Two New Sterol Sulfates from Marine Spider Conch Lambis Lambis Linnaeus,1758

Abstract

Two new (1 and 2) and six known (3-8) compounds were isolated from the methanolic extract of the Vietnamese marine spider conch Lambis lambis Linnaeus, 1758. Based on comprehensive spectroscopic analyses, particularly HRESIMS, and 1D- and 2D-NMR spectra, their chemical structures were determined to be 25,26,27-tri-nor-cholest-5-ene-2β,3α,24-triol-2,24-disulfate (1), 25,26,27-tri-nor-cholesta-2β,3α,24-triol-3,24-disulfate (2), 25,26,27-tri-nor-cholesta-2β,3α,24-triol-2,3,24-trisulfate (solomonsterol A, 3), 1-O-arachidonoyl-3-O-stearylglycerol (4), eicosapentaenoic acid (5), adenosine (6), adenine (7), and uracil (8). This is the first report of compounds 3-8 from L. lambis.

Introduction

Lambis Linnaeus, 1758, commonly known as the spider conch, is a species of large sea snail, a marine gastropod mollusk in the family Strombidae. ¹ The meat of L. lambis provides many nutrients, such as minerals (macro minerals, Na, K, Ca, P; micro minerals, Fe, ZN, Cu), protein, and vitamins. ² Fucoidanase was isolated from the digestive glands of Lambis sp. producing sulfated fucooligosaccharides. ³ The mucus extract of L. lambis exhibited cytotoxic activity. ⁴ Continuing our program of study on Vietnamese marine organisms,^5-8 we report in this paper the isolation and structural elucidation of two new sterol sulfates (1 and 2) and six known compounds (3-8) from the methanol extract of the sea spider conch L. lambis.

Results and Discussion

Compound 1 was obtained as a white amorphous solid with a molecular formula of C₂₄H₃₈O₉S₂Na_2, according to its HRESIMS ion at m/z 581.1844 [M + H]⁺ (calcd. for [C₂₄H₃₉O₉S₂Na₂]⁺: 581.1825, Δ =  + 3.3 ppm) and at m/z 603.1637 [M + Na]⁺ (calcd. for [C₂₄H₃₈O₉S₂Na₃]⁺: 603.1645, Δ = −1.3 ppm). The ¹H NMR spectrum of 1 showed the presence of two methyl singlets at δ_H 0.75 and 1.19 (each 3H), one methyl doublet at δ_H 0.99 (3H, d, J = 7.0 Hz), and one olefinic proton at δ_H 5.37 (1H, dd, J = 2.5, 2.5 Hz), along with two methine carbinol groups at δ_H 4.04 and 4.46 (each, 1H, br d, J = 2.5 Hz) and one oxygenated methylene group at δ_H 3.98 (2H, td, J = 7.0, 2.5 Hz). The ¹³C NMR and HSQC spectra of 1 showed the presence of 24 carbons, including three quaternary carbons (δ_C 37.8, 43.5, 139.6), nine methylenes (δ_C 21.9, 25.3, 27.2, 29.2, 32.9, 33.2, 35.8, 39.0, 41.2), eight methines (δ_C 32.8, 36.9, 51.8, 57.5, 58.1, 69.6, 78.3, 123.5), and three methyls (δ_C 12.1, 19.1, 22.2) (Table 1). The above information suggested that 1 was a sulfated sterol compound having one double bond, three oxygenated carbons, and which was structurally similar to solomonsterol A (3) (Figure 1).^9,10 In the HSQC spectrum of 1, methyl protons at δ_H 0.75, 0.99, and 1.19 had cross peaks to carbons at δ_C 12.1, 19.1, and 22.2, respectively, while protons at δ_H 3.98, 4.40, 4.46, and 5.37 correlated with carbons at δ_C 69.7, 69.6, 78.3, and 123.5, respectively. In the ¹H-¹H COSY spectrum of 1, correlations of H-1/H-2/H-3/H-4, H-7/H-8/H-9/H-11/H-12, H-8/H-14/H-15/H-16/H-17/H-20/H-21, and of H-20/H-22/H-23/H-24 (Figure 2) were observed. This evidence suggested that the A, B, C, and D rings of 1 were similar to that of (20R)-cholesta-5,24-diene-2β,3α,21-triol-2,21-disulfate, ¹¹ and the side chain was similar to that of solomonsterol.^9,10 These suggestions were confirmed by the HMBC spectrum. HMBC correlations were observed from H₃-19 (δ_H 1.19) to C-1 (δ_C 39.0)/C-5 (δ_C 139.6)/C-9 (δ_C 51.8)/C-10 (δ_C 37.8), from H₃-18 (δ_H 0.75) to C-12 (δ_C 41.2)/C-13 (δ_C 43.5)/C-14 (δ_C 58.1)/C-17 (δ_C 57.5), from H₃-21 (δ_H 0.99) to C-17/C-20 (δ_C 36.9)/C-22 (δ_C 33.2), and from H₂-24 (δ_H 3.98) to C-22 (δ_C 33.2). In addition, the lower field signals of C-2 (δ_C 78.3) and C-24 (δ_C 69.7), together with the complete match of the NMR data of the A and B rings of 1 with those of (20R)-cholesta-5,24-diene-2β,3α,21-triol 2,21-disulfate, ¹¹ and of the side chain of 1 with that of solomonsterol A (3)^9,10 suggested two sulfate groups were at C-2 and C-24, and the hydroxy group was at C-3. These were further confirmed by the interpretation of ¹H NMR, ¹³C NMR, ¹H-¹H COSY, HSQC, HMBC, and NOESY spectra. The small proton coupling constants of H-3 and H₂-4 (J = 2.0∼2.5 Hz), H-2 and H₂-1 (J = 2.0∼2.5 Hz), and of H-2 and H-3 (J = 2.5 Hz) confirmed that H-3 and H-2 were β/equatorial and α/equatorial, respectively. This were further indicated from the observation of the NOE correlation of H-2 (δ_H 4.46) and H-3 (δ_H 4.04). The other key NOE correlations of 1 are shown in Figure 3 indicating that its relative configuration was similar to that of (20R)-cholesta-5,24-diene-2β,3α,21-triol 2,21-disulfate. ¹¹ The CH₃-20 group was suggested as having an α-orientation due to the biosynthetic characteristics of all sterols. ¹² From the above evidence, compound 1 was determine to be a new compound, 25,26,27-tri-nor-cholest-5-ene-2β,3α,24-triol-2,24-disulfate.

Figure 1.

Chemical structures of compounds 1-8.

Figure 2.

Key ¹H-¹H COSY and HMBC correlations of 1 and 2.

Figure 3.

Key NOE correlations of 1 and 2.

Table 1.

NMR Spectroscopic Data for 1–3 (in CD₃OD).

Pos.	1		2		3
	δ C	δH (mult, J in Hz)	δ C	δH (mult, J in Hz)	δ C	δH (mult, J in Hz)
1	39.0	1.60 (dd, 14.0, 2.5) 2.14 (dd, 14.0, 2.0)	41.1	1.37 (14.0, 2.5) 1.77 (14.0, 2.0)	39.3	1.44 (m), 2.09 (m)
2	78.3	4.46 (br d, 2.5)	70.0	4.09 (br d, 2.5)	76.6	4.72 (br s)
3	69.6	4.04 (br d, 2.5)	78.5	4.42 (br d, 2.5)	76.2	4.75 (br s)
4	35.8	2.82 (dt, 14.5, 2.5) 1.97 (dd, 14.5, 2.0)	30.3	1.60 (m), 1.84 (m)	30.8	1.61 (m), 1.28 (m)
5	139.6	-	40.6	1.06 (m)	40.2	1.59 (m)
6	123.5	5.37 (dd, 2.5, 2.5)	29.2	1.25 (m), 1.30 (m)	29.5	1.32 (m), 1.91 (m)
7	32.9	1.62 (m), 1.97 (m)	33.2	0.95 (m), 1.70 (m)	33.2	1.12 (m), 1.72 (m)
8	32.8	1.50 (m)	36.4	1.41 (m)	36.3	1.44 (m)
9	51.8	1.06 (m)	56.6	0.72 (m)	56.5	0.73 (m)
10	37.8	-	36.3	-	36.3	-
11	21.9	1.21 (m), 1.54 (m)	22.0	1.33 (m), 1.55 (m)	22.0	1.47 (m), 1.45 (m)
12	41.2	1.20 (m), 2.06 (m)	41.4	1.16 (m), 2.02 (m)	41.4	1.18 (m), 2.02 (m)
13	43.5	-	43.8	-	43.8	-
14	58.1	1.06 (m)	57.9	1.07 (m)	57.8	1.08 (m)
15	25.3	1.10 (m), 1.62 (m)	25.2	1.29 (m), 1.60 (m)	25.2	1.09 (m), 1.51 (m)
16	29.2	1.32 (m), 1.90 (m)	29.3	1.33 (m), 1.88 (m)	29.2	1.32 (m), 1.91 (m)
17	57.5	1.13 (m)	57.6	1.12 (m)	57.6	1.14 (m)
18	12.1	0.75 (s)	12.5	0.71 (s)	12.5	0.71 (s)
19	22.2	1.19 (s)	14.6	1.02 (s)	14.1	0.99 (s)
20	36.9	1.47 (m)	36.8	1.46 (m)	36.8	1.44 (m)
21	19.1	0.99 (s)	19.1	0.97 (s)	19.6	0.97 (d, 6.5)
22	33.2	1.13 (m), 1.56 (m)	33.1	1.12 (m), 1.55 (m)	33.1	0.99 (m), 1.54 (m)
23	27.2	1.55 (m), 1.76 (m)	27.2	1.57 (m), 1.77 (m)	27.2	1.09 (m), 1.51 (m)
24	69.7	3.98 (t, 7.0)	69.7	3.97 (td, 7.0, 2.5)	69.7	3.97 (m)

The NMR spectra of 2 were similar to those of 1 and 3 suggesting that 2 was a sterol. The double bond signals of 1 were absent from the NMR spectra of 2. In addition, the molecular formula of 2 was determine as C₂₄H₄₀O₉S₂Na₂ from the exhibition of ions at m/z 583.1977 [M + H]⁺ (calcd C₂₄H₄₁O9S₂Na₂ = 583.1982 Δ = −0.9 ppm) and m/z 605.1808 [M + Na]⁺ (calcd C₂₄H₄₀O9S₂Na₃ = 605.1801 Δ =  + 1.2 ppm). The above data suggested that 2 was a disulfate sterol, similar to compounds 1 and 3.^9,10 The NMR data of 2 were elucidated based on analyzing its ¹H, ¹³C NMR, HSQC, ¹H-¹H COSY, HMBC, and NOESY spectra in comparison with the corresponding data of 1 and solomonsterol A (3), measured in the same solvent (Table 1).^9,10 In the HSQC spectrum, protons H-1 (δ_H 1.16/2.02), H-2 (δ_H 4.09), H-3 (δ_H 4.42), H-4 (δ_H 1.60/1.84) and H-5 (δ_H 1.60) correlated to carbons at δ_C 41.4, 70.0, 78.5, 30.3, and 40.6, respectively. In addition, COSY correlations of H-1/H-2/H-3/H-5/H-5 were observed. This evidence indicated two oxygenated carbons were at C-2 and C-3. The complete agreement of the NMR data of the A, B, and C rings of 2 with those of (20R)-5α-cholest-24-ene-2β,3α,21-triol-3,21-disulfate (in the same solvent, CD₃OD) ¹² suggested that one sulfate moiety was at C-3 and one hydroxy group was at C-2. The NMR data of the side chain of 2 were compared to those of 1 and solomonsterol A (3) and found to match perfectly, suggesting that the other sulfate group was at C-24. The key HMBC correlations of 2 are shown in Figure 2. HMBC correlations from H-3 (δ_H 4.42) to C-5 (δ_C 40.6), from H-2 (δ_H 4.09) to C-10 (δ_C 36.3), from H₃-21 (δ_H 0.97) to C-22 (δ_C 33.1), and from H-24 (δ_H 3.97) to C-22 (δ_C 33.1)/C-23 (δ_C 27.2) further confirmed the positions of the hydroxy group at C-2 and two sulfate groups at C-3 and C-24. Similar to 1, the small proton coupling constants between H₂-1 and H-2, and H-2 and H-3, as well as the observed NOE correlation between H-2 and H-3 clearly indicated equatorial positions of both H-2 and H-3. Thus, compound 2 was elucidated as a new compound, 25,26,27-tri-nor-cholesta-2β,3α,24-triol-3,24-disulfate.

Compounds 3-5 were identified as 25,26,27-tri-nor-cholesta-2β,3α,24-triol-2,3,24-trisulfate (solomonsterol A, 3),^9,10 1-O-arachidonoyl-3-O-stearylglycerol (4), ¹³ and eicosapentaenoic acid (5) ¹⁴ by the coincidence between their NMR spectral data and those of the corresponding reported data, and further confirmed by HSQC and HMBC spectra. Compounds 6-8 were identified as adenosine (6), adenine (7), and uracil (8) ¹⁵ by analyzing their ¹H NMR data, and comparison with authentic samples by HPLC analysis. Compounds 3-8 are reported for the first time from L. lambis. The sulfated sterol solomonsterol A was isolated from a sponge for the first time. This compound has been reported to exhibit anti-inflammatory activity and attenuated systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. ¹⁶ The isolated new compounds are its analogs and it seems to be obvious that substances 1–3 have a dietary origin from a sponge eaten by L. lambis. Hence the found substances are food markers.

The NMR spectra for compounds 1-8 are shown in Supplemental Figures S1-S31. The NMR data of 4 and 5 are shown in Tables S1 and S2.

Materials and Methods

General Experimental Procedures^17,18

Optical rotation was measured on a Jasco P-2000 polarimeter. HRESIMS were measured on an Agilent 6530 Accurate Mass Q-TOF system, and NMR spectra on a Bruker Avance III 500 MHz spectrometer. For preparative HPLC, an Agilent 1260 Infinity II system was used equipped with a YMC J′sphere ODS-H80 (20 × 250 mm, 4 µm) HPLC column. Flash column chromatography was performed using either silica gel or reversed phase (RP-18) resins as adsorbent. Thin layer chromatography was carried out on pre-coated silica gel 60 F₂₅₄ and/or RP-18 F_254S plates.

Animal Materials

The samples of Lambis Linnaeus, 1758 were collected from Truong Sa archipelago, Vietnam (8°51′53″N 112°15′27″E, 8°51′53″N 112°15′27″E, 8°38′39″N 111°55′21″E, 8°58′30″N 113°42′23″E, 10°22′30.9″N 114°28′55.4″E, 112°15′27″E, 8°51′53″N 112°15′27″E, 8°38′39″N 111°55′21″E, 8°58′30″N 113°42′23″E, 10°10′41″N 114°21′50″E, 10°01′23″N 113°51′19″E) during June 2021 and identified by Dr Nguyen Thi Bich Nga, Institute of Biotechnology, Vietnam Academy of Science and Technology. The voucher specimen (TS07-08) has been deposited at Vietnam - Russia Tropical Center, Vietnam.

Extraction and Isolation

L. lambis (2.5 kg) was defrosted, then extracted with MeOH in an ultrasonic bath (3 times × 10.0 L MeOH, 40 ^oC, 3 h). The MeOH extract (121.1 g) was suspended in distilled water (2.5 L) and extracted with CH₂Cl₂ (2.5 L) to obtained the CH₂Cl₂ extract (LL1, 10.3 g). LL1 (10.0 g) was chromatographed on a silica gel column, eluting with a gradient solvent system of CH₂Cl₂ in MeOH (100:0, 40:1, 20:1, 0:100) to obtain four fractions, LL1A-LL1D. LL1A (1.8 g) was chromatographed on a silica gel column, eluting with n-hexane/EtOAc (7/1, v/v) to obtain compounds 4 (13.5 mg) and 5 (17.1 mg). LL1B (1.2 g) was chromatographed on a RP-18 column, eluting with acetone/water (4/1, v/v) to give compound 6 (23.1 mg). LL1C (4.3 g) was chromatographed on a RP-18 column, eluting with acetone/water (3.5/1, v/v) to obtain 5 fractions, LL1C1- LL1C5. LL1C2 (736 mg) was purified by HPLC (J’sphere ODS H-80, 250 mm × 20 mm column), eluting with 35% acetonitrile (ACN) in H₂O to yield 3 (6.1 mg, t_R 37.8). LL1C3 (679 mg) was chromatographed on an HPLC (J’sphere ODS H-80, 250 mm × 20 mm column), eluting with 45% acetonitrile (ACN) in H₂O to yield 1 (5.2 mg, t_R 42.5) and 2 (4.7 mg, t_R 44.6). LL1D (2.7 g) was chromatographed on a RP-18 column, eluting with acetone/water (2/1, v/v) to give compounds 7 (6.1 mg), 8 (4.6 mg), and 9 (7.2 mg).

3.3.1. 25,26,27-Tri-nor-cholest-5-ene-2β,3α,24-triol-2,24-disulfate (1)

White amorphous solid, : + 8.7 (c 0.1, MeOH); HRESIMS m/z 581.1844 [M + H]⁺ (calcd. for [C₂₄H₃₉O₉S₂Na₂]⁺: 581.1825, Δ =  + 3.3 ppm); m/z 603.1637 [M + Na]⁺ (calcd. for [C₂₄H₃₈O₉S₂Na₃]⁺: 603.1645, Δ = −1.3 ppm). ¹H NMR (CD₃OD, 500 MHz) δ (ppm), ¹³C NMR (CD₃OD, 125 MHz) δ (ppm) data: see Table 1.

3.3.1. 25,26,27-Tri-nor-cholesta-2β,3α,24-triol-3,24-disulfate (2)

White amorphous solid, : + 12.3 (c 0.1, MeOH); HRESIMS m/z m/z 583.1977 [M + H]⁺ calcd C₂₄H₄₁O9S₂Na₂ = 583.1982 Δ = -0.9 ppm; m/z 605.1808 [M + Na]⁺ calcd C₂₄H₄₀O9S₂Na₃ = 605.1801 Δ =  + 1.2 ppm. ¹H NMR (CD₃OD, 500 MHz) δ (ppm), ¹³C NMR (CD₃OD, 125 MHz) δ (ppm) data: see Table 1.

Conclusions

From the methanol extract of the Vietnamese marine spider conch Lambis Linnaeus, 1758, two new (1 and 2) and six known compounds (3-8) were isolated. Their chemical structures were elucidated as 25,26,27-tri-nor-cholest-5-ene-2β,3α,24-triol-2,24-disulfate (1), 25,26,27-tri-nor-cholesta-2β,3α,24-triol-3,24-disulfate (2), and 25,26,27-tri-nor-cholesta-2β,3α,24-triol-2,3,24-trisulfate (3), 1-O-arachidonoyl-3-O-stearylglycerol (4), eicosapentaenoic acid (5), adenosine (6), adenine (7), and uracil (8) based on extensive spectroscopic methods, including HRESIMS, 1D- and 2D-NMR spectra. Compounds 3-8 are reported herein from L. lambis for the first time.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221113184 - Supplemental material for Two New Sterol Sulfates from Marine Spider Conch Lambis Lambis Linnaeus, 1758

Supplemental material, sj-docx-1-npx-10.1177_1934578X221113184 for Two New Sterol Sulfates from Marine Spider Conch Lambis Lambis Linnaeus, 1758 by Nguyen Trong Dan, Le Thi Giang, Cu Nguyen Dinh, Truong Ba Hai, Nguyen Dang Hoi, Vu Thi Loan, Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Bui Huu Tai and Phan Van Kiem in Natural Product Communications