J. prakt. Chem. 337 (1995) 397-400

Journal fur praktische Chemie C hemi ker -Zei tung

0 Johann Ambrosius Barth 1995

Chemical Analysis of the Sponge Suberites Massa Coming from the Lagoon of Venice: Isolation of a New Diacetylenic Carotenoidl)

Anna Aiello, Ernesto Fattorusso, and Marialuisa Menna Naples (Italy), Dipartimento di Chimica delle Sostanze Naturali

Maurizio Pansini Genova (Italy), Istituto di Zoologia

Received December 7th, 1994

Abstract. A new carotenoid pigment (1) containing a di- acetylenic function was isolated from a sample of Suberites mama collected in the Lagoon of Venice. Its structure and absolute configuration have been elucitated by spectroscopic

Within a scientific project devoted to study the peculiar ecological habitat of the lagoon of Venice, we are inves- tigating the marine invertebrates present in the lagoon basin. As reported in a previous paper [l], we have car- ried out the analysis of the chemical composition of the three most common species of Porifera present in the la- goon (Tedania anhelans, Hymeniacidon sanguinea, Hal- icondria bowerbanki).This analysis pointed to a plank- tonic origin of some metabolites isolated from the spec- imens coming from the lagoon. In the course of our study we have now analyzed the chemical composition of the sponge Suberites massa on which no chemical data were available. We wish to report here the analy- sis of the most abundant lipophylic metabolites of this organism which resulted to be sterols and fatty acids. Furthermore, the sponge elaborates a carotenoid pig- ment, 1 (suberixanthin), which has not been previously reported as a naturally occurring compound. In this pa- per, we report the isolation and structure determination of 1.

Suberites mama (Class: Demospongia, Order: Hadro- merida, Family: Suberitidae) described by Nardo [2] from the canals of the lagoon of Venice, has generally a massive form, characterized by the presence of finger- like lobate processes, which do not diverge from the sponge body. The basal part of the sponge is yellow- orange while the upper parts, more exposed to light, become brownish. The species thrives in very shallow waters, as those of estuaries, coastal ponds and lagoons and are known from the Mediterranean and the At-

l) This work was performed within the scientific project "Sis- tema Lagunare Veneziano", research line n. 3.02.

method including 2D-NMR experiments. Also reported here is the analysis of the most abundant lipophylic metabolites of this organism.

lantic. A very close species, Suberites carnosus Johnston, which was alternatively considered to be synonymous of S. massa, was definitely distinguished as a separate species by Levi [3] just in force of its different typically marine habitat.

The sponge S. massa, collected in lagoon of Venice, was extracted with acetone and the EtOAc soluble ma- terial was chromatographed on a SO2 column using in- creasing concentrations of ethyl acetate in hexane as eluent. The crude sterol band, eluted with hexane/ethyl acetate 7:3, was acetylated and analyzed with GC-MS to determine the sterol profile of the sponge. The ob- tained results (see Table 1) showed that cholestanol was the major sterol in the sponge.

Table 1 Sterol composition of S. massa

Compound %

cholesterol 24-methylcholest-5-en-3P-ol cholestanol 24-methylcholest-24(28)-en-3P-ol 24-methyl-3P-cholestanol

12.6 3.8

76.1 3.4 4.1

The EtOAc extract of S. mama revealed to contain a high percentage of 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid (EPA). This compound was found to be also the major component of the fatty acid fraction of T an- helans, H. sanguinea and H. bowerbanki [l]. Analo- gously to the analysis carried out on these sponges, we separately examined two samples of Suberites mama collected in different periods of the year (spring and

J. prakt. Chem. 337 (1995) 398

4'

20' 19'

4

1

4' OH

H 4

2

Fig. 1 Structures of suberixanthin (1) and alloxanthin (2). Table 2 13C and I H NMR data of compound la) and 2

autumn) in order to verify if the content of EPA in the 1 2 sponge is related to the seasonal variation of microflora and /or microfauna of the lagoon of Venice. The ob- served decrease of the content of EPA in the autumn sample (Spring sample: 0.4%; Autumn sample: 0.3% dry weight) is in good agreement with the hypothesis of an exogenous origin of this metabolite.

The fraction eluted by a MPLC column with light petroleum/EtOAc 8:2 showed to contain carotenoid pig- ments. Purification of this fraction on reverse phase HPLC gave 10 mg of pure suberixanthin (1).

The HREIMS spectrum of 1 showed a molecular ion at m/z 592.4279 appropriate for a molecular formula C42H5602 (calcd. 592.4283).The UV spectrum of suber- ixanthin showed absorption maxima at 428 and 480 nm, typical for carotenoids; features of th; IR (u,,,, 2165 cm-I) and 13C NMR spectrum [ S 89.17 (s) , 98.54 (s)] pointed to the presence of a disubstituted acetylenic group.The signals in the I3C NMR spectrum of 1 match closely the corresponding ones of alloxanthin (2) [4], a diacetylenic carotenoid isolated from flagellates of the algal class Cryptophycaee [5, 61. Differences were con- fined in the resonances of C-3, which was shifted down- field from 6 64.80 to 73.46, and of C-2 and C-4 shifted upfield from S 46.7 and 41.5 to S 42.76 and 38.29 , re- spectively (see Table 2).

An extra signal at S 55.72 was also present, indicating that 1 contained a methoxy functionality; this was con- firmed by the presence in the lH NMR spectrum of a singlet (3H) at 6 3.37.

Position

1 2

3 4

5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20

OCH3

13c

36.2 42.8

73.5 38.3

137.4 124.3 89.2 98.5

119.1 138.0 124.3 135.1 136.4 133.4 130.3 28.7 30.5 22.6 18.0 12.8

55.8

'H -

Hax: 1.38, dd (12, 11.5) Heq: 1.89, dd (12, 4)

3.49, m Hax: 2.05b)

Heq: 2.46, dd (18, 7)

- 6.35, d (15)

6.52, dd (15, 12) 6.45, d (12)

6.27, bd (11.5) 6.63, bd (11.5)

1.19, s 1.14, s 1.92, s 2.00, s 1.96, s 3.37, s

13 c 36.9 46.7

64.8 41.5

137.2 124.3 89.1 98.8

119.1 138.0 124.3 135.2 136.4 133.4 130.3 28.8c) 3 0 3 ) 22.4 18.0 12.7 -

1H

1.19:s 1.14:s 1.90, s 1.99, s 1.94, s

-

a) Assignments were determined by analogy to model com- pounds and by lH-'H COSY, HMQC, and HMBC NMR experiments. IH (500 MHz) and I3C (125 MHz) NMR spec- tra were recorded in CDC13. Proton coupling constants (J) are given in Hertz. b, Submerged by other signals. c,d) The resonances with the same superscript may be reversed.

A. Aiello et al., Isolation of a New Diacetylenic Carotenoid 399

All the above data strongly suggested that 1 was 3,3'-dimethoxy-7,8,7',8'-tetradehydro-P,p-carotene; this structure was amply confirmed through an extensive two dimensional analysis (lH-lH COSY, 13C-lH shift corre- lation via lJ and 2,3J) which allowed us t o assign the resonances of the 13C and 'H NMR spectra (see Table 2). The 3s - and 3's-chirality of 1 were suggested by its CD-spectrum which showed opposite features to that of the structurally related alloxanthin (Fig. 2), possessing 3R,3'R-chirality [7].

Mass spectral data were provided by "Servizio Spettrometria di Massa del CNR e dell'universith di Napoli".The assistance of the staff is greatly appreciated. N.m.r. spectra were per- formed at "Centro Interdipartirnentale di Analisi Strumen- tale", University of Naples "Federico 11". We thank Mr. G. Scognamiglio, Istituto per la Chimica di Molecole di Interesse biologic0 del CNR, Arc0 Felice (Naples), Italy, for the CD spectrum.

Experimental

General Methods

The CD spectrum was recorded in EtzO/Zpentane/EtOH 5:5:2 solution at 20°C on a JASCO 5710 spectropolarime- ter. The UV spectrum was recorded on a Beckman DU 70 instrument (diethyl ether solution ) and the FT-IR spectrum on a Bruker IFS-48 spectrophotometer in a KBr pellet. The high resolution mass spectrum (HRMS) was obtained by elec- tron impact at 70eV on a Kratos MS 50 mass spectrometer.

'H and 13C NMR spectra were run on a Bruker AMX-500 spectrometer in CDC13 and the solvent was used as an inter- nal standard (lH: 6 7.26; I3C: 6 77.0). The multiplicities of the 13C resonances were determined by DEFT experiments which were performed using polarization transfer pulses of 90" and 135", obtaining in the first case only signals for CH groups and in the other case positive signals for CH and CH3 and negative ones for CH2.

The proton-proton correlation (COSY) spectrum was col- lected as 2048 x 1024 points and processed using a sinusoidal multiplication before the Fourier transformation of both do-

mains.The l H detected multiple quantum heteronuclear cor- relation (HMQC) spectrum was performed using a pulse se- quence developed by Bax and Subramanian [8] using a BIRD pulse of 0.35 seconds before each scan to suppress the 'H res- onances not directly bonded to 13C. The data matrix of 2048 x 512 points was processed using an exponential multiplica- tion in both F2 and F1 domains. Zero filling in F1 before the Fourier transformation led to the final data matrix of 2048 x 1024 points. The interpulse delays were adjusted for an aver- age ~Jc-H of 125 Hz. The l H detected heteronuclear multiple bond correlation (HMBC) spectrum was performed according to Bax and co-workers [9]. The data processing was identical to that used for the HMQC experiment.

Medium pressure liquid chromathography (MPLC) was performed on a Buchi 861 apparatus using a Si02 (Merck, 230-400 mesh) column.

High performance liquid chromatography (HPLC) was per- formed on a Varian 5000 apparatus equipped with a RI-3 refractive index detector using a Hibar RP-18 LiChrospher super 100 column.

Combined GLC-MS analysis was performed on a Hewlett- Packard 5890 chromatograph with a mass selective detector MSD HP 5970 MS and a splitkplitness injector for capillary columns using a fused -silica column, 25m x 0.2mm HP 5 (cross-linked 5 % PhMe Silicone, 0.33 pm film tickness).

Collection and Extraction

Specimens of Suberites massa were collected by scraping the solid surfaces present in the lagoon of Venice in dif- ferent locations ( Isola Sacca Sessola, Isola Poveglia, Riva del Fontego dei Turchi) during two expeditions (March 1992; October 1992). They were frozen immediately after collec- tion; a voucher specimen is deposited at the Dipartirnento di Chimica delle Sostanze Naturali-Universith di Napoli "Fed- erico 11".

The fresh thawed sponge (15.8 g dry weight after extrac- tion) was homogenized and extracted with acetone (300 ml x 5 ) at room temperature. The extracts were evaporated in vacuo to give an aqueous phase which was extracted with EtOAc (100 ml x 3). Evaporation of the combined EtOAc extracts afforded 2 g of a brown residue which was chro- matographed by MPLC on a Si02 column eluting with sol- vents of increasing polarity (hexane -+ EtOAc + MeOH).

c

/ - 8 / /

- / - -4

I / '. /

/ /

1 WL Cnml 400.0

-6.000E-01 iv , , , I , , , , , , ' , , , , , , , , , , , 200.0

Fig. 2 CD spectra of 1 (-) and 2 (---)

400 J. prakt. Chem. 337 (1995)

Analysis of the Sterol Fraction

Fractions eluted with hexane/EtOAc 7:3 from the Si gel col- umn of the EtOAc extract provided the crude sterol mixture (1.2% dry weight). Sterols were characterized by GLC-MS analysis of their acetate derivatives prepared by treating with acetic anhydride and pyridine (1:l) for 18h at room temper- ature.

The steryl acetates were filtered on a Si gel column and eluted with hexane containing increasing amounts of Et2O. The fraction eluted with Et20/hexane 8:2 was further ana- lyzed by GLC-MS. The identification of the steryl acetates was based on comparison of the GC-MS spectrum with those of authentic specimens. The quantitation of sterols was per- formed by a programmable integrator using Sa-cholestane as an internal standard. Individual sterols and their percentage are reported in Table 1.

Isolation of 5Z,8Z,11 Z,14Z,17Z-eicosapentaenoic Acid (EPA).

EPA was obtained pure from the Si gel chromatography of the EtOAc extract eluting with hexane ethyl acetate 1:l. It was identified by comparison of its spectroscopic data with those reported in the literature [lo 3.

Additional amounts from a more lipophylic fraction eluted with hexane EtOAc 9:l as methyl ester.

Isolation of 3,3'-dimethoxy-7,8,7',8'-tetrahydro-p, p-carotene

Fractions (132 mg) eluted with hexane/EtOAc 8:2 were fur- ther purified by HPLC on a RP-18 column (MeOH/H20 98:2) thus obtaining 10 mg of pure (1): UV Amax (Et20) 428 (sh), 452, 480; IR urnax (KBr) 3300, 2165 and 960 cm-l; HREIMS d z 592.4279 [M'] calcd. 592.4283 for

(1)

C42H5602; CD (Et2012-pentaneEtOH) nm (As) 232 (+0.59), 250 (+0.62), 277 (+0.32), 393 (+0.6);'H and 13C NMR data : see Table 2.

References

A.Aiello, E.Fattorusso, M.Menna, M. Pansini, Biochem. Syst. Ecol. 21 (1993) 655 Nardo,"Prospetto della fauna del veneto estuario. Es- tratto dall'opera: Venezia e le sue 1agune.Venezia" 1-45 (1847) C.Levi, Remarques sur la faune des spongiaires de Roscoff. Arch. Zool. Exp.Gen.,Paris, N.R. 87 (1950) 10 G.P. Moss, Pure and Appl. Chem. 47 (1976) 97 D. J. Chapman, F. T. Haxo, Plant Cell Physiol. 4 (1963) 56; M. B. Allen, L. Fries, T. W. Goodwin, D. M. Thomas, J. Gen. Microbiol. 34 (1964) 259 D. J. Chapman, Phytochemistry 5 (1966) 1331 T. Matsuno, M. Ookubo, T. Komori, J. Nat. Prod. 48 (1985) 606 A. Bax, S. Subramanian, J Magn. Res. 67 (1986) 565 A. Bax, F. Summers, J. Am. Chem. SOC. 108 (1986) 2093 ; A. Bax, A. Aszalos, Z . Dinya, K. Sudo,J.Am.Chem. SOC. 108 (1986) 8056 P. Ciminiello, E. Fattorusso, S. Magno, A. Mangoni, A. Ialenti, M. Di Rosa, Experientia 47 (1991) 739

Address for correspondence: Prof. E. Fattorusso Dipartimento di Chimica delle Sostanze Naturali Via D. Montesano 49 1-80131 Naples, Italy