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Styela plicata (pleated sea squirt, Omandungi in Korean language) is a species of sea squirts (Ascidian), having ovular, greyish to tannish white solitary benthic tunicate and distribute from subtropics to temperate seawaters (http://en.wikipedia.org/wiki/Sea_pineapple). It has been culturing together other two species such as Halocynthia roretzi and Styela clava in the southern coast of Korea as a resource of food material and produced about 6,400 tons in 2017(https://www.mof.go.kr, 2018). They are using as a major ingredient with another tunicates for some cooked seafood due to their peculiar flavor and taste.

Many antimicrobial sulfated alkane and alkenes have been isolated from the ascidians. C-11 alkene (4,8-dimethyl nonenyl sulfate) was found from the cultured S. clava(Yun et al., 2007). And, C-9 alkane (2,6-dimethylheptyl sodium sulfate) and sulfated C-10 alkenes (4,7-decadienyl sulfate and 3,6,9-decatrienyl sulfate) were isolated as the antimicrobial active compounds from the hepatopancreas of the Japanese tunicate (ascidian), H. roretzi and H. aurantium (Tsukamoto et al., 1994). They presumed those similar compounds had been contained in the S.plicata, but, not confirmed. 2,6-dimethylheptyl sulfate, was also found in the ascidian, Policitor adriaticus, collected at Croatia (De Rosa et al., 1997) and from the Mediterranean ascidian, H. papillosa(Aiello et al., 2000). 6-methylheptyl sulfate and 5-octenyl sulfate were also found in the same P. adriaticus with cytotoxic activity in vitro(De Rosa et al., 1997).

Differently from alklyl or alkenyl sulfates, many peptides were reported from tunicates (Tincu et al., 2004). Plicatamide was found as anti microbial octapeptide from the S. plicata(Tincu et al., 2003) and halocyamines having bromo-tetrapeptides from H. roretzi(Azumi et al., 1990), dicynchaurin and halocidin from H. aurantium(Lee et al., 2001a), clavanins, clavaspirin and styelins from H. styela(Lee et al., 1997, Lee et al., 2001b).

In the course of our studies evaluating potential bio-active materials in the cultured pleated sea squirt (S. plicata), we found that those methanol extract showed antibacterial activity against Bacillus subtilis. We isolated a known polar antibacterial sulfated alkane (1) and new two unknown antibacterial compounds. In this paper, we described the isolation and structural determination of those compounds as sulfated alkenes (2, 3).

Compound 1 was obtained amorphous colorless powder and the structure was elucidated by comparing ¹H, ¹³C and MS spectral data with known 2,6-dimethylheptyl sodium sulfate (Tsukamoto et. al., 1994). Negative FAB-MS of compound 1 exhibited an [M - Na]^-ion peak at m/z 223, corresponding to a molecular formula C₉H₁₉O₄S. The ¹H NMR spectrum showed three doublet methyl signals at δ = 0.73 ppm (3H, d, J= 6.4 Hz, CH₃-2), δ = 0.74 ppm (6H, d, J= 6.4 Hz, CH₃-7 and CH₃-9), two methylene protons of CH₂-1 bearing as sulfate group appeared at δ = 3.45 and δ = 3.55 ppm, a methine proton of CH-2 at 1.62 ppm, CH-6 at δ = 1.55 ppm, and six methylene protons at δ = 1.01 ~ 1.29 ppm <Table 1>. The connectivity of protons and neighboring carbon atoms on the Heteronuclear Multiple Bond Correlation (HMBC) spectrum for the compound 1 led us the known structure, 2,6-dimethylheptyl sodium sulfate. Indubitably, all of those assignments were well coincided with those of 2,6-dimethyl heptyl sodium sulfate reported by Tsukamoto et al.(1994).

Compound 2 was isolated amorphous pale yellowish powder with ascidian smell.

Negative FAB-MS showed [M - Na]^- ion at m/z 235 [Fig. 1], and positive FAB-MS gave an [M + Na]⁺m/z 281, HRFAB-MS m/z [M - Na]^-m/z 235.0891 (obsd. -11.3 mmu) corresponding to the molecular formula C₁₀H₁₉O₄S.

[Fig. 1] 
FAB-MS(negative mode) spectrum of compound 2.

The structure of compound 2 was elucidated by ¹H, ¹³C, 2D NMR spectra such as HMBC, Heteronuclear Multiple-Quantum Correlation (HMQC), Nuclear Overhauser Spectroscopy (NOESY) spectral data and comparing the structure obtained from the 5-octenyl sulfate (Aiello et al., 2000). On the ¹H NMR spectrum, a terminal methyl proton observed at δ = 0.84 ppm (3H, t, J = 6.2Hz, CH₃-10), two methylene proton signals neighboring a sulfate group (CH₂-1) at δ = 3.65 ppm, four methylene protons connected methane (CH₂-3 and CH₂-4) at δ = 1.98 ppm (2H, q, J = 6.2 Hz CH₂-5) and δ = 2.22 ppm (2H, q, J = 8.2 Hz, CH₂-2). Four methylene protons(CH₂-6, CH₂-7, CH₂-8 and CH₂-9) gave overlapped signals at δ = 1.25 ppm and two methine protons of CH-3 and CH-4 were observed at δ = 5.31 ppm and δ = 5.39 ppm, respectively <Table 1>. Those assignment of proton signals with corresponding carbon signals and correlations between each proton and carbon on the HMBC spectrum [Fig. 2, 5] allowed us to establish compound 2 as 3-decenyl sulfate [Fig. 6].

[Fig. 2] 
HMBC spectrum of antibacterial compound 2 (400 MHz, CD₃OD).

The Z configuration of the double bond was assigned on the basis of the value of the H-3/H-4 coupling constant (12 Hz) and of the ¹³C NMR chemical shifts of the allylic methylenes CH₂-2 and CH₂-5 and compared with those of 5-octenyl sulfate.

Compound 3 was also gained as amorphous pale yellow powder. EI-MS of compound 3 exhibited molecular ion peak at m/z 256 [Fig. 3]. The molecular weight of compound 3 was same as known 4,7-decadienyl sulfate (Tsukamoto et al., 1994).

[Fig. 3] 
EI-MS spectrum of antibacterial compound 3.

[Fig. 4] 
¹H- ¹H COSY spectrum of antibacterial compound 3 (400 MHz, CD₃OD).

All the proton and carbon signals of compound 3 on the NMR spectra were strictly compared with those of 4,7-decadienyl sulfate. ¹H-NMR (400 MHz, CD₃OD) revealed a methyl group at δ = 1.00 ppm (3H, t, J= 6.0 Hz, CH₃-10), 4 methine protons at δ 5.35 ppm and 5.46 ppm, two methylene protons connected sulfate group at δ = 4.01 ppm (2H, t, J=8.0 Hz, CH₂-1) and 8 methylene protons such as δ = 2.36 ppm (2H, m, CH₂-2),δ 2.48 (2H, m CH₂-5), δ = 2.11 ppm (2H, m, CH₂-8), δ = 1.33 ppm (2H, m, CH-9) as <Table 1>. ¹H-¹H COSY spectrum of compound 3 [Fig. 5] showed those assignments were suitable to the structure as 3,6-decadienyl sulfate [Fig. 6], differently the position of double bond at C-4 and C-7 at 4,7-decadienyl sulfate.

[Fig. 5] 
HMBC correlations of antibacterial compound 2 and ¹H-¹H COSY correlations of 3.

[Fig. 6] 
Structures of antibacterial compound 1, 2 and 3 isolated from S. plicata.

The stereochemistry of both double bonds in compound 3 assumed Z configurations due to those upfield shifted chemical shifts of allylic methylenes observed on the ¹³C NMR spectrum (Ciminiello et al., 1991, Gunstone, 1993), similarly to the two double bonds of 4,7-decadienyl sulfate (Tsukamoto et al.,1994).

During the isolation procedure, the antimicrobial placatamide (Tincu et al., 2003) was not detected from our species. MIC of purified 1, 2, and 3 against to the Gram positive B. subtilis showed 200 μg/mL. However, in the same concentration, they exhibited very weak antimicrobial activities against to the gram negative Escherichia coli, pathogenic yeast, Candida albicans and the mold, Epidermophyton floccosum. They showed different antimicrobial activity depends on the bacterial strain, Some sulfated alkenes have cytotoxicity (Aiello et al., 2000, Rosa et al., 1997) or ability to inhibit the matrix metalloproteinase 2 (MMP2, Fujita et al., 2002). Therefore, sulfated alkane and alkene have significant value for drug development. Details about in vitro antimicrobial activity to pathogenic bacteria, yeasts, fungi and another biological activities such as anti-oxidant and antitumor including physico-chemical properties will be published elsewhere.

Many alkyl sulfates having similar structure are using important material for surfactant. Sodium Lauryl Sulfate (SLS), as one of the Linear Alkyl Sulfate (LAS), made by sulfatation of lauryl alcohol and neutralization with sodium hydroxide, industrially. SLS is an lower toxic anionic surfactants used widely in cosmetics as cleansing agents (http://ijt.sagepub.com/content/2/7/127) and also, effective as microbicide against enveloped and non enveloped viruses (Piret et al., 2002) and HIV (Piret et al., 2000).

Some kind of alkyl sulfate sulfactants such as sodium octyl sulfate (SOS), sodium decyl sulfate (SDecS) and sodium dodecyl sulfate (SDS), were known as effective to the chemical shark repellent (Joseph and Nelson, 2001).

After 7-decene-1-ol was firstly reported from the unsafonifiable fraction of the H. roretzi (Kita, 1957), this unique flavor of ascidian were known that it was originated from the presence of many C-8 to C-10 saturated and unsaturated alcohols and named 2,7 decadienol as cynthiaol by Suzuki (1960) and the formation of peculiar ascidian flavors by octanol was reported(Fujimoto et al., 1982a). Furthermore, Fujimoto et al., (1982b) reported as those precursors of peculiar ascidian flavor in H. roretzi was C-8 to C-10 alkyl sulfates because they gave C-8 to C-10 alcohols by hydrolysis. From these fact, those alkyl sulfates may convert to those alcohols by some enzyme such as sulfatase in the body of ascidian.

From these results, sodium alkyl sulfates contained in ascidian show not only the role of peculiar flavor but also have other biological activities. It is need further study on the Industrial and medicinal utilization of those alkyl sulfates.