Acetylenes and fatty acids from Codonopsis pilosula.

Four new acetylenes (1-4) and one new unsaturated ω-hydroxy fatty acid (5), together with 5 known analogues, were isolated from an aqueous extract of Codonopsis pilosula roots. Their structures were determined by spectroscopic and chemical methods. The new acetylenes are categorized as an unusual cyclotetradecatrienynone (1), tetradecenynetriol (2), and rare octenynoic acids (3 and 4), respectively, and 3 and 4 are possibly derived from oxidative metabolic degradation of 1 and/or 2. The absolute configuration of 1 was assigned by comparison of the experimental circular dichroism (CD) spectrum with the calculated electronic circular dichroism (ECD) spectra of stereoisomers based on the quantum-mechanical time-dependent density functional theory, while the configuration of 2 was assigned by using modified Mosher׳s method based on the MPA determination rule of Δδ RS values for diols.

Introduction

Codonopsis pilosula (Franch.) Nannf., a perennial plant of the Campanulaceae family, is widely cultivated in the northwest provinces of China to meet the demand of pharmaceutical and food industries. The root of this plant, known as “Dang shen” in Chinese, is one of the most common traditional Chinese medicines used for the treatment of body weakness, poor appetite, thirsty, indigestion, chronic diarrhea, archoptoma, chronic anemia and leukemia. It exhibits similar therapeutic effects of Panax ginsengas as tonic agents and is used in many cases as a substitute of the much more costly Panax ginseng. Previous studies showed that extracts of the C. pilosula roots exhibited pharmacological effects in protecting against peptic ulceration and promoting its healing, enhancing immunity, and improving learning and memory behavior, as well as inhibiting inducible NO synthase and protein oxidation and attenuating the cardiac-impaired insulin-like growth factor II receptor pathway2, 3, 4, 5, 6. Meanwhile, different types of chemical constituents were isolated from the extracts, such as phytosteroids, sesquiterpenes, triterpenes, alkaloids, alkyl alcohol glycosides, phenylpropanoid glycosides, polyacetylene glycosides and polysaccharides7, 8, 9, 10, 11, 12, of which only polysaccharides were biologically evaluated13, 14, 15, 16, 17. As part of a program to systematically study the chemical diversity of traditional Chinese medicines and their biological effects, focusing on minor small molecules18, 19, 20, 21, 22, 23, 24, 25, an aqueous decoction of the C. pilosula roots was investigated since a variety of formulations containing “Dang shen” are practically used by decocting with water. Herein, reported are the isolation and structural elucidation of four new acetylenes (1–4), a new unsaturated ω-hydroxy fatty acid (5) (Fig. 1), and five known analogues, as well as their preliminary bioassays.

Figure 1

The structures of compounds 1−5.

Results and discussion

Compound 1 was obtained as a yellowish amorphous powder with [α]D20 +10.8 (c 0.07, MeOH). Its IR spectrum showed absorption bands for hydroxyl (3395 cm−1), double bond (3011 cm−1), triple bond (2191 and 2062 cm−1), and conjugated carbonyl (1720, 1685 and 1647 cm−1) functionalities. The molecular formula of 1, C14H16O3, was indicated by HR-ESI-MS at 255.0999 [M+Na]+ (Calcd. for C14H16O3Na, 255.0992) and NMR data (Table 1). The 1H NMR spectrum of 1 in CD3OD showed signals attributed to a conjugated cis-disubstituted double bond at δH 7.13 (ddd, J=9.6, 6.0 and 2.4 Hz, H-4) and 6.07 (ddd, J=9.6, 3.0 and 0.6 Hz, H-5); a trans-disubstituted double bonds at δH 6.19 (d, J=15.6 Hz, H-8) and 5.99 (dd, J=15.6 and 2.4 Hz, H-9); and a terminal trans-propenyl at δH 5.61 (dq, J=15.6 and 1.8 Hz, H-12), 6.11 (dq, J=15.6 and 6.6 Hz, H-13), and 1.78 (dd, J=6.6 and 1.8 Hz, H3-14). In addition, the 1H NMR spectrum displayed signals assigned to an oxygen-bearing methylene at δH 3.86 (dd, J=10.8 and 4.8 Hz, H-1a) and 3.45 (dd, J=10.8 and 8.4 Hz, H-1b) and an aliphatic methylene at δH 2.73 (ddd, J=10.8, 6.0 and 1.0 Hz, H-3a) and 2.35 (ddd, J=10.8, 6.0 and 2.4 Hz, H-3b), as well as a multiplet due to a methine at δH 2.28 (m, H-2), which is vicinal to both the methylenes based on the splitting patterns and coupling constants. The 13C NMR and DEPT spectra of 1 exhibited 14 resonances corresponding to proton-bearing carbons of the above units and four quaternary carbons including a conjugated carbonyl (δC 199.6, C-6), a triple bond (δC 86.4 (C-10) and 91.2 (C-11)), and an oxygen-bearing (δC 80.2, C-7) carbon (Table 1). Together, the above spectroscopic data indicated that 1 is a monocyclic C14 hydroxytrienynone with an unusual structure feature, which was further elucidated by 2D NMR experimental data analysis. The assignments of proton and proton-bearing carbon signals in the NMR spectra were confirmed by cross-peaks in the 1H-1H COSY and HSQC spectra. The vicinal coupling cross-peaks H2-1↔H-2↔H2-3↔H-4↔H-5 in the 1H-1H COSY spectrum and three-bond hetero nuclear correlations (Fig. 2) from H2-1 to C-3 and C-7; from H-2 to C-4 and C-7; from H2-3 to C-1, C-5, and C-7; from H-4 to C-2 and C-6; and from H-5 to C-3 and C-7 in the HMBC spectrum, together with the chemical shifts of these proton and carbon resonances, revealed that the oxygen-bearing quaternary carbon (C-7) was connected with both the methine (CH-2) and the carbonyl (C-6) to form a cyclohexenone moiety in 1. The 1H-1H COSY cross-peaks H-8↔H-9 and H-12↔H-13↔H3-14, together with the HMBC correlations from H-8 to C-10, from H-9 to C-11, from H-12 to C-14, from H-13 to C-11, and from H3-14 to C-12, in combination with their chemical shifts, confirmed the presence of a 8,12-dien-10-yn-8-yl chain moiety in 1. In addition, the HMBC correlations from H-2 to C-8, from H-8 to C-6 and C-7, and from H-9 to C-7 indicated a C-7–C-8 bond connection between the two moieties. To match requirement of the molecular composition, two hydroxyl groups must occur at the oxygen-bearing carbons (C-1 and C-7) in 1. Accordingly the planar structure of 1 is elucidated as shown in Fig. 2. In the NOE difference spectrum of 1, irradiation of H-3b enhanced H-2 and H-8 (Fig. 3), indicated that these protons were cofacial and that the chain moiety had a quasi-axial orientation on the cyclohexenone ring, which was supported by conformational analysis with the MMFF94 molecular mechanics force field using molecular operating environment (MOE) software package. The circular dichroism (CD) spectrum of 1 displayed a positive Cotton effect at 299 nm (Δε +1.41) for n→π* and a negative Cotton effect at 256 nm (Δε –4.71) for π→π*. Based on the octant rule for cyclohexenones, these Cotton effects suggest that 1 possesses the 2R,7S-configuration (Fig. 4). The suggestion was further supported by comparing the experimental CD spectrum with the electronic circular dichroism (ECD) spectrum predicted from the quantum-mechanical time-dependent density functional theory (TDDFT) calculations (Fig. 5). Therefore, compound 1 was determined as (+)-(2R,7S)-1,7-dihyrdroxy-2,7-cyclotetradeca-4,8,12-trien-10-yn-6-one.

Table 1

1H and 13C NMR spectral data (δ) for compounds 1–5.a

No.	1		2		3
	δH	δC	δH	δC	δH	δC
1a	3.86 dd (10.8, 4.8)	62.8	3.49 t (6.5)	63.2		170.1
1b	3.45 dd (10.8, 8.4)
2	2.28 m	48.2	1.48 m	33.9	6.08 d (16.0)	131.9
3a	2.73 ddd (10.8, 6.0, 1.0)	29.9	1.34 m	27.3	6.74 d (16.0)	126.2
3b	2.35 ddd (10.8, 6.0, 2.4)		1.47 m
4	7.13 ddd (9.6, 6.0, 2.4)	152.4		27.1		85.9
5	6.07 ddd (9.6, 3.0, 0.6)	127.8	1.39 m	34.2		98.3
6		199.6	3.34 q (8.0)	75.3	5.66 d (16.0)	111.7
7		80.2	3.46 m	74.2	6.23 dq (16.0, 7.0)	144.0
8a	6.19 d (15.6)	138.2	1.62 m	33.6	1.77 d (7.0)	19.2
8b			1.57 m
9	5.99 dd (15.6, 2.4)	113.6	2.34 m	16.9
10		86.4		88.8
11		91.2		80.6
12	5.61 dq (15.6, 1.8)	111.9	5.39 dd (16.5, 1.5)	112.6
13	6.11 dq (15.6, 6.6)	140.8	5.93 dq (16.5, 7.0)	138.9
14	1.78 dd (6.6, 1.8)	18.7	1.67 dd (7.0, 1.5)	18.7

NMR data (δ) were measured in MeOH-d4 for 2, 3, and 4 at 500 MHz for 1H NMR and at 125 MHz for 13C NMR and 1, 5 at 600 MHz for 1H NMR and at 150 MHz for 13C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H-1H COSY, HSQC, and HMBC experiments.

Figure 2

The 1H-1H COSY (thick line) and key HMBC (arrows) correlations of compound 1.

Figure 3

The NOE enhancements induced by irradiation of H-3b (dashed arrows) for compound 1.

Figure 4

Absolute configuration of compound 1.

Figure 5

The experimental CD spectrum of 1 (black) and the calculated ECD spectra of (2R,7S)-1 (dashed blue) and (2S,7R)-1 (dashed red).

Compound 2, a yellowish amorphous powder with [α]D20 +41.5 (c 0.06, MeOH), has the molecular formula C14H24O3 as indicated by HR-ESI-MS at m/z 241.1807 [M+H]+ (Calcd. for C14H25O3, 241.1798) and 263.1620 [M+Na]+ (Calcd. for C14H24O3Na, 263.1618). The IR and NMR spectroscopic data of 2 (Section 4 and Table 1) demonstrated that it was a C14 enynetriol derivative with the same terminal unit trans-12-en-10-ynyl as that of 1. Comparison of the NMR spectroscopic data between 2 and 1 indicated replacement of the 2,7-cyclo-4,8-dien-6-one moiety in 1 by a linear diol moiety consisting of two hydroxyl-bearing methines δH 3.34 (q, J=4.0 Hz, H-6) and 3.46 (q, J=4.0 Hz, H-7), δC 75.3 (C-6) and 74.2 (C-7) and six aliphatic methylenes δH1.34–1.48 (8H, partially overlapped, H2-2–H2-5), 1.62 (m, H-8a), 1.57 (m, H-8b), and 2.34 (m, H2-9); δC 16.9 (C-9), 27.1 (C-4), 27.3 (C-3), 33.6 (C-8), 33.9 (C-2), and 34.2 (C-5) in 2. In addition, the two double doublets of inequivalent H-1a and H-1b in 1 were replaced by a hydroxymethylene triplet at δH 3.49 (J=6.5 Hz, H2-1) in 2, while the triple bond carbon resonances were shifted from δC 86.4 (C-10) and 91.2 (C-11) in 1 to δC 88.8 (C-10) and 80.6 (C-11) in 2. These data suggest that 2 differs from 1 in the 2,7-cyclo-4,8-dien-6-one moiety with cleavage of the C-2–C-7 bond and saturation of the 4,8-dien-6-one. This was further confirmed by 2D NMR data analysis, especially by the 1H-1H COSY cross-peaks H-12↔H-13↔H3-14 and the HMBC correlations from H2-8 to C-6 and C-10; from H2-9 to C-7 and C-11; from H-13 to C-11 and C-14; and from H2-14 to C-11, C-12, and C-13, combined with their chemical shifts. The absolute configuration the 6,7-diol moiety in 2 was determined using the modified Mosher׳s method since the primary alcohol at C-1 is away from and has less shielding/deshielding effects on protons around the chiral centers. Treatment of 2 with (−)-(R)- and (+)-(S)-α-methoxyphenylacetic acid (R- and S-MPA) in anhydrous CH2Cl2 afforded the tri-(R)-and tri-(S)-MPA esters of 2. The Δδ values of H-6 and H-7 were calculated by NMR data measurement of the esters. According to the Mosher׳s model of vicinal diols, the Δδ values predict that 2 has the 6R,7R-configuration (Fig. 6). This was supported by Mo2(OAc)4-induced circular dichroism (ICD) spectrum of 2, which displayed a negative Cotton effect at 316 nm (Fig. S17, Supporting information). On the basis of the empirical rule proposed by Snatzke30, 31, the Cotton effect indicate that the O–C–C–O torsion angle of the diol moiety in 2 is negative and also predicts 6R,7R configuration. Thus, compound 2 was determined as (+)-(6R,7R,12E)-tetradeca-12-en-10-yne-1,6,7-triol.

Figure 6

Diagnostic Δδ values (δ−δ, blue data in ppm, left upper) and applied model for tri-MPA esters of compound 2.

Compound 3, a yellowish amorphous powder, has the molecular formula C8H8O2 as indicated by HR-ESI-MS m/z 137.0595 [M+H]+ (Calcd. for C8H9O2, 137.0597) and 159.0413 [M+Na]+ (Calcd. for C8H8O2Na, 159.0417). Its IR spectrum showed the presence of OH (3395 cm−1), C=C (3011 cm−1), C C (2185 cm−1), and carbonyl (1698 cm−1, sh) functionalities. The 1H NMR data of 3 (Table 1) demonstrated occurrence of a terminal trans-propenyl (δH 6.23 (dq, J=16.0 and 7.0 Hz, H-7), 5.66 (d, J=16.0 Hz, H-6), and 1.77 (d, J=7.0 Hz, H-8)) and a trans-disubstituted double bond (δH 6.08 (d, J=16.0 Hz, H-2) and 6.74 (d, J=16.0 Hz, H-3)). Besides the resonances corresponding to the proton-bearing carbons, the 13C NMR and DEPT spectral data confirmed the presence of the triple-bond (δC 85.9 (C-4) and 98.3 (C-5)) and carbonyl (δC 170.1 (C-1)) units. These spectroscopic data reveal that 3 is (2E,6E)-octa-2,6-dien-4-ynoic acid, which was further confirmed by 2D NMR data analysis, particularly by HMBC correlations from H-2 to C-1 and C-4 and from H-3 to C-1 and C-5 combined with their chemical shifts. Thus, compound 3 was determined as (2E,6E)-octa-2,6-dien-4-ynoic acid.

Compound 4 was isolated as a yellowish amorphous powder. The IR and HR-ESI-MS data (Section 4) indicated that 4 was a dihydro analogue of 3. Comparison of the NMR spectral data of 4 and 3 (Table 1, Table 2) demonstrated that the 2-en in 3 was saturated in 4. Therefore, compound 4 was determined as (E)-oct-6-en-4-ynoic acid, which was also verified by 2D NMR data analysis.

Table 2

1H and 13C NMR spectral data (δ) for compounds 4 and 5.a

No.	4		5
	δH	δC	δH	δC
1a		176.8		178.9
1b
2	2.40 t (7.5)	35.4	2.25 t (7.2)	35.7
3a	2.47 t (7.5)	16.4	1.59 quin (7.2)	26.3
3b			1.33 m
4		87.6		29.9
5		80.7	1.33 m	30.1
6	5.39 dd (16.0, 1.5)	112.4	1.39 quin (7.2)	30.3
7	5.95 dq (16.0, 6.5)	139.4	2.07 q (7.2)	33.5
8a	1.67 dd (6.5, 1.5)	18.7	5.65 dt (15.0, 7.2)	131.1
8b
9			6.04 dd (15.0, 4.2)	131.0
10			6.18 dd (15.0, 4.2)	132.5
11			5.68 dt (15.0, 6.0)	135.6
12			4.05 d (6.0)	63.4
13
14

NMR data (δ) were measured in MeOH-d4 for 4 at 500 MHz for 1H NMR and at 125 MHz for 13C NMR and 5 at 600 MHz for 1H NMR and at 150 MHz for 13C NMR. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H-1H COSY, HSQC and HMBC experiments.

Compound 5 has the molecular formula C12H20O3 as indicated by HR-ESI-MS at m/z 235.1296 [M+Na]+ (Calcd. for C14H16O3Na, 235.1305). The IR spectrum showed the presence of OH (3200 cm−1), CC (3017 cm−1), and CO (1723 cm−1) functionalities. The 1H NMR spectral data of 5 (Table 2) revealed the presence of two double bonds, an oxygen-bearing methylene, and six aliphatic methylenes. These spectroscopic data suggested that 5 is (8E,10E)-12-hydroxydodeca-8,10-dienoic acid though an expected carboxylic carbon resonance was extremely diminished and almost not recognized in the 13C NMR spectrum. The suggestion was confirmed by 2D NMR experiments. Especially, the HMBC spectrum exhibited correlations from H2-2 and H2-3 to a carbon resonated at δC 178.9, leading to an ambiguous assignment of the carboxylic carbon in 5. In addition, the HMBC correlations from H-10 to C-8 and C-12 and from H2-12 to C-10 and C-11 confirmed positions of the hydroxyl and double bonds. Therefore, compound 5 was determined as (8E,10E)-12-hydroxydodeca-8,10-dienoic acid.

The known compounds were identified by comparing their spectroscopic data with the reported data as (6R,7R,4E,8E,12E)-tetradeca-4,8,12-trien-10-yn-1,6,7-triol, (10E)-12-hydroxydodeca-10-enoic acid, hexadecanoicacid-2′,3′-dihydroxy propyl ester, fulgidic acid, and pinellic acid.

Various biological activities of acetylene and unsaturated fatty acid derivatives were reported, such as adjuvant activity, cytotoxicity and antidiabetic activity. This indicates that these types of compounds might play some biological roles in clinical effects of this herbal medicine. In the preliminary in vitro assays carried out in this study, the isolates were assessed for inhibitory activity against LPS-induced NO production in BV2-cells, protein tyrosine phosphatase 1B (PTP1B) , HIV-1 replication and several human cancer cell lines, as well as antioxidant activity, but all were inactive at a concentration of 10 μmol/L. Therefore, their potential biological activity is still expected from a further in depth evaluation on other biological models.

Conclusions

From the aqueous extract of C. pilosula roots, four new acetylenes (1–4) and one new unsaturated ω-hydroxy fatty acid (5), together with five known analogues, were isolated. The new acetylenes are categorized as an unusual cyclotetradecatrienynone (1), tetradecenynetriol (2), and rare octenynoic acids (3 and 4), respectively, of which 3 and 4 are possibly derived from oxidative metabolic degradation of 1 and/or 2. Although the new compounds were inactive in the assays carried out in this study, the results provide a clue for further studies of synthesis, chemical transformation, structural modification and biosynthesis, as well as biological evaluations on other pharmacological models, of the diverse ployacetylene derivatives from the C. pilosula.

Experimental

General experimental procedures

Optical rotations were measured on P-2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were measured on a V-650 spectrometer (JASCO, Tokyo, Japan). IR spectra were recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR microscope transmission) (Thermo Electron Corporation, Madison, USA). NMR spectra were obtained at 500 MHz or 600 MHz for 1H NMR, and 125 MHz or 150 MHz for 13C NMR, respectively, on Inova 500 or SYS 600 (Varian Associates Inc., Palo Alto, USA) or Bruker 600 NMR spectrometers (Bruker Corp. Switzerland) in MeOH-d4 with solvent peak used as references. ESI-MS and HR-ESI-MS data were measured using an AccuToFCS JMS-T100CS spectrometer (Agilent Technologies, Ltd., Santa Clara, USA). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), and CHP 20P (Mitsubishi Chemical Inc., Tokyo, Japan). HPLC separation was performed on an instrument consisting of an Agilent ChemStation for LC system, an Agilent 1200 pump, and an Agilent 1100 single-wavelength absorbance detector (Agilent Technologies, Ltd.) with a YMC-Pack Ph (250 mm×10 mm, i.d.) column packed with Phenyl-silica gels (5 μm) (YMC Co., Ltd., Kyoto, Japan) or a Grace (250 mm×10 mm, i.d.) semipreparative column packed with C18 reversed phase silica gel (5 μm) (W.R Grace & Co., USA). TLC was carried out with glass precoated silica gel GF254 plates (Qingdao Marine Chemical Inc.). Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% EtOH followed by heating. Unless otherwise noted, all chemicals were obtained from commercially available sources and were used without further purification.

Plant material

The roots of C. pilosula were collected in October 2012 from the culture field in Weiyuan, Gansu Province, China. Plant identity was verified by Mr. Lin Ma (Institute of Materia Medica, Beijing, China). A voucher specimen (No. ID-S-2503) was deposited at the herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Beijing, China.

Extraction and isolation

The dried and minced roots of C. pilosula (50 kg) were extracted with H2O (150 L, 3×1 h). The aqueous extracts were evaporated under reduced pressure to yield a dark brown residue (26 kg). The residue was dissolved in H2O (100 L), loaded on a macroporous adsorbent resin (HPD-110, 20 L) column (20 cm×200 cm), and eluted successively with H2O (100 L), 50% EtOH (120 L), and 95% EtOH (80 L) to yield three corresponding fractions, A, B and C. Fraction C (31.0 g) was subjected to CC over silica gel, with elution by a gradient of increasing acetone concentration (0–100%) in petroleum ether, to yield fractions C1–C18 based on TLC analysis. Fraction C10 (1.0 g) was separated by CC over Sephadex LH-20, eluted with petroleum ether–CHCl3–MeOH (5:5:1, v/v/v), to give C10-1–C10-8. Hexadecanoicacid-2′-3′-dihydroxy propyl ester (7.0 mg) was precipitated from C10-3 (MeOH). Fraction C10-6 (80 mg) was separated by reversed-phase (Phenyl-silica gel) semipreparative HPLC, using MeOH–H2O (40:60, v/v) as the mobile phase (1.5 mL/min, UV 254 nm), to yield 1 (1.0 mg, tR=36 min). Fraction C12 (0.8 g) was separated by CC over Sephadex LH-20, eluted with petroleum ether-CHCl3-MeOH (5:5:1, v/v/v), to give C12-1–C12-8, of which C12-6 (100 mg) was separated by reversed-phase (phenyl-silica gel) semipreparative HPLC, using MeOH–H2O (42:58, v/v) as the mobile phase (1.5 mL/min, UV 254 nm), to yield 4 (1.1 mg, tR=23 min). Fraction C12-8 (60 mg) was isolated by preparative TLC, using petroleum ether-ethyl acetate (1:1, v/v) containing 0.1% HOAc as the mobile phase, to give (10E)-12-hydroxydodeca-10-enoic acid (6.9 mg). Fraction C14 (1.0 g) was separated by CC over Sephadex LH-20, eluted with petroleum ether-CHCl3-MeOH (5:5:1, v/v/v), to give C14-1–C14-8. Fraction C14-4 (80 mg) was separated by reversed-phase (C18) semipreparative HPLC, using MeOH-H2O (62:38, v/v) as the mobile phase (1.5 mL/min, UV 220 nm), to yield 2 (1.0 mg, tR=26 min) and 3 (20 mg, tR=23 min). Fraction C14-6 (60 mg) was purified by reversed-phase (C18) semipreparative HPLC, using MeOH-H2O (63:37, v/v) containing 0.1% HOAc as the mobile phase (1.5 mL/min, UV 220 nm), to yield 5 (1.7 mg, tR=42 min). Fraction C17 (1.1 g) was separated by CC over Sephadex LH-20, eluted with CHCl3-MeOH (1:1, v/v), giving C17-1–C17-6. Fraction C17-4 (300 mg) was separated by CC over silica gel, eluted by a gradient of increasing MeOH concentration (0–100%) in CHCl3, yielding C17-4-1–C17-4-8 based on TLC analysis. Fulgidic acid (13.0 mg) was precipitated from C17-4-7 (MeOH). Pinellic acid (15.0 mg) was precipitated from C17-6 (MeOH). Purification of B3-2-1–2 (100 mg) by RP HPLC (C18) (49% CH3OH in H2O), afforded (6R,7R,4E,8E,12E)-tetradeca-4,8,12-trien-10-yn-1,6,7-triol (2.0 mg, 1.5 mL/min, UV 220 nm, tR=51 min).

(+)-(2R,7S)-1,7-dihyrdroxy-2,7-cyclotetradeca-4,8,12-trien-10-yn-6-one (1)

Yellowish amorphous powder; [α]D20 +10.8 (c 0.07, MeOH); UV (MeOH) λmax (logε): 270 (3.22) nm; CD (MeOH) 256 (Δε −4.71), 299 (Δε +1.41) nm; IR νmax 3395, 3189, 2921, 2850, 2191, 2062, 1721, 1684, 1647, 1469, 1420, 1134, 1120, 1092, 957, 648 cm−1; 1H NMR (CD3OD, 600 MHz) data, see Table 1; 13C NMR (CD3OD, 150 MHz) data, see Table 1; (+)-ESI-MS m/z 255 [M+Na]+, 271 [M+K]+; HR-ESI-MS m/z 255.0999 [M+Na]+(Calcd. for C14H16O3Na, 255.0992).

(+)-(6R,7R,12E)-tetradeca-12-en-10-yn-1,6,7-triol (2)

Yellowish amorphous powder; [α]D20 +41.5 (c 0.06, MeOH); UV (MeOH) λmax (logε): 227 (3.60), 265 (2.28 sh), 279 (2.10) nm; IR νmax 3389, 3294, 3197, 2926, 2851, 2217, 1646, 1468, 1419, 1112, 1073, 1045, 954, 723, 654 cm−1; 1H NMR (CD3OD, 500 MHz) data, see Table 1; 13C NMR (CD3OD, 125 MHz) data, see Table 1; (+)-ESI-MS m/z 241 [M+H]+, 263 [M+Na]+; HR-ESI-MS m/z 241.1807 [M+H]+ (Calcd. for C14H25O3, 241.1798), 263.1620 [M+Na]+ (Calcd. for C14H24O3Na, 263.1618).

(2E,6E)-octa-2,6-dien-4-ynoic acid (3)

Yellowish amorphous powder; UV (MeOH) λmax (logε) 207 (3.73), 284 (3.97) nm; IR νmax 3395, 3187, 3011, 2922, 2850, 2185, 1647, 1468, 1420, 1302, 1273, 1201, 1120, 971, 946, 722, 648 cm−1; 1H NMR (CD3OD, 500 MHz) data, see Table 1; 13C NMR (CD3OD,125 MHz) data, see Table 1; (+)-ESI-MS m/z 137 [M+H]+, 159 [M+Na]+, 175 [M+K]+; (−)-ESI-MS m/z 135 [M−H]–; HR-ESI-MS m/z 137.0595 [M+H]+(Calcd. for C8H9O2, 137.0597), 159.0413 [M+Na]+ (Calcd. for C8H8O2Na, 159.0417).

(E)-oct-6-en-4-ynoic acid (4)

Yellowish amorphous powder; UV (MeOH) λmax (logε): 190 (3.56), 226 (4.02) nm; IR νmax 3393, 3031, 2962, 2852, 2216, 1722, 1558, 1489, 1435, 1413, 1356, 1301, 1020, 955, 837 cm−1; 1H NMR (CD3OD, 500 MHz) data, see Table 2; 13C NMR (CD3OD, 125 MHz) data, see Table 2; (+)-ESI-MS m/z161 [M+Na]+, 177 [M+K]+; (–)-ESI-MS m/z 173 [M+Cl]−; HR-ESI-MS m/z 137.0601 [M−H]− (Calcd. for C8H9O2, 137.0608).

(8E,12E)-12-hydroxydodeca-8,10-dienoic acid (5)

Yellowish amorphous powder; UV (MeOH) λmax (logε): 195 (3.77), 229 (4.30) nm; IR νmax 3200, 3017, 2922, 2847, 1723, 1571, 1462, 1439, 986, 725 cm−1; 1H NMR (CD3OD, 600 MHz) data, see Table 2; 13C NMR (CD3OD, 150 MHz) data, see Table 2; (+)-ESI-MS m/z 235 [M+Na]+, 251 [M+K]+; (−)-ESI-MS m/z 211 [M−H]−; HR-ESI-MS m/z 235.1296 [M+Na]+ (Calcd. for C14H16O3Na, 235.1305).

ECD calculation of 1

Conformational analysis of 1 was performed with the MMFF94 molecular mechanics force field using Molecular Operating Environment (MOE) software package. The lowest-energy conformers having relative energies within 2 kcal/mol were optimized with the Gaussian09 programat the B3LYP/6–31+G(d) level in MeOH (Fig. S1, Supporting information). The stabilities of these conformers were confirmed by harmonic vibrational frequency calculations at the B3LYP/6–31+G(d) level. The energies, oscillator strengths, and rotational strengths of the electronicexcitations of the lowest-energy conformers were calculated using the TDDFT method at the B3LYP/6–311++ G(2d,2p) level in MeOH, and ECD spectra were then simulated by the Gaussian function. The final ECD spectrum of 1 was obtained according to Boltzmann weighting of each conformer.

Synthesis of tri-(R)- and tri-(S)-MPA esters of 2

Compound 2 (0.3 mg) was stored in 10 mL round-bottomed flask and dried under vacuum. Anhydrous CH2Cl2 (3.0 mL) and R-MPA or S-MPA (5.3 mg), 1-ethyl-3-(3- dimethyllaminopropyl)carbodiimide hydrochloride (EDCI) (6.1 mg), and 4-dimethylaminopyridine (DMAP) (3.9 mg) were added to the round-bottomed flask. The reaction round-bottomed flask was permitted to stand at room temperature for 15 min. The residue obtained after evaporation of the solvent was applied to preparative TLC (petroleum ether-acetone (3:1, v/v)) to give 2-tri-(R)-MPA and 2-tri-(S)-MPA. 2-tri-(R)-MPA: 1H NMR (600 MHz, CD3OD) δH: 7.28–7.37 (15H, m, aromatic H), 5.96 (1H, dq, J=15.6, 6.6 Hz, H-13), 5.39 (1H, d, J=15.6 Hz, H-12), 4.95 (1H, m, H-7), 4.80 (1H, overlapped, H-6), 4.79 (1H, s, α-H), 4.77 (1H, s, α-H), 4.74 (1H, s, α-H), 3.98 (1H, dt, J=4.2, 12.6 Hz, H-1a), 3.90 (1H, dt, J=12.6, 6.6 Hz, H-1b), 3.33 (3H, s, α-OCH3), 3.31 (3H, s, α-OCH3), 3.30 (3H, s, α-OCH3), 1.95 (2H, m, H-9), 1.69 (3H, d, J=6.6 Hz, H-14), 1.23 (10H, m, H-2, 3, 4, 5, 8); 2-tri-(S)-MPA: 1H NMR (600 MHz, CD3OD) δH 7.24–7.40 (15H, m, aromatic H), 5.95 (1H, dq, J=15.6, 6.6 Hz, H-13), 5.35 (1H, dd, J=15.6, 1.8 Hz, H-12), 5.08 (1H, m, H-7), 4.89 (1H, m, H-6), 4.78 (1H, s, α-H), 4.77 (1H, s, α-H), 4.74 (1H, s, α-H), 3.89 (1H, dt, J=12.6, 4.8 Hz, H-1a), 3.84(1H, dt, J=12.6, 6.0 Hz, H-1b), 3.33 (6H, s, α-OCH3), 3.31 (3H, s,α-OCH3), 1.67 (3H, dd, J=6.6, 1.8 Hz, H-14), 1.50 (2H, m, H-9), 1.15 (10H, m, H-2, 3, 4, 5, 8).

Measurement of Mo2(OAc)4-induced circular dichroism (ICD) spectrum of 2

According to the published approach, a solution of 2 (0.5 mg) in dry DMSO (1 mL) was mixed with dimolybdenumtetraacetate (1.0 mg). The first CD of the mixture (ca. 1:1 diol/dimolybdenumtetraacetate) was recorded immediately after mixing, and its time evolution was monitored until stationary. The observed sign of the diagnostic band at 316 nm in the ICD was correlated to the 6R,7R-configuration of the 6,7-diol moiety in 2.