Methyl 3,4-Dicarboxy-3-hydroxyeicosanoate

Abstract

Methyl 3,4-dicarboxy-3-hydroxyeicosanoate (1) was isolated from the acetone extract of the fruiting bodies of Ciborinia camelliae Kohn. The planar structure of 1 was elucidated by spectroscopic methods.

Introduction

Camellia flower blight, one of the most serious diseases of Camellia spp., is caused by the fungus Ciborinia camelliae Kohn [1]. Generally, this disease is most frequently observed in early spring. At first, ascospores released from apothecia, saucer-shaped fruiting bodies, of C. camelliae adhere to the surface of camellia petals and rapidly infect the flowers, causing the development of many small brown spots. Then, the infected flowers turn brown and fall to the ground prematurely. The fungus forms sclerotia in the fallen flowers and remains throughout winter until the next season. In spring, apothecia that are produced from the sclerotia release the ascospores, which are then spread through air. An apothecium, measuring 1 cm in diameter, has a slender stipe up to ca. 1–5 cm long. This is the characteristic cycle of C. camelliae Kohn. In a previous phytochemical investigation, a lectin was isolated from apothecia of C. camelliae Kohn [2]. However, very little is known about the secondary metabolites related to host/parasite relationships [1,3]. In our research on the chemical constituents of apothecia of C. camelliae Kohn, we isolated 1 from an acetone extract of this fungus. As a dicarboxylic acid with hexadecyl alkyl side chain, agaricic acid has been reported [4]. Agaricic acid has been isolated from fungi including basidiomycetes, Polyporus officinalis, and P. igniarius, and it can participate in the transport of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) and function as an inducer of mitochondrial permeability transition [5]. Here, we describe the isolation and structure elucidation of 1 (Figure 1).

Results and Discussion

High resolution electrospray ionization time-of-flight mass spectrometry (HRESITOFMS) and NMR data showed that 1 has the molecular formula C₂₃H₄₂O₇, implying that 1 has three degrees of unsaturation. The ¹³C NMR (

Table 1. NMR Spectroscopic Data (400 MHz, CD₃OD) for 1.

Compound 1
No.	δC, type	δH (J in Hz)	HMBC
1	172.3 C
2	23.7 CH2	2.72, d (15.9)	1, 4, 21
		3.10, d (15.9)	1, 4, 21
3	76.9 C
4	54.8 CH	2.65, dd, (9.0, 2.5)	3, 5, 21
5	28.2 CH2	1.73, m
		2.21, m	22
6–19	*	1.26–1.28, (m)
20	14.5 CH3	0.89, t (7.4)	18, 19
21	176.4 a C
22	176.2 a C
OMe	52.1 CH3	3.64, (s)	1

* 28.8 t, 30.5 t, 30.7 t, 30.8 t, 33.1 t, 42.5 t, 48.4 t; ^a interchangeable.

) and DEPT spectra of 1 revealed the presence of 16 signals for 23 carbons that included one methyl, one methoxy, 16 methylene, one methine, one quaternary, and three carbonyl carbons. The IR spectrum showed the absorption band characteristic of hydroxyl (3463 cm⁻¹) and carbonyl (1737 and 1698 cm⁻¹) groups. ¹³C NMR (δ_C 172.3, 176.2, 176.4) of 1 and formation of trimethyl esters via treatment with trimethylsilyldiazomethane supported the presence of two carboxyl groups. The ¹H NMR spectrum (Table 1) of 1 showed a methoxy proton [δ_H 3.64 (3H, s)], long-chain alkyl protons [δ_H 0.90 (3H, t, J = 7.4 Hz, H3-20), 1.26–1.28 (approximately 28H, m)], isolated methylene protons [δ_H 2.72 (1H, d, J = 15.9 Hz, H-2), 3.10 (1H, d, J = 15.9 Hz, H-2)], and a methine proton [δ_H 2.65 (1H, dd, J = 9.0, 2.5 Hz, H-4)]. The structure of 1 was elucidated based on the results of ¹H-¹H COSY (correlation spectroscopy), ¹³C-¹H COSY, and HMBC experiments. In the HMBC analysis (Figure 2), the correlations from H-2 to C-1, C-3, and C-21, from H-4 to C-21, and from H-5 to C-22 indicated the structure of a 3,4-dicarboxy-3-hydroxymethyl butanoate moiety substituted by the alkyl moiety. From the above data, the structure of 1 was determined as shown in Figure 1. To our knowledge, this represents the first report of the isolation of 1 from the natural source. In addition, with TLC analysis, 1 was detected from the acetone extract. Thus, the methyl ester of 1 was not formed as an artifact from free acids during the extraction and isolation procedures.

The fungal pathogen C. camelliae has been known to cause camellia flower blight disease. Initially, droplets (20 μL) of a solution (mg/mL) of 1 were applied to the surface of the camellia petals. However, the symptoms of disease progression and the lesions seen in camellia flower blight were not observed for 24 h. Thus, in order to evaluate the potential phytotoxicity, 1 was tested on lettuce seeds. The effects of aqueous solutions of 1, with concentrations ranging between 50 μg/mL and 500 μg/mL, on seed germination, root growth, and shoot length of the lettuce were evaluated. Compound 1 inhibited the root growth to 40% of the control at a concentration of 300 μg/mL. At the highest concentration (500 μg/mL), 1 displayed an inhibitory effect on seed germination.

Experimental

General

Optical rotation value was measured with Horiba SEPA-300 polarimeter (Kyoto, Japan), and IR spectrum was recorded with Jasco J-20A spectrophotometer (Tokyo, Japan). Mass spectra were obtained with Jeol JMS-700 (FABMS) and Synapt G2 (HRESITOFMS) mass spectrometers (Tokyo, Japan). NMR data were recorded on a Jeol EX-400 spectrometer at 400 MHz for ¹H and 100 MHz for ¹³C (Tokyo, Japan). Chemical shifts are given on a δ (ppm) scale with TMS as an internal standard. ¹H, ¹³C, DEPT, COSY, ¹³C-¹H COSY and HMBC spectra were recorded using standard Jeol standard pulse sequences. Chemical shifts are given on a δ (ppm) scale with CD₃OD as an internal standard. Column chromatography was conducted on silica gel 60 (Merck, MercK KGaA, Darmstadt, Germany) and ODS (Chromatolex ODS, Fuji Silysia). TLC was carried out using precoated silica gel plates (0.5 mm; Merck and Macherey-Nagel and Co., MercK KGaA, Darmstadt, Germany), and spots were detected by spraying with 10% vanillin in H₂SO₄ followed by heating, or by UV irradiation.

Biological Material

The fruiting bodies of Ciborinia camelliae Kohn were collected at Tsuruoka city, Yamagata Prefecture, Japan, in Autumn 2008 and identified by one of authors (Y.S.). A voucher specimen has been deposited in the Faculty of Agriculture, Yamagata University, Yamagata, Japan.

Extraction and Isolation

Dried fruiting bodies (450 g) of Ciborinia camelliae Kohn were extracted with acetone. The acetone extract was evaporated in vavuo and the acetone extract was concentrated to give a residue (10.5 g). The resulting residue concentrate was partitioned into n-hexane and EtOAc layers. The purification of the EtOAc layer was guided by the intense brown characteristic coloration with vanillin-sulfuric acid solution on TLC plates. The EtOAc layer (8.8 g) was chromatographed on a silica gel column using stepwise elution with n-hexane–EtOAc (100:0–0:100) followed by stepwise elution with EtOAc–MeOH (100:0–0:100) to give fractions 1–13 (Fr. 1-1–1-13). Fractions 1-6 (1.05 g, n-hexane–EtOAc, 50:50) was chromatographed on silica gel using a stepwise gradient of CHCl₃–EtOAc (100:0–0:100) to afford fractions 2-1~2-11. Fraction 2-3 (CHCl₃–EtOAc, 80:20) was rechromatographed by ODS column chromatography by eluting with H₂O and an increasing ratio of MeOH to afford fractions 3-1~3-11. Compound 1 (28.0 mg) was isolated from Fr. 3-7 (H₂O–MeOH, 40:60) as an amorphous powder;

IR (KBr, cm⁻¹): 3463 (OH), 1737 (C=O), 1698 (C=O).

¹H (400 MHz, CD₃OD) and ¹³C (100 MHz, CD₃OD) NMR data see Table 1.

${[\mathsf{\alpha }]}_{\mathrm{D}}^{20}$ +84° (c 0.04, CH₃OH)

FABMS: m/z 417 (M+H)⁺.

HRESITOFMS: m/z 431.2910 (calcd for C₂₃H₄₃O₇, 431.3009).

Methyl Ester Derivative of Compound 1

Compound 1 (10 mg) was dissolved in a solution of MeOH, and trimethylsilyldiazomethane (2.0 M in diethylether, 0.05 mL), was added to the solution. The mixture was stirred at 0 °C for 5 min and evaporated to dryness. The residue (9.2 mg) was subjected to silica gel column chromatography with mixtures of n-hexane–EtOAc to afford a trimethyl ester (6.0 mg).

¹H-NMR (400 MHz; CD₃OD): δ_H 0.88 (3H, t, J = 7.4 Hz, Me-20), 1.80 (1H, m, H-5), 1.24–1.26 (28H, m), 1.47 (1H, m, H-5), 2.68 (1H, m, H-4), 2.72 (1H, d, J = 16.0 Hz, H-2), 3.10 (1H, d, J = 16.0 Hz, H-2), 3.67, 3.71, 3.82 (each 3H, OCH₃).

¹³C-NMR (100 MHz; CD₃OD): δ_H 14.1 (CH₃, C-20), 22.7 (CH₂, C-2), 27.1, 27.7, 29.4, 29.5, 29.6, 29.7, 31.9, 40.8 (each CH₂, C-5-C19), 51.9, 52.0, 53.2 (each CH₃, OCH₃), 53.8 (CH, C-4), 75.9 (C, C-3), 174.1 172.9, 171.7, (each C, C-1, C-21, C-22).

HRESITOFMS: m/z 459.3382 (calcd for C₂₅H₄₇O₇, 459.3322).