We thank Dr. Z. Lin (University of Utah, USA) for the suggestion of the plausible biosynthetic pathway and Dr. R. A. Keyzers (Victoria University of Wellington, New Zealand) for helping with the manuscript preparation.

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