(10E,15Z)-12-(Dimethylsulfonio)-9,13-dihydroxyoctadeca-10,15-dienoate

Abstract

A novel oxylipin, okeanoate (1), was isolated from the Okinawan cyanobacterium Okeania hirsuta. The structure of 1 was elucidated based on spectroscopic data including 1D and 2D NMR, as well as high-resolution mass spectrometry. This is the first oxylipin with a dimethylsulfonium moiety in the middle of the hydrocarbon chain.

1. Introduction

Oxylipins are a group of oxygenated natural products formed from fatty acids [1]. It has been reported that many oxylipins perform physiologically important activities in many organisms [2]. Cyanobacteria are known to produce numerous bioactive substances and are recognized as valuable biochemical resources [3,4]. A few oxylipins have been reported as natural products from cyanobacteria so far [5,6,7,8,9,10]. Our research group has recently reported two known isolated oxylipins, malyngic acid, 15,16-dihydro malyngic acid, and a new oxylipin, okeanic acid–A [10], from the marine cyanobacterium Okeania hirsuta. In this study, further research on the cyanobacterium O. hirsuta extracts led to the isolation of a novel oxylipin, (10E,15Z)-12-(dimethylsulfonio)-9,13-dihydroxyoctadeca-10,15-dienoate (1, Figure 1). Only a few oxylipins with a dimethylsulfonium group at the end of the hydrocarbon chain have been reported from nature [11,12]. To the best of our knowledge, there are no reports of oxylipins having a dimethylsulfonium group in the middle of the hydrocarbon chain so far. This is the first finding of an oxylipin with a dimethylsulfonium moiety in the middle of the hydrocarbon chain from nature. The compound (1) was designated as okeanoate.

2. Results and Discussion

The cyanobacterium Okeania hirsuta, identified using 16S rRNA sequence analysis, was collected from Kuba Beach, Okinawa, Japan, in 2010. A frozen sample of O. hirsuta was extracted with methanol (MeOH). The compound (1) was purified using reversed-phase column chromatography. From 9.7 kg of algal cells, 1.1 mg of 1 was isolated.

Compound (1) was isolated as a colorless amorphous material with no UV absorption above 210 nm. The molecular formula of 1 was C₂₀H₃₆O₄S (m/z 373.2408 [M + H]⁺, calcd. for C₂₀H₃₇O₄S m/z 373.2407) via high-resolution electron spray ionization mass spectrometry (HR-ESI-MS), suggesting the presence of a sulfur atom in the molecule. ¹H, ¹³C NMR, and HSQC spectra revealed the presence of a triplet methyl group (δ_H 0.97) and two singlet methyls (δ_H 2.79 and 2.80), nine aliphatic methylenes (δ_C 39.0, 27.6, 30.5, 30.4, 30.6, 26.3, 38.1, 34.4, 21.9), three methines (δ_H 4.23, 4.12, 4.16), four olefin signals (δ_C 149.9, 115.7, 123.7, 136.3), and a carbonyl (δ_C 180.1) (

Table 1. ¹H (600 MHz) and ¹³C (150 MHz) NMR data of compound 1 in methanol-d₄.

Position	δC (ppm)	δH (ppm), Multiplicity (J in Hz)
1	180.1	–
2	39.0	2.14 t (7.4, 2H)
3	27.6	1.57–1.62 m (2H)
4	30.5	1.32–1.37 m (2H)
5	30.4	1.32–1.37 m (2H)
6	30.6	1.32–1.37 m (2H)
7	26.3	1.32–1.37 m (2H)
8	38.1	1.57–1.62 m (2H)
9	72.4	4.23 ddd (6.3, 6.1, 5.7, 1H)
10	149.9	6.13 dd (15.5, 5.5, 1H)
11	115.7	5.87 dd (15.5, 10.4, 1H)
12	64.7	4.12 dd (10.5, 2.6, 1H)
13	72.0	4.16 ddd (14.2, 7.0, 2.6, 1H)
14	34.3	2.34 dt (4.3, 6.6, 1H), 2.25 dt (14.7, 7.4, 1H)
15	123.7	5.31 dd (8.4, 7.5, 1H)
16	136.3	5.55 dd (8.6, 7.6, 1H)
17	21.9	2.04–2.08 m (2H)
18	14.5	0.97 t (7.5, 3H)
19	23.9	2.79 s (3H)
20	23.1	2.80 s (3H)

). Considered from MS and NMR analysis, the unsaturation of 1 was three, which accounted for a carbonyl and two double bonds. Thus, 1 was a C18:2 fatty acid derivative that contained a sulfonium group, two singlet methyls bonding a hetero atom, two hydroxy groups, and a carboxylate in the molecule. Similar to malyngic acid [5] and okeanic acid–A [10], 1 was deduced to be an ω-3 fatty acid derivative by ¹H-¹H COSY correlation, from H₃-18 (δ_H 0.97) to H-15 (δ_H 5.31). Another proton connectivity around the hydroxy groups and the double bonds H-15–H₂-14–H-13–H₂-12–H-11–H-10–H-9–H₂-8 was also determined based on the detailed analyses of ¹H-¹H COSY and TOCSY. The rest of the six methylenes were assigned into a linear alkyl chain from H₂-2 to H₂-7. An HMBC correlation from H₂-2 (δ_H 2.14) to C-1 (δ_C 180.1) was observed. Therefore, the skeletal structure of 1 was elucidated. The geometry of Δ¹⁰ was deduced to be E from ³J_H-10,H-11 (15.5 Hz), and the ¹³C chemical shift of C-17 at δ_C 21.7 suggested that the configuration of C-15=C-16 was Z [10]. Two singlet methyls observed at δ_H 2.80 and 2.79 and δ_C 23.1 and 23.9, respectively, indicated a dimethylsulfonium group in the molecule [11,12]. The ¹³C chemical shift (δ_C 64.7) at C-12 and the HMBC correlation from the singlet methyls to C-12 indicated that the dimethylsulfonium group resided on C-12 (Figure 2). The presence of two hydroxy groups was deduced from the molecular formula obtained from HR-MS results and the ¹H and ¹³C NMR chemical shifts of CH-9 (δ_H 4.23, δ_C 72.4) and CH-13 (δ_H 4.16, δ_C 72.0). The position of the hydroxy groups was deduced to be at C-9 (δ_C 72.4) and C-13 (δ_C 72.0). These MS and NMR analyses allowed us to determine that the structure of okeanoate (1) was (10E,15Z)-12-(dimethylsulfonio)-9,13-dihydroxyoctadeca-10,15-dienoate. Okeanoate (1) was unstable in methanol at 5 °C under dark condition. Within a week, more than half of the isolated 1 changed to other unknown compounds under these conditions. Therefore, biological activity tests could not be performed for 1.

3. Material and Methods

3.1. Collection of Cyanobacteria

The cyanobacterium Okeania hirsuta was collected at Kuba Beach, Nakagusuku, in Okinawa Prefecture, Japan, in 2010. O. hirsuta was the dominant cyanobacterial species in all samples. The identification of O. hirsuta was performed based on 16S rRNA sequence analysis [13]. A voucher specimen (20100713-a) was deposited at the Tokyo University of Marine Science and Technology.

3.2. Isolation and Structural Characterization of 1

The frozen O. hirsuta sample (wet weight: 9.7 kg) was extracted with MeOH thrice. After the elimination of MeOH under reduced pressure, the extracts were partitioned between ethyl acetate and H₂O. The H₂O layer was extracted with 1-butanol (BuOH). After the evaporation of 1-BuOH, the extract was fractionated by MPLC, using ODS gel (Cosmosil 75C18-OPN, Nacalai Tesque Inc., Kyoto, Japan) with stepwise elution with 50%, 70%, 90%, and 100% MeOH [size of the column, 20 × 300 mm; each eluant volume, 200 mL]. The 70% MeOH eluate was subjected to an ODS column (Cosmosil 5C18-AR-II, 10 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) with 70% MeOH for 10 min, then a gradient elution from 70% to 100% MeOH for 20 min [flow rate, 3.0 mL/min; detection at 210 nm]. The fraction eluted from 5 to 8 min was combined. Final purification was conducted via HPLC using the reversed-phase column (Cosmosil 5C18-AR-II, 4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) with gradient elution from 45% to 50% MeOH for 0–5 min, then 50% MeOH for 5–15 min [flow rate, 1.0 mL/min; detection at 210 nm]. Okeanoate (1, 1.1 mg) was isolated at a retention time of 7.0 min.

HR ESI-MS was measured with an micrOTOF-QII (Bruker, MA, USA) Quadrupole time-of-flight mass spectrometer (QTOFMS) with high-performance liquid chromatography of the UltiMate 3000 Series (Thermo Fisher Scientific, Billerica, MA, USA). NMR spectra were measured in methanol-d₄ at 300 K using a Bruker AVANCE III 600 spectrometer (Bruker Biospin AG, Fällanden, Switzerland).

4. Conclusions

Okeanoate (1), a novel oxylipin, was isolated from the cyanobacterium Okeania hirsuta collected in Okinawa, Japan. In this study, the stereochemistry of the two double bonds could be determined, but only the planar structure could be determined for the other three chiral carbon atoms. To the best of our knowledge, okeanoate (1) is the first oxylipin with a dimethylsulfonium moiety in the middle of the hydrocarbon chain from nature. The biosynthetic pathway to this chemically unique compound is of interest.