Steroids from a Far-Eastern Glass Sponge Aulosaccus sp.

Abstract

This report concerns the identification of steroid constituents of a Far-Eastern glass sponge Aulosaccus sp. (the Sea of Okhotsk), belonging to one of the least chemically investigated group of sponges. Steroid mixtures, isolated from the extract of Aulosaccus sp., were fractionated by high-performance liquid chromatography on normal-phase and/or reversed-phase columns, and the obtained fractions were analyzed by nuclear magnetic resonance spectroscopy and gas chromatography–mass spectrometry. In result, 64 compounds, including 32 3-keto derivatives of sterols with saturated, Δ⁷-, Δ⁸⁽¹⁴⁾-, Δ⁴-, and Δ^4,6-tetracyclic systems and 32 sterols (stanols, 4α-methyl-stanols, Δ⁵ -, Δ⁷-, 4α-methyl-Δ⁸⁽¹⁴⁾-sterols), were identified. Most of these steroids were not found in glass sponges earlier. The structures of 2 new steroids, 24-propyl-5α-cholest-24(28)Z-en-3-one and 24-nor-cholest-4-en-3-one, were elucidated. Sterol esters were not found. 3-Keto derivatives of stanols were major components in the steroids of Aulosaccus sp. It is probable that association of Aulosaccus sp. with bacteria, capable of oxidizing sterols, led to the transformation of a significant part of sponge sterols into their 3-keto derivatives.

Keywords

steroid ketones 3-ketosteroids sterols Hexactinellida glass sponge

Most sponges (phylum Porifera) are sessile colonial filter-feeding animals that feed on bacterioplankton and phytoplankton. These invertebrates are distributed in 4 classes: demosponges (Demospongiae), calcareous (Calcarea), homoscleromorph (Homoscleromorpha), and glass (Hexactinellida) sponges. Glass sponges may be considered as one of the least chemically investigated class of Porifera.

Different sterols are common in sponges, but 3-keto derivatives of sterols are less frequently found in these animals.^1
–3 A single comparative study of steroids from glass sponges⁴ showed that hexactinellids, in general, contained Δ⁵-sterols, stanols, 3-keto derivatives of stanols, and Δ⁴-3-ketosteroids in varying proportions. In these invertebrates, sterols, as a rule, were presented at higher concentrations than their 3-keto derivatives, but sometimes these compounds were found in comparable concentrations.⁴ In contrast, we observed an unusually high content of 3-keto derivatives of sterols in the steroids isolated from a Far-Eastern glass sponge Aulosaccus sp. (the Sea of Okhotsk). Although the maximum number of the 3-ketosteroids, reported earlier to occur in a hexactinellid sponge, was 4,⁴ preliminary gas chromatography–mass spectrometry (GC–MS) employed by us for the tentative characterization of the 3-keto derivatives of sterols of Aulosaccus sp. showed many more components. This prompted us to obtain detailed information on the steroid composition of this sponge. In addition, steroids of glass sponges from the North-West Pacific have not been investigated previously.

The extractable material of Aulosaccus sp. was separated by column chromatography on Sephadex LH-20 and silica gel. Then, most of the obtained mixtures, containing steroids, were fractionated by high-performance liquid chromatography (HPLC) on normal-phase and/or reversed-phase columns. The obtained fractions were analyzed by nuclear magnetic resonance (NMR) spectroscopy and GC–MS (electron impact ionization). As a result, 64 compounds, namely, 32 3-keto derivatives of sterols (including the 2 new ones) and 32 sterols, were identified (Figure 1).

Figure 1

Steroids from Aulosaccus sp.

The least polar fraction I (Table 1) was a major steroid fraction isolated from Aulosaccus sp. A part of fraction I was separated by HPLC in the reversed-phase mode (RP-HPLC). In result, several subfractions, containing compounds 1b, 1e, 1 h, 1i, and 1 m as main components, were obtained whereas other steroids were constituents of more complex RP-HPLC subfractions. Another part of fraction I was separated by HPLC in the normal-phase mode that gave subfractions, enriched in major saturated compounds and minor monoenes (3-keto derivatives of stanols with monounsaturated side chains, Δ⁷- and Δ⁸⁽¹⁴⁾-3-ketosteroids).

Table 1

Compositions of Four Steroid Fractions Isolated from Aulosaccus sp.

Steroid	RRT^a	% in fraction	Steroid	RRT	% in fraction
Fraction I: 3-ketosteroids with tetracyclic systems 1‒3
C₂₆Δ²² (1a)	0.82	tr.	(24R)-C₂₈Δ⁰ (1i)	1.15	20.4
C₂₆Δ⁰ (1b)	0.87	9.6	C₂₉Δ²² (1 j)	1.17	tr.
C₂₇Δ²² (1 c)	0.93	tr.	C₂₈Δ^7,24(28) (2 h)	1.19	tr.
C₂₇Δ²² (1d)	0.95	0.8	C₂₈Δ⁷ (2i)	1.21	tr.
C₂₇Δ⁸⁽¹⁴⁾ (3e)	0.98	tr.	C₂₉Δ^7,22 (2k)	1.25	tr.
C₂₇Δ⁰ (1e)	1.00	50.2	(24R)-C₂₉Δ⁰ (1 m)	1.29	4.1
C₂₇Δ⁷ (2e)	1.06	tr.	C₂₉Δ^24(28)E (1 n)	1.29	tr.
C₂₈Δ^7,22 (2g)	1.10	tr.	C₂₉Δ^24(28)Z (1o)	1.32	0.9
(24R)-C₂₈Δ²² (1 g)	1.09	tr.	C₂₉Δ⁷ (2 m)	1.36	tr.
(24S)-C₂₈Δ⁸⁽¹⁴⁾ (3i)	1.12	tr.	C₃₀Δ^24(28)Z (1 p)	1.45	tr.
C₂₈Δ²⁴⁽²⁸⁾ (1 h)	1.13	5.7	Unidentified components		8.3
Fraction II: Δ⁴-3-ketosteroids (structures 4 and 5)
C₂₆Δ^4,22 (4a)	0.88	1.0	C₂₈Δ^4,22 (4g)	1.16	7.1
C₂₆Δ⁴ (4b)	0.95	10.0	C₂₈Δ^4,24(28) (4 h)	1.26	0.9
C₂₇Δ^4,22 (4c)	1.02	1.9	(24R)-C₂₈Δ⁴ (4i)	1.27	8.2
C₂₇Δ^4,22 (4d)	1.04	2.3	C₂₉Δ^4,22 (4k)	1.32	3.1
C₂₇Δ⁴ (4e)	1.11	49.6	C₂₉Δ⁴ (4 m)	1.45	6.6
C₂₇Δ^4,6 (5e)	1.14	2.1	Unidentified components		7.2
Fraction III: sterols (structures 6‒8)^b
C₂₆Δ⁵ (7b)	0.87	0.8	C₂₈Δ^5,24(28) (7 h)	1.14	0.8
C₂₆Δ⁰ (6b)	0.88	12.3	C₂₈Δ⁵ (7i)	1.15	0.9
C₂₇Δ^5,22 (7d)	0.95	0.8	C₂₈Δ²⁴⁽²⁸⁾ (6 h)	1.17	3.2
C₂₇Δ²² (6d)	0.97	0.9	C₂₈Δ⁰ (6i)	1.18	10.0
C₂₇Δ⁵ (7e)	1.00	5.1	C₂₈Δ⁷ (8i)	1.26	2.1
C₂₇Δ⁰ (6e)	1.03	52.5	C₂₉Δ⁵ (7 m)	1.31	4.9
C₂₈Δ^7.22 (8g)	1.14	0.9	C₂₉Δ⁰ (6 m)	1.34	1.9
			Unidentified components		2.9
Fraction IV: 4α-methyl-sterols (structures 9‒11)^b
4-Me-C₂₆Δ²² (9a)	0.87	3.3	4-Me-C₂₈Δ⁸⁽¹⁴⁾ (10i)	1.24	1.3
4-Me-C₂₆Δ⁰ (9b)	1.00	1.5	4-Me-C₂₈Δ²⁴⁽²⁸⁾ (9 h)	1.26	5.5
4-Me-C₂₇Δ²² (9 c)	1.01	1.7	4-Me-C₂₈Δ⁰ (9i)	1.28	9.4
4-Me-C₂₇Δ²² (9d)	1.04	3.1	4-Me-C₂₉Δ²² (9j)	1.31	9.2
4-Me-C₂₇Δ⁸⁽¹⁴⁾ (10e)	1.07	10.0	4-Me-C₂₉Δ⁸⁽¹⁴⁾ (10e)	1.40	3.2
4-Me-C₂₇Δ⁰ (6e)	1.09	3.8	4-Me-C₂₉Δ⁸⁽¹⁴⁾ (10 m)	1.42	2.8
4-Me-C₂₈Δ²² (9f)	1.12	4.0	4-Me-C₂₉Δ⁰ (9 I)	1.43	1.8
(24R)-C₂₈Δ²² (9 g)	1.17	16.6	4-Me-C₂₉Δ⁰ (9 m)	1.46	5.0
4,14-diMe-9,10-methylene-C₂₇Δ⁰ (11e)	1.19	7.5	4-Me-C₂₉Δ^24(28)Z (9o)	1.49	2.6
4,14-diMe-9,10-methylene-C₂₇Δ⁰ (11e)	1.19	7.5	Unidentified components		7.7

The weight ratio of fractions I, II, III, and IV was ≈100:1:3:1.7.

RRT, relative retention time. 3-Ketosteroids: RRT = 1.00 for 5α-cholestan-3-one (1e). Sterols: RRT = 1.00 for acetate of cholesterol (acetate of compound 5e).

Sterols were analyzed as acetylated derivatives; in case of some 4α-methyl-sterols, identifications were confirmed by GC–MS of free sterols according to mass spectrometry data in the literature.

3-Keto derivatives of stanols (1a–e, 1g–j, 1m–p) with side chains, typical of benthic marine organisms, were abundant in fraction I. The ¹H and ¹³C NMR spectra (CDCl₃) of these 5α−3-ketosteroids displayed the characteristic signals of C-3 (δ _C 212.1), H₃C-18 (δ _H 0.68, s; δ _C 12.1), H₃C-19 (δ _H 1.005, s; δ _C 11.5), axial H-4 (δ _H 2.26, dd, J = 13.9; 15.0 Hz), and H-5α (δ _H 1.52, m).^5
–7 In the mass spectra of these compounds, the prominent fragment ion peak at m/z 231 (a base peak for saturated 3-ketosteroids 1b, 1e, 1i, and 1 m), produced by ring D cleavage,^6,8,9 was observed. The mass spectra of Δ²²- (1a, 1 c, 1d, 1g, 1 j) and Δ²⁴⁽²⁸⁾- (1 h, 1 n, 1o, 1 p) 3-ketosteroids showed distinctive peaks at m/z 300 and 314, respectively, which indicated the corresponding positions of the double bonds in the unsaturated side chains.⁶ Among the 3-keto derivatives of stanols, a C₂₇ component (1e, 50.2%) was dominant, and compounds with saturated side chains (84.3%) were presented at higher concentrations than monoenes. (24R)-Configuration was assigned to compounds 1 g, 1i, and 1 m according to their ¹Н NMR spectra so these steroids possessed the side chains of phytosterols: brassicasterol, campesterol, and sitosterol, respectively.¹⁰

Among minor monounsaturated 3-keto derivatives of sterols (fraction I), an unknown compound 1 p (24-propyl-5α-cholest-24(28)Z-en-3-one) was found. The mass spectrum of 1 p showed distinctive peaks at m/z 426 [M]⁺, 314 (formed by cleavage of C-22–C-23 bond and located a double bond in position 24(28)), 271 [M–side chain–2 H]⁺, and 231. Apart from the signals of saturated 5α-3-ketosteroid nucleus, the¹Н NMR spectra of HPLC fraction, enriched in 1 p, showed the resonances of olefinic H-28 (δ _H 5.01, t, J = 7.0 Hz), allylic H₂C-29 (δ _H 2.02, m), and H-25 (δ _H 2.80, m), characteristic of (24Z)-24-n-propylidenecholesterol side chain.¹¹ Accordingly, the signal of allylic H-25 displayed a cross-peak with H₃C-26 and H₃C-27 (δ _H 0.97, 6H, d, J = 7.0 Hz), whereas H-28, H₂C-29, and H₃C-30 (δ _H 0.94, t, J = 7.5 Hz) formed a linear spin system in the ¹H–¹H COSY spectrum.

Earlier, 3-ketosteroids 1b, 1e, 1i, and 1 m have been reported to occur in hexactinellids.⁴ In addition, compound 1е was identified as a component of the extracts of a variety of Antarctic and two Arctic glass sponges.¹² However, 3-keto derivatives of stanols have been found not only in glass sponges, but also in demosponges^6,13 and in marine and lacustrine sediments.^14
–16

In fraction I, 3-keto derivatives of Δ⁷- and Δ⁸⁽¹⁴⁾-sterols were present in trace amounts. In the mass spectra of these compounds, typical fragmentation patterns were observed according to reference spectra and GC–MS data described in Iida et al⁸ and Smith and Brooks.⁹ In addition, the ¹H NMR spectra of subfractions, containing Δ⁷-components (2e, 2 g‒i, 2 k, 2 m), showed the characteristic signals of H-7 (δ _H 5.18, m) and H₃C-18 (a relatively high-field chemical shift, δ _H 0.56–0.58, s).^5,17 3-Keto derivatives of Δ⁷-sterols have never been detected in sponges. However, these compounds have been found earlier in some terrestrial plants and fungi (2 g‒i, 2 k, 2 m),^18

–22 a marine dinoflagellate (2е, 2i),²³ and a starfish (2е).¹⁷ As for Δ⁸⁽¹⁴⁾-3-ketosteroids 3e and (24S)-3i, their ¹H NMR spectra showed the characteristic signals of H₃C-18 (δ _H 0.875, s) and H₃C-19 (δ _H 0.90, s). In addition, H₃C-18 displayed a cross-peak with C-14 (δ _C 143.4) of a tetrasubstituted double bond in HMBC experiment. We did not find any information concerning the isolation of compounds 3e and 3i from natural sources.²⁴ Perhaps, in the case of Aulosaccus sp., their precursors were C₂₇ and (24S)-C₂₈ Δ⁸⁽¹⁴⁾-sterols that may be found in marine dinoflagellates.²⁵

Fraction II (Table 1), consisting of Δ⁴-3-ketosteroids 4a–e, 4g–i, 4 k, 4 m, and 5e, was isolated using HPLC in the normal-phase mode and separated by RP-HPLC. The GC profiles of fractions I and II were similar. In particular, these fractions contained significant amounts of С₂₆ components with saturated side chain b (9.6% and 10.0%, respectively) that gave us the opportunity to isolate compounds 1b and 4b using RP-HPLC. Steroid 1b, previously found in trace amounts in two hexactinellids, has been described as “a compound with a tentatively assigned 24-nor-cholesterol side chain” based on its GC–MS data.⁴ However, the ¹H and ¹³C NMR data of 1b have not been reported, a gap that was filled by us (Table 2). The structure elucidation of another C₂₆ component, new Δ⁴-3-ketosteroid 4b, is described below.

Table 2

¹H (700 MHz, CDCl₃) and ¹³C (176 MHz, CDCl₃) NMR Spectra^a of 24-Nor-3-Ketosteroids 1b and 4b.

Position	1b		4b
	δ _H (mult., J Hz)	δ _C	δ _H (mult., J Hz)	δ _C ^b
1	1.34 (m), 2.01 (m)	38.6	1.69 (m), 2.02 (m)	35.8
2	2.29 (m),2.38 (ddd, 6.6; 14.0; 15.4)	38.2	2.335 (m), 2.42 (m)	34.1
3		212.1
4	2.08 (ddd, 2.3; 4.0; 15.0), 2.26 (dd, 13.9; 15.0)	44.75	5.72 (s)	123.8
5	1.52 (m)	46.7		172.6
6	1.33 (2H, m)	29.0	2.26 (m), 2.37 (m)	33.0
7	0.90 (m), 1.70 (m)	31.7	1.02 (m), 1.83 (m)	32.1
8	1.39 (m)	35.4	1.52 (m)	35.7
9	0.725 (m)	53.85	0.92 (m)	53.9
10		35.7		38.8
11	1.375 (m), 1.52 (m)	21.5	1.43 (m), 1.52 (m)	21.1
12	1.14 (m), 1.99 (m)	39.9	1.15 (m), 2.02 (m)	39.7
13		42.6		42.5
14	1.00 (m)	56.3	1.01 (m)	56.0
15	1.06 (m), 1.58 (m)	24.2	1.11 (m), 1.60 (m)	24.3
16	1.25 (m), 1.83 (m)	28.2	1.29 (m), 1.855 (m)	28.3
17	1.10 (m)	56.2	1.12 (m)	56.1
18	0.675 (s)	12.05	0.71 (s)	12.0
19	1.005 (s)	11.5	1.18 (s)	17.45
20	1.375 (m)	35.9	1.37 (m)	36.0
21	0.90 (d, 6.5)	18.7	0.905 (d, 6.5)	18.65
22	1.01 (m), 1.37 (m)	33.5	1.01 (m), 1.37 (m)	33.5
23	1.07 (m), 1.18 (m)	35.3	1.075 (m), 1.18 (m)	35.3
24	‒	‒	‒	‒
25	1.45 (m)	28.4	1.455 (m)	28.5
26	0.87 (d, 6.6)	22.4	0.87 (d, 6.6)	22.4
27	0.85 (d, 6.5)	23.1	0.855 (d, 6.5)	23.1

All signals were assigned based on ¹H‒¹H COSY, HSQC, and HMBC experiments.

¹³C NMR spectrum was obtained through CH correlations in HSQC and HMBC experiments; δ_C of C-3 was not determined due to the paucity of compound 4b.

The mass spectrum of 4b showed peaks at m/z 370 [M]⁺, 271 [M–side chain]⁺, 247 [M–“ring A ion”]⁺, 229 [M–ring D with side chain]⁺, 124 (“ring A ion,” 100%), and others that allowed for the identification of a monounsaturated 3-ketosteroid nucleus and a saturated side chain (7 carbon atoms)^9,26 in this compound. In the ¹H and ¹³C NMR spectra of Δ⁴-3-ketosteroid 4b (Table 2), the characteristic signals of H-4 (δ _H 5.72, s; δ _C 124.0), CH₃-18 (δ _H 0.71, s; δ _C 12.2), and CH₃-19 (δ _H 1.18, s; δ _C 17.6)^5,27,28 were observed. In addition, the NMR spectra of this steroid showed the signals of 2 methyl groups (δ _H 0.855, d, J = 6.5 Hz, δ _C 23.1, and δ _H 0.87, d, J = 6.6 Hz, δ _C 22.5) and 1 methine group (δ _H 1.46, m; δ _C 28.5), the protons of which formed a spin system of the isopropyl group according to ¹H–¹H COSY correlations. HMBC experiments confirmed that the side chains of 24-nor-cholestan-3-one (1b) and 24-nor-cholest-4-en-3-one (4b) were identical. It should be noted that, among living organisms, 24-nor-3-ketosteroids 1b and 4b were found only in glass sponges. As for other ∆⁴-3-ketosteroids found in the present study, compound 4e has only been reported to occur in hexactinellids.⁴ In addition, known ∆⁴-3-ketosteroids, reported here (including Δ^4,6-3-ketosteroid 5e), have been isolated from several benthic organisms, including demosponges.^{27,29,30,31,32,33,34,35} Furthermore, compound 4e and/or its several homologs were detected in marine and lacustrine sediments.^15,16,36

Fraction III (Table 1), containing major sterols of Aulosaccus sp., was obtained using repeated column chromatography on silica gel. This fraction, analogous to fraction I, was rich in saturated components, especially in C₂₇ compound. Namely, a high content (76.7%) of saturated sterols, including stanols 6b, 6е (major), 6i, and 6 m, was observed in fraction III. Stanols 6d and 6 h with monoenoic side chains, Δ⁵-sterols (7b, 7d, 7e, 7 h, 7i, 7 m), and Δ⁷-sterols (8 g, 8i) were minor constituents. Most of these compounds (except for Δ⁷-sterols) have been reported previously in the study of steroids from glass sponges.⁴ A mixture of 4α-methyl-sterols (fraction IV), possessing saturated (9а–j, 9 l, 9 m, 9o), Δ⁸⁽¹⁴⁾- (10e, 10i, 10 l, 10 m), and cyclopropane-containing (11e) polycyclic systems, was isolated from Aulosaccus sp. using HPLC in the normal-phase mode. These sterols (except for 11e) were considered typical of dinoflagellates.^23,37,38,39 As we did not find any documented case of an association between deep-sea hexactinellids and dinoflagellates, the above-mentioned 4α-methyl-sterols, known as dinoflagellate markers, were apparently of phytoplanktonic (food) origin. Two saturated 4α-methyl-sterols, 9i and 9 m, have been found previously in some hexactinellid species.⁴ Furthermore, several 4α-methyl-sterols have been isolated earlier from some demosponges.^40,41 31-Nor-cycloartanol (11e), known as a metabolite in the biosynthesis of plant sterols,²² has been found not only in land plants (see, e.g., refs. ^42,43) but also in red algae.^44,45

It was reported that steroids of the glass sponge Aulosaccus cf. mitsukuri (Galapagos) included stanols (54%), Δ⁵-sterols (4%), and 3-keto derivatives of stanols (42%).⁴ Similarly, stanols found in the present study were major sterols of a related Aulosaccus sp. In addition, both the sponges of the genus Aulosaccus contained significant amounts of steroid ketones. However, the total amount of 3-keto derivatives of sterols in Aulosaccus sp. was more than 20 times higher than the total amount of its sterols (Table 1). We have observed a similar trend in our previous study on steroids from some sponges. Namely, an Antarctic demosponge Haliclona sp. was shown to contain Δ⁴-3-ketosteroids and Δ⁵-sterol esters although this invertebrate was characterized by the almost total absence of free sterols (only cholesterol was found in trace amount).⁴¹ We tried to find sterol esters in Aulosaccus sp., but our attempt was unsuccessful. Indeed, the characteristic signals (including multiplet of esterified НС-3 group) of these compounds were not observed in the ¹Н NMR spectrum of the least polar fraction, which has been expected to contain sterol esters. Probably, as a result of the intensive oxidation of sterol precursors, 3-ketosteroids became dominant in the steroids of Aulosaccus sp. whereas biosynthesis of sterol esters was blocked. Perhaps, this metabolic shift induced the substitution of sterols by their 3-keto derivatives in sponge membranes. It seems to be interesting that, in the ¹Н NMR spectra of cholesterol and 5α-cholestan-3-one, the virtually indistinguishable chemical shifts of Н₃С-18 (δ _Н 0.681 and 0.678, respectively) and Н₃С-19 (δ _Н 1.009 and 1.005, respectively) may imply a certain similarity in the molecular features of these two compounds.

Our earlier study⁴⁶ indicated that Aulosaccus sp. contained significant amounts of fatty acids of microbial origin. Namely, the occurrence of some branched-chain, cyclopropane-containing fatty acids and their monoenoic precursors in Aulosaccus sp. suggested that this sponge was associated with mycobacteria or related microbes.⁴⁶ Notably, the bacteria of this group (phylum Actinobacteria) are not only known as sponge specific microorganisms,⁴⁷ but these microbes are also potent steroid degraders, particularly active in the catabolism of sterols.⁴⁸ The proposed oxidation of sterols in Aulosaccus sp. is reminiscent of oxidation by cholesterol oxidase or NAD(P)-dependent dehydrogenase, produced by a variety of Actinobacteria including Mycobacterium (see review⁴⁸ and references cited herein). These enzymes transform Δ⁵-sterols into 3-keto-4-enes that is considered as the first step in the sterol catabolism pathway. In addition, it was shown that cholesterol oxidase not only transformed Δ⁵-sterols, but also catalyzed the transformations of stanols, Δ⁷- and Δ⁸⁽¹⁴⁾-sterols into corresponding 3-ketosteroids,⁹ including those found in the present study. Therefore, we suggested that association of Aulosaccus sp. with bacteria, capable of oxidizing sterols, led to the transformation of the significant part of sponge sterols into their 3-keto derivatives. Similarly, the presence of 3-ketosteroids in different sponges (as a rule, in species from cold waters of polar or temperate latitudes and in deep-water species) may be the result of the bacterial degradation of sterols. For example, such degradation may explain the presence of biologically active, hydroxylated Δ⁴-3-ketosteroids in a deep-water Alaska demosponge⁴⁹ because some bacteria can not only oxidize C-3 in the sterol but also introduce a hydroxy group into the sterol side chain followed by deeper transformations of steroid structure.⁴⁸

To explain the co-occurrence of Δ⁵-sterols, stanols, 3-keto derivatives of stanols, and Δ⁴-3-ketosteroids in glass sponges, a stereoselective route of Δ⁵-sterol conversion into 5α-stanols was proposed, where the steroid ketones were intermediates in the stanol formation.⁴ In contrast to this proposal, we suggest that the 3-keto derivatives of sterols are products of catabolism rather than biosynthesis of sponge sterols. However, further studies of marine sponge-associated bacteria are needed to confirm or reject our suggestion.

Experimental

General Experimental Procedures

¹H NMR, ¹Н− ¹Н-COSY, DEPT, HSQC, and HMBC spectra (CDCl₃) were recorded on Bruker Avance III HD 500 and Bruker Avance III 700 spectrometers at 500 and 700 MHz. ¹³C NMR spectra (CDCl₃) were recorded on Bruker Avance III 700 spectrometer at 176 MHz. GC analyses were done on an Agilent 6850 Series GC System chromatograph (Agilent, Germany) equipped with an HP-1 (Agilent technology, Inc., USA) capillary column (30 m × 0.32 mm), the carrier gas was helium (flow rate 1.7 mL/min), the detector temperature was 300°C. GC–MS analyses were carried out on a Hewlett Packard HP6890 GC System (Hewlett Packard Company, USA) with an HP-5MS (J&W Scientific, USA) capillary column (30.0 m × 0.25 mm), helium as the carrier gas, and 70 eV ionizing potential. The GC–MS analyses of acetylated sterols, free sterols and their 3-keto derivatives were done using the injector temperature of 270°C and the temperature program 100°C (1 min)–20 °C/min–270°C (30 min). Column chromatography was performed using Sephadex LH-20 (GE Healthcare, Sweden) and silica gel (50/100 µm, Sorbpolimer, Russia). HPLC separations were performed using a Du Pont Series 8800 Instrument (Du Pont Company, USA) with a RIDK-102 refractometer (Laboratorni pristroje Praha, Czechoslovakia). An Agilent ZORBAX SB-C18 column (4.6 × 150 mm; Agilent Technologies, USA) and an Altex Ultrasphere^TM-Si column (10 × 250 mm; Beckman Instruments, Inc., USA) were used in the HPLC separations.

Animal Material

The sponge sample of the genus Aulosaccus (phylum Porifera, class Hexactinellida, order Lyssacinosida, family Rossellidae), 930 g (wet weight), was collected in July 2011 by dredging (with a small Sigsbee trawl) from 505 m depth near Iturup Island (the Sea of Okhotsk, Kuril Islands, 45°01′5″ N, 147°00′3″ E) during a cruise onboard the r/v “Academik Oparin.” The species was identified by Dr A.L. Drozdov (National Scientific Center of Marine Biology, Far-Eastern Branch of RAS, Vladivostok, Russia). A voucher specimen (041-030) is on deposit in the collection of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia.

Extraction and Isolation

The collected sponge (930 g, wet weight) was frozen, stored at −15°C, then cut and extracted with EtOH at room temperature. A mixture, containing cerebrosides, fatty acids, and steroids (895 mg), was isolated from the EtOH extract as described in our previous report.⁵⁰ This mixture was subjected to column chromatography (SiO₂:hexane/ethyl acetate, 10:1 → 5:1). The least polar fraction (79 mg: phthalates, minor 3-ketosteroids), a sum of 3-ketosteroids 1– 3 (150 mg; fraction I), and a mixture of Δ⁴-3-ketosteroids and 4α-methyl-sterols (20 mg) were eluted consecutively with hexane/ethyl acetate 10:1. Elution with hexane/ethyl acetate (5:1) gave a mixture containing more polar sterols (13 mg). The RP-HPLC separation of a part of fraction I (≈50 mg) on an Agilent ZORBAX SB-C18 column in EtOH:H₂O:CHCl₃ (90:10:5) yielded subfractions, containing 1b (2.7 mg), 1 h (1.6 mg), 1e (9.5 mg), (24R)-1i (2.0 mg), 1 p (1.0 mg), and (24R)−1 m (0.5 mg). The subfraction containing 1 p was purified using an Altex Ultrasphere^TM-Si column (hexane/ethyl acetate 20:1). Another part of fraction I (≈ 100 mg) was separated by HPLC in the normal-phase mode (an Altex Ultrasphere^TM-Si column, hexane/ethyl acetate 20:1). In result, some subfractions, enriched in minor Δ⁸⁽¹⁴⁾-3-ketosteroids (0.3 mg), 3-keto derivatives of stanols with monounsaturated side chains, and Δ⁷-3-ketosteroids (0.7 mg), were obtained. The mixture of Δ⁴-3-ketosteroids and 4α-methyl-sterols (20 mg) was also separated by HPLC in the normal-phase mode (hexane/ethyl acetate 8:1); Δ⁴-3-ketosteroids (fraction II, 1.5 mg) and 4α-methyl-sterols (fraction IV, 2.5 mg) were isolated. Fraction II (1.5 mg) was separated by RP-HPLC (Agilent ZORBAX SB-C18 column, 95% EtOH) to obtain subfractions enriched in 4b (0.2 mg), 4e (1.0 mg), and 4i (0.1 mg). The mixture, containing more polar sterols (20 mg), was chromatographed on silica gel column (4.0 cm × 1.2 cm) using hexane/ethyl acetate (40:1 → 20:1 → 10:1) system; elution with hexane/ethyl acetate (10:1) gave a sterol sum (fraction III, 4.4 mg). The sterols were acetylated with Ac₂O in pyridine (1:1, 0.4 ml, overnight).

24-Propyl-5α-Cholest-24(28)Z-En-3-One (1p)

¹H NMR (700 MHz, CDCl₃): 0.68 (3H, s, H₃C-18), 0.73 (1H, m, H-9), 0.94 (3H, d, J = 6.5 Hz, H₃C-21), 0.94 (3H, t, J = 7.5 Hz, H₃C-30), 0.97 (3H, d, J = 7.0 Hz, H₃C-26, 27), 0.94 (3H, t, J = 7.5 Hz, H₃C-30), 0.92‒2.05 (m, CH and CH₂ pool), 2.02 (2H, m, H₂C-29), 2.08 (1H, m, H-4a), 2.26 (1H, “pseudotriplet,” J = 14.5 Hz, H-4b), 2.29 (1H, m, H-2b), 2.38 (1H, m, H-2a), 2.80 (1H, m, H-25), 5.01 (1H, t, J = 7.0 Hz, H-28).

GC–MS: m/z (%) = 426 [M]⁺ (6), 411 (2), 314 (100), 299 (27), 271 (16), 258 (9), 245 (7), 231 (19), 217 (7).

24-Nor-Cholest-4-En-3-One (4b)

IR (CDCl₃): 1661 (C = O) cm⁻¹.

¹H and ¹³C NMR (CDCl₃): Table 2.

GC–MS: m/z (%) = 370 [M]⁺ (46), 355 (13), 328 (21), 313 (10), 299 (3), 285 (10), 271 (10), 247 (29), 229 (56), 149 (21), 147 (24), 124 (100).

Footnotes

Acknowledgments

The authors express their gratitude to Drs O.P. Moiseenko and L.P. Ponomarenko for their kind help in the GC–MS and GC analyses. The study was carried out on the equipment of the Collective Facilities Center "The Far-Eastern Center for Structural Molecular Research (NMR/MS) PIBOC FEB RAS".

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by Grant No. 17-04-00034 A from the Russian Foundation for Basic Research.

Supplemental Material

References

Bergquist

PR.

Hofheinz

Oesterhelt

. Sterol composition and the classification of the Demospongiae. Biochem Syst Ecol. 1980;8(4):423–435.doi:10.1016/0305-1978(80)90045-9

Bergquist

PR.

Lavis

Cambie

. Sterol composition and classification of the Porifera. Biochem Syst Ecol. 1986;14(1):105–112.doi:10.1016/0305-1978(86)90094-3

Aiello

Fattorusso

Menna

. Steroids from sponges: recent reports. Steroids. 1999;64(10):687–714.doi:10.1016/S0039-128X(99)00032-X

Blumenberg

Thiel

Pape

Michaelis

. The steroids of hexactinellid sponges. Naturwissenschaften. 2002;89(9):415–419.doi:10.1007/s00114-002-0343-x

Iida

Tamura

Wainai

Mashimo

Matsumoto

. Substituent effects on the PMR signals of methyl groups in methyl steroids. Chem Phys Lipids. 1977;19(2):169–178.doi:10.1016/0009-3084(77)90097-4

Seldes

AM.

Deluca

ME.

Gros

EG.

Rovirosa

San-Martín

Darias

. Steroids from aquatic organisms, XIX. New sterols from the Antarctic sponge Artemisina apollinis . Z Naturforsch, B. 1990;45(1):83–86.doi:10.1515/znb-1990-0116

Chodounská

Buděšínský

Šídová

Šíša

Kasal

Kohout

. Simple NMR determination of 5α/5β configuration of 3-oxosteroids. Collect Czech Chem Commun. 2001;66(10):1529-1544.doi:10.1135/cccc20011529

Iida

Tamura

Satomi

et al . Characterization of methyl steroids by gas chromatography-mass spectrometry. II. Characteristic fragmentations of methyl oxosteroids. Yukagaku. 1974;23(3):546-551.

Smith

AG.

Brooks

CJW

. Mass spectra of Δ⁴- and 5α-3-ketosteroids formed during the oxidation of some 3β-hydroxysteroids by cholesterol oxidase. Biomed Mass Spectrom. 1976;3(2):81–87.doi:10.1002/bms.1200030208

10.

Rubinstein

Goad

LJ.

Clague

ADH.

Mulheirn

. The 220 MHz NMR spectra of phytosterols. Phytochemistry. 1976;15(1):195–200.doi:10.1016/S0031-9422(00)89083-4

11.

Rohmer

Kokke

WCMC.

Fenical

Djerassi

. Isolation of two new C₃₀ sterols, (24E)-24-n-propylidenecholesterol and 24-ξ-n-propylcholesterol, from a cultured marine Chrysophyte. Steroids. 1980;35(2):219–231.doi:10.1016/0039-128X(80)90104-X

12.

Núñez-Pons

Carbone

Paris

et al . Chemo-ecological studies on hexactinellid sponges from the Southern Ocean. Naturwissenschaften. 2012;99(5):353–368.doi:10.1007/s00114-012-0907-3

13.

Rovirosa

Quezada

San-Martin

. 3-Keto-esteroids from sponge, Iophon proximun . Rev Latinoam Quim. 1990;21(3-4):99-100.

14.

Gagosian

RB.

Smith

. Steroids ketones in surface sediments from the south-west African shelf. Nature. 1979;277(5694):287–289.doi:10.1038/277287a0

15.

Boudou

JP.

Trichet

Robinson

Brassell

. Lipid composition of a recent Polynesian microbial mat sequence. Org Geochem. 1986;10(4-6):705–709.doi:10.1016/S0146-6380(86)80006-7

16.

Serebrennikova

OV.

Strel’nikova

EB.

Russkikh

. Composition of organic compounds in bottom sediments of lakes in the taiga and steppe zones in Siberia. Geochem Int. 2017;55(12):1090–1104.doi:10.1134/S0016702917090087

17.

Kiem

PhV.

Thanh

NV.

Cuong

NX.

Minh

ChV

. Cholest-7-en-3-one, cholesterol and batilol from the starfish Archaster typicus . Tap Chi Hoa Hoc (Vietnam Journal of Chemistry). 2007;45(4):513-517.

18.

Khuroo

MA.

Qureshi

MA.

Razdan

TK.

Nichols

. Sterones, iridoids and a sesquiterpene from Verbascum thapsus . Phytochemistry. 1988;27(11):3541-3544.doi:10.1016/0031-9422(88)80764-7

19.

Shen

. Chemical constituents from resin of Dracaena cochinchinensis . Zhongcaoyao. 2004;35(7):728-730.

20.

Cui

Song

Sh.

Sun

. Chemical constituents of Lasiosphaera fenzlii and primary filtration of their anti-tumor activity. Zhongyaocai. 2006;29(7):703-705.

21.

Rösecke

König

. Constituents of various wood-rotting basidiomycetes. Phytochemistry. 2000;54(6):603–610.doi:10.1016/S0031-9422(00)00165-5

22.

Pascal

Taton

Rahier

. Plant sterol biosynthesis. Identification and characterization of two distinct microsomal oxidative enzymatic systems involved in sterol C4-demethylation. J Biol Chem. 1993;268(16):11639–11654.

23.

Harvey

HR.

Bradshaw

SA.

O'Hara

SCM.

Eglinton

Corner

EDS

. Lipid composition of the marine dinoflagellate Scrippsiella trochoidea . Phytochemistry. 1988;27(6):1723–1729.doi:10.1016/0031-9422(88)80432-1

24.

SciFinder

Database

. https://scifinder.cas.org. Accessed 10 December, 2018.

25.

Miao

Yan

X-J.

J-L.

Hou

Y-D

. Study on the regular mass spectrometry pattern of the TMS derivatives of sterols from microalga. Zhipu Xuebao. 2009;30(3):139-147.

26.

Brown

FJ.

Djerassi

. Mass spectrometry in structural and stereochemical problems. 254. Elucidation of the course of the electron impact induced fragmentation of α, β-unsaturated 3-keto steroids. J Am Chem Soc. 1980;102(2):807–817.doi:10.1021/ja00522a061

27.

Sheikh

YM.

Djerassi

. Steroids from sponges. Tetrahedron. 1974;30(23-24):4095–4103.doi:10.1016/S0040-4020(01)97391-0

28.

Santalova

EA.

Denisenko

VA.

Chernyshev

AV.

Gavagnin

Sanamyan

. Ketosteroids from the Far-East marine prosobranch mollusk Onchidiopsis variegata . Chem Nat Compd. 2007;43(1):86–89.doi:10.1007/s10600-007-0038-9

29.

Migliuolo

Piccialli

Sica

. Steroidal ketones from the sponge Geodia cydonium . J Nat Prod. 1990;53(5):1262–1266.doi:10.1021/np50071a019

30.

Tam Ha

TB.

Kokke

WCMC.

Djerassi

. Minor sterols of marine invertebrates 37. Isolation of novel coprostanols and 4α-methyl sterols from the tunicate Ascidia nigra . Steroids. 1982;40(4):433–453.

31.

Ayer

SW.

Andersen

. Steroidal antifeedants from the dorid nudibranch Aldisa sanguinea cooperi. Tetrahedron Lett. 1982;23(10):1039-1042.doi:10.1016/S0040-4039(00)87016-1

32.

Tischler

Ayer

SW.

Andersen

RJ.

Mitchell

JF.

Clardy

. Anthosterones A and B, ring A contracted steroids from the sponge Anthoracuata graceae . Can J Chem. 1988;66(5):1173–1178.doi:10.1139/v88-192

33.

Aiello

Fattorusso

Magno

Menna

Pansini

. Steroids of the marine sponge Cinachyra tarentina: isolation of cholest-4-ene-3,6-dione and (24R)-24-ethylcholest-4-ene-3,6-dione. J Nat Prod. 1991;54(1):281–285.doi:10.1021/np50073a035

34.

Sheu

JH.

Huang

SY.

Wang

GH.

Duh

. Study on cytotoxic oxygenated desmosterols isolated from the red alga Galaxaura marginata . J Nat Prod. 1997;60(9):900–903.doi:10.1021/np9701844

35.

Sheu

JH.

Wang

GH.

Sung

PJ.

Duh

. New cytotoxic oxygenated fucosterols from the brown alga Turbinaria conoides . J Nat Prod. 1999;62(2):224–227.doi:10.1021/np980233s

36.

Gagosian

RB.

Smith

SO.

Nigrelli

. Vertical transport of steroid alcohols and ketones measured in a sediment trap experiment in the equatorial Atlantic Ocean. Geochim Cosmochim Acta. 1982;46(7):1163–1172.doi:10.1016/0016-7037(82)90002-3

37.

Kokke

WCMC.

Fenical

Djerassi

. Sterols of the cultured dinoflagellate Pyrocystis lunula . Steroids. 1982;40(3):307–318.doi:10.1016/0039-128X(82)90042-3

38.

Kokke

WCMC.

Fenical

Djerassi

. Sterols with unusual nuclear unsaturation from three cultured marine dinoflagellates. Phytochemistry. 1981;20(1):127–134.doi:10.1016/0031-9422(81)85231-4

39.

Kerr

RG.

Baker

. Marine sterols. Nat Prod Rep. 1991;8(5):465–497.doi:10.1039/np9910800465

40.

Elenkov

Dragova

Andreev

Popov

. 4α-Methyl sterols from the sponges Haliclona cinerea and Haliclona flavescens . Comp Biochem Physiol B: Biochem Mol Biol. 1997;118(1):155-157.doi:10.1016/S0305-0491(97)00029-1

41.

Santalova

EA.

Makarieva

TN.

Ponomarenko

et al . Sterols and related metabolites from five species of sponges. Biochem Syst Ecol. 2007;35(7):439–446.doi:10.1016/j.bse.2006.10.020

42.

Berti

Bottari

Marsili

Morelli

Polvani

Mandelbaum

. 31-Norcycloartanol and cycloartanol from Polypodium vulgare . Tetrahedron Lett. 1967;8(2):125–130.doi:10.1016/S0040-4039(00)90500-8

43.

Itoh

Ishii

Tamura

Matsumoto

. Four new and other 4α-methylsterols in the seeds of Solanaceae. Phytochemistry. 1978;17(5):971–977.doi:10.1016/S0031-9422(00)88659-8

44.

Ferezou

JP.

Devys

Allais

JP.

Barbier

. C_₂₆ sterols. IX. C_₂₆ sterol from the red alga Rhodymenia palmata . Phytochemistry. 1974;13(3):593-598.

45.

Shameel

Afaq-Husain

Zarina

. Phycochemical studies on seven species of Rhodophycota from Karachi coast of Pakistan. Int J Algae. 2013;15(3):285–290.doi:10.1615/InterJAlgae.v15.i3.80

46.

Santalova

EA.

Denisenko

. Fatty acids from a glass sponge Aulosaccus sp. Occurrence of new cyclopropane-containing and methyl-branched acids. Lipids. 2017;52(1):73–82.doi:10.1007/s11745-016-4214-1

47.

Taylor

MW.

Radax

Steger

Wagner

. Sponge-associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev. 2007;71(2):295–347.doi:10.1128/MMBR.00040-06

48.

Kreit

. Microbial catabolism of sterols: focus on the enzymes that transform the sterol 3β-hydroxy-5-en into 3-keto-4-en. FEMS Microbiol Lett. 2017;364(3):1-9.doi:10.1093/femsle/fnx007

49.

Park

HB.

Tuan

NQ.

et al . Sesterterpenoid and steroid metabolites from a deep-water Alaska sponge inhibit Wnt/β-catenin signaling in colon cancer cells. Mar Drugs. 2018;16(9):297/1–297/15.doi:10.3390/md16090297

50.

Santalova

ЕА.

Denisenko

VА.

Dmitrenok

PS.

Drozdov

АL.

Stonik

. Cerebrosides from a Far-Eastern glass sponge Aulosaccus sp. Lipids. 2015;50(1):57-69.doi:10.1007/s11745-014-3974-8

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

3.00 MB

0.00 MB