New Glycerides from a Two-Sponge Association of Jaspis sp. and Poecillastra sp.

Abstract

A chemical investigation of the MeOH extract of a two-sponge association (Jaspis sp. and Poecillastra sp.), collected from Jeju Island, Korea, led to the isolation of two new lysophosphatidylglycerols (1 and 2) along with known lysophosphatidylglycerol (3) and glycolipids (4 and 5). Their structures were elucidated on the basis of NMR and MS spectroscopic data.

Keywords:

• Glycolipids
• Jaspis sp.
• lysophosphatidylglycerols
• Poecillastra sp.

Introduction

Sponges of the genus Jaspis are reported to contain many biologically active molecules, which include macrolides [jaspisamides (Kobayashi et al., 1993)], cyclic peptides [jaspamides (Gala et al., 2007) or jasplakinolide (Crews et al., 1986)], amino acid derivatives [bengamides (Thale et al., 2001) and bengazoles (Groweiss et al., 1999)], and triterpenes (Tang et al., 2006). Similarly, macrolide lactams [poecillastrins (Takada et al., 2007)] and sesquiterpene derivatives (Killday et al., 1993) have been isolated from sponges of the genus Poecillastra. Two-sponge association of Jaspis sp. and Poecillastra sp. was known to contain bisimidazoles [wondonins (Shin et al., 2001)].

As a part of our continuing research aimed at the discovery of biologically active secondary metabolites from marine organisms (Mansoor et al., 2007a,b; Shinde et al., 2007), we had isolated pectenotoxin II and psammaplins from the 90% MeOH extract of a two-sponge association of Jaspis sp. and Poecillastra sp. by bioactivity-guided fractionation (Jung et al., 1995; Park et al., 2003). In a continuing study, two new (1 and 2) and one known (3) lysophosphatidylglycerols and two known glycolipids (4 and 5) were isolated from the MeOH extract. In this paper, we describe the isolation and structural elucidation of 1– 5 (Fig. 1).

Figure 1.  Structures of compounds 1–7.

Materials and Methods

General experimental methods

Optical rotations were measured with a Jasco (Japan) P-1020 polarimeter using a 1-dm path length cell. The¹H and 2D NMR spectra were recorded at 500 MHz using a Varian INOVA (USA) 500 spectrometer. The¹³C NMR spectra were recorded at 125 MHz and 75 MHz using Varian INOVA 500 and Varian UNITY 300 spectrometers. Fast Atom Bombardment Mass Spectrometry (FABMS) data were obtained on a JEOL (USA) JMS SX-102A spectrometer. High Resolution Fast Atom Bombardment Mass Spectrometry (HRFABMS) data were obtained on a JEOL JMS SX-101A spectrometer. Chemical shifts were reported with reference to the respective solvent peaks and residual solvent peaks (δ_H 3.30 and δ_C 49.0 for CD₃OD). HPLC was performed on a Gilson (USA) 370 pump with a Shodex C18M 10E (preparative, 250 × 10 mm, i.d., 5 μm, and 100 Å) column using Shodex RI-71 and RI-101 detectors.

Animal material

The sponges, collected in November 2002 off the Coast of Jeju Island, South Korea, using SCUBA, were frozen immediately after collection and stored at −20°C until extraction. This specimen was identified as an association of sponges Jaspis sp. (Jaspidae) and Poecillastra sp. (Pachastrellidae) by Prof. Chung Ja Sim of Hannam University. A voucher specimen was deposited in the Natural History Museum, Hannam University, Daejon.

Extraction and isolation

The freeze-dried animal material was cut into small pieces and extensively extracted with MeOH at room temperature. The MeOH extract showed toxicity against brine shrimp larvae (LD₅₀ 43 μ g/mL). The MeOH extract was partitioned between CH₂Cl₂ and H₂O. The CH₂Cl₂ layer was further partitioned between 90% MeOH and n-hexane. The aqueous MeOH fraction was selected for further separation on the basis of its toxicity (LD₅₀ 16 μ g/mL) in brine shrimp lethality assay (Meyer et al., 1982) and subjected to a reversed-phase flash column chromatography (YMC Gel ODS-A, 60 Å 500/400 mesh), eluting with a gradient solvent system of 50% to 100% MeOH to yield 19 fractions (DCM1–DCM19). Fractions DCM11 to DCM16 were subjected to repeated reversed-phase chromatographic separation to afford 10 compounds. Compound 1 was obtained from fraction DCM13 and compound 2 was isolated from fraction DCM14. Compounds 3 and 4 were obtained from fraction DCM11, and fraction DCM16 yielded compound 5.

Methoxy-trifluoromethyl-phenyl-acetate (MTPA) ester of compound 3: Each 0.3 mg of compound 3 (0.61 μ mol) was dissolved in CDCl₃ (300 μL), followed by treatment with (R)-(−)- and (S)-(+)-α -methoxy-α -(trifluoromethyl)phenylacetyl chloride (0.46 μL) in pyridine-d₅ (10 μL) to yield (S)-MTPA ester (6) and (R)-MTPA ester (7), respectively. After being kept at room temperature for 24 h, the reaction mixtures were evaporated and NMR spectra measured.

1-O-Heptadecanoyl-sn-glycero-3-phosphoglycerate (1): Light yellow oil; [α]²³_D −5.4° (c 0.10, MeOH); ¹H NMR (CD₃OD, 500 MHz) δ 4.16 (1H, dd, J = 12.0, 5.0 Hz, H-1a), 4.09 (1H, dd, J = 12.0, 5.0 Hz, H-1b), 3.96 (1H, m, H-2), 3.88 (4H, m, H-3, H-1′), 3.75 (1H, m, H-2′), 3.59 (1H, dd, J = 12.0, 5.5 Hz, H-3′a), 3.54 (1H, dd, J = 12.0, 5.5 Hz, H-3′b), 2.34 (2H, t, J = 7.5 Hz, H-2″), 1.60 (2H, m, H-3″), 1.25–1.31 (26H, br s, H-4″–H-16″), 0.89 (3H, t, J = 7.0 Hz, H-17″); ¹³C NMR (CD₃OD, based on HSQC and HMBC experiments, 500 MHz) δ 174.0 (C-1″), 71.3 (C-2′), 68.7 (C-2), 66.3 (C-3), 66.3 (C-1′), 65.0 (C-1), 62.6 (C-3′), 33.6 (C-2″), 29.4–32.8 (C-4″–C-16″), 24.6 (C-3″), 12.8 (C-17″); LRFABMS m/z 497 [M – H]⁻; HRFABMS m/z 497.2865 [M – H]⁻ (calcd for C₂₃H₄₆O₉P, 497.2879).

1-O-(15-M e t h y l h e x a decanoyl)-sn-glycero-3-phospho-glycerate (2): Light yellow oil; [α]²²_D –0.88° (c 0.09, MeOH); ¹H NMR (CD₃OD, 500 MHz) δ 4.16 (1H, dd, J = 12.0, 5.0 Hz, H-1a), 4.09 (1H, dd, J = 12.0, 5.0 Hz, H-1b), 3.96 (1H, m, H-2), 3.88 (4H, m, H-3, H-1′), 3.75 (1H, m, H-2′), 3.59 (1H, dd, J = 12.0, 5.5 Hz, H-3′a), 3.54 (1H, dd, J = 12.0, 5.5 Hz, H-3′b), 2.34 (2H, t, J = 7.5 Hz, H-2″), 1.60 (2H, m, H-3″), 1.52 (1H, m, H-15″), 1.25–1.31 (20H, br s, H-4″–H-13″), 1.16 (2H, m, H-14″), 0.86 (6H, d, J = 6.5 Hz, H-16″, H-17″); ¹³C NMR (CD₃OD, based on Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear Multiple Bond Correction (HMBC) experiments, 500 MHz) δ 174.0 (C-1″), 71.3 (C-2′), 68.7 (C-2), 66.3 (C-3), 66.3 (C-1′), 65.0 (C-1), 62.6 (C-3′), 38.0 (C-14″), 33.6 (C-2″), 29.4–32.8 (C-4″–C-13″), 28.8 (C-15″), 24.6 (C-3″), 19.9 (C-16″, 17″); Low Resolution Fast Atom Bombardment Mass Spectrometry (LRFABMS) (− ve mode) m/z 497 [M – H]⁻, (+ ve mode) m/z 521 [M + Na]⁺, m/z 543 [M + 2Na − H]⁺;HRFABMS m/z 543.2656 [M + 2Na − H]⁺(calcd. for C₂₃H₄₆O₉PNa₂, 543.2675).

Results and Discussion

The MeOH crude extract of a two-sponge association of Jaspis sp. and Poecillastra sp. was subjected to activity-guided fractionation using solvent partition and reversed-phase flash column chromatography followed by repeated reversed-phase HPLC to yield compounds 1– 5. The structures of these metabolites were deduced using NMR (¹H, ¹³C, COSY, HSQC, and HMBC) and MS analysis, optical rotation data, and by direct comparison with those reported.

Compound 1 was isolated as light yellow oil. Its molecular formula was established as C₂₃H₄₇O₉P on the basis of HRFABMS and NMR data. The exact mass of the [M – H]⁻ ion (m/z 497.2865) matched well with the expected molecular formula C₂₃H₄₆O₉P (Δ − 1.4 mmu). The ¹H NMR spectrum of 1 displayed signals typical for glycerol moieties, which included multiplets at δ 3.96 (H-2), 3.75 (H-2′), and 3.88 (H₂-3, H₂-1′), and doublets of doublet at δ 4.16 (H-1a), 4.09 (H-1b), 3.59 (H-3′a), and 3.54 (H-3′b). The presence of two glycerol moieties was also confirmed from the analysis of COSY spectrum. A large peak at δ 1.28–1.40 indicated the presence of linear aliphatic hydrocarbon chain that was coupled with methyl signal at δ 0.89 (t, J = 7.0 Hz, H₃-17″) in the COSY spectrum. The ¹³C NMR data of six oxygenated carbons at δ71.3 (C-2′), 68.7 (C-2), 66.3 (C-3), 66.3 (C-1′), 65.0 (C-1), 62.6 (C-3′) in conjunction with one ester carbonyl group at δ 174.0 (C-1″), aliphatic alkyl chain at δ 24.6–33.6 (C-2″–C-16″), and one methyl carbon at δ 12.8 (C-17″) supported the above assignments. Furthermore, HMBC correlations from H-1a (δ 4.16) and H-1b (δ 4.09) to the carbonyl carbon at C-1″ (δ 174.0) confirmed the ester linkage in the molecule. The presence of phosphate group was secured from the careful comparison of obtained NMR data with those reported (Tamehiro et al., 2002). The absolute configuration at C-2 was presumed to be R (vide infra). Thus, the chemical structure of compound 1 was established as 1-O-heptadecanoyl-sn-glycero-3-phosphoglycerate. To our knowledge, this is the first report on isolation of lysophosphatidylglycerol containing heptadecanoic acid moiety.

Compound 2 was isolated as light yellow oil. The HRFABMS of 2 supported the molecular formula C₂₃H₄₇O₉P, the same as 1. The exact mass of the [M + 2Na − H]⁺ ion (m/z 543.2656) matched well with the expected molecular formula C₂₃H₄₆O₉PNa₂ (Δ − 1.9 mmu). The negative and positive FABMS spectra of 2 showed the [M – H]⁻, [M + Na]⁺ and [M + 2Na − H]⁺ ions at m/z 497, 521, and 543, respectively. ¹H and ¹³C NMR data of 2 were almost identical to those of 1, with only difference in signals of the fatty acid residue. The ¹H NMR spectrum of 2 exhibited signals for an isopropyl group (Mansoor et al., 2005), indicated by a two-methyl doublet at δ 0.86 (J = 6.5 Hz, H₃-16″, H₃-17″), which in turn showed coupling with a methine signal at δ 1.52 (H-15″), which was coupled with methylene protons at δ 1.16 (H₂-14″) in the COSY spectrum. The absolute configuration at C-2 was presumed to be R (vide infra). Hence, compound 2 was deduced to be 1-O-(15-methylhexadecanoyl)-sn-glycero- 3-phospho-glycerate. Compound 2 represents the first example of lysophosphatidylglycerol containing terminal isopropyl group.

Known metabolites, lysophosphatidylglycerol (3), α-quinovosylglycerol (4), and β-galactosylglycerol (5), were also isolated in the current study. Their structures were identified by comparison of NMR and MS data with those reported (Rho et al., 1996; Sakamoto et al., 2000; Yu et al., 2006).

The absolute stereochemistry at C-2 in compound 3 was determined as R by Mosher′s method (Dale & Mosher, 1973). The (S)- and (R)-MTPA esters (6 and 7) were prepared and Δ δ (δ_S−δ_R) values for all assignable protons were observed (Fig. 2). However, the absolute configuration at C-2′ could not be defined due to hidden signals of H₂-3′ with MTPA signals. As the chemical shift values of 1 and 2 are similar with that of compound 3, and from the same sign (−ve) of optical rotation, the same stereochemistry (2R) can be assumed for compounds 1 and 2.

Figure 2.  Δδ(δ_S−δ_R) values for (S)- and (R)-MTPA esters of compound 3.

Lysophosphatidylglycerols may be biosynthesized from phosphatidylglycerol through acyl ester hydrolysis as last step (Fig. 3) (De et al., 1993; Tamehiro et al., 2002). Glycerol 3-phosphate upon esterification with the first fatty acyl-CoA gives 1-acylglycerol 3-phosphate, which undergoes esterification with the second fatty acyl-CoA to form phosphatidic acid (1,2-diacylglycerol 3-phosphate). This phosphatidic acid will be converted into phosphatidylphosphoglycerate on addition of a glycerol 3-phosphate molecule, which forms phosphatidylglycerol upon removal of a phosphate molecule. Enzyme lysophospholipase carries out hydrolysis of phosphatidylglycerol to yield lysophosphatidylglycerol (De et al., 1993; Tamehiro et al., 2002).

Figure 3.  Plausible biosynthetic pathway for lysophosphatidylglycerols (1– 3).

Bacilysocin, a novel lysophosphatidylglycerol, isolated from Gram-positive bacteria (Bacillus subtilis), is reported to possess antimicrobial activity against bacteria and certain fungi (Tamehiro et al., 2002). One recent study showed that lysophosphatidylglycerols inhibit β -glucuronidase and can be used for prevention of colon and bladder cancers and also as drugs (oral or topical), health foods, and cosmetics for inhibiting unpleasant body odor (Shiojiri, 2007). Small amounts of the isolated metabolites (1– 3) prevented us from carrying out any biological evaluation.

Phosphatidylglycerols are usually major components of phospholipids in microorganisms. Lysophosphatidylglycerols are common components of bacterial membrane and, as we know, there is a close relationship between sponges and bacteria; the isolation of these types of compounds from the methanol extract of a two-sponge association of Jaspis sp. and Poecillastra sp. is not surprising and might be the result of presence of bacteria in sponge bodies. Therefore, it would be interesting to study metabolites of the bacteria associated with these sponges to determine the real producer of interesting metabolites such as macrolides, peptides, and psammaplins that were previously reported from these sponges.

Acknowledgements

This study was financially supported by a grant from Marine Bio 21, Ministry of Maritime Affairs and Fisheries, Korea. Our thanks are due to Prof. Chung Ja Sim of Hannam University for the identification of the sponge material.