Variations in the halogenated metabolites of Laurencia obtusa from eastern Sicily

Biochemical SystemadcsandEco/ogy, Vol. 9, No. 4, pp. 241-246, 1981. Printed in Great Britain.

0305-1978/81/040241~36 $02.00/0 ©1981 PergamonPress Ltcl

Variations in the Halogenated Metabolites of Laurencia obtusa from Eastern Sicily SALVATORE CACCAMESE, ROBERTO AZZOLINA, ROSA MARIA TOSCANO and KENNETH L. RINEHART, JR.* Department of Chemistry, University of Catania, 95127 Catania, ~taly; *Roger Adams Laboratory, University of Illinois, Urbana, IL 61801, USA Key Word Index - Laurencia; halogenated metabolites; gas chromatography/mass spectrometry. Abstract - Lipid extracts of Laurencia obtusa from eastern Sicily were examined by gas chromatography/mass spectrometry in order to investigate their composition in halogenated products and a marked variability has been found in specimens from different sites, while life history or season had a minor influence. In different habitats and types of substrate and flora, within a few kilometres distance, a completely different array of metabolites has been observed. Some of these compounds, namely obtusin, obtusadiol and obtusenyne, were reported in L. obtusa from other Mediterranean locations. A new compound, laurencienyne, is a major component in individual specimens collected at Castelluccio, whereas it is absent in specimens from other sites.

Introduction The red alga Laurencia obtusa (Huds.) Lamour., whose type locality is England, is widely distributed in other geographical areas, such as the western Pacific [1], Indian [2] and Atlantic [3] Oceans and Mediterranean Sea [4-6] and dozens of varieties are reported on the basis of morphological keys. This species is a rather prolific source of halo: genated metabolites [7] and from the Mediterranean Sea several sesquiterpenoids, diterpenoids and non-terpenoid acetylenes have been recently isolated [8-14]. A marked variation in the major chemical constituents from this species collected at different locations has been observed. In the course of an extensive screening for antimicrobial activity in algae from eastern Sicily [15], we became interested in Laurencfa obtusa because of the good antimicrobial activity, invariably exhibited by its extracts, regardless of collection site or sexual stage. In the chemical investigation for isolating the active components from the crude extracts, we soon observed a marked variation in the TLC and GC profiles of specimens collected at different sites, and thus we resorted to gas chromatography/mass spectrometry (GC/MS) to investigate this peculiarity further. In this paper, some results on the chemical variations of the halogenated metabolites in L. obtusa from eastern S icily coasts will be presented.

Results and discussion Combined GC/MS is a valuable tool in characterizing individual plants, providing not only a fingerprint but also structural information on the individual GC peaks. Chemical ionization GC/MS is a useful ancillary technique to provide the molecular ion in such cases where this information is lacking, as in naturally occurring non-aromatic compounds, with unusual side-chains or strained alicyclic rings, possessing an intrinsic lability to the heat or to the electron impact (El). Moreover, GC alone could be inconclusive for the identification of a peak since different compounds can give identical retention times. Thus, for chemical identification we employed GC/MS and careful GC co-injections with authentic standards. Using these criteria, we extensively examined a large number of extracts of individual specimens of Laurenc/a obtusa. This species was identified by examination of obvious morphological characters. Specimens from different locations did not show, at visual observation or under magnification, differences in arrangement and shape of branchlets, apices and tetrasporangia. The only noticeable difference was in the length of the thalli, the plants from Capo Murro di Porco appearing as small (2-3 cm) dense orange-brown clumps, while those from Brucoli were broad orange-green erect forms (10-15 cm). Specimens from Castelluccio and Portopalo were

(Received 26 November 1980; received for pub#cation 9 April t981 ) 241

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of intermediate size, possessing the same morphological keys. The GC/MS traces of the L. obtusa samples from the four above-mentioned sites are shown in Fig. 1 (lettering of the peaks is specific for each location). Although no morphological differences could be observed among the four samples, beside thalli dimensions and colour, significant differences in their G C/M S profiles were observed. In the sample from Castelluccio, component E is one of its major constituents whereas it is absent in samples from the other locations. It has been identified as taurencienyne (1) by comparison of the retention time to that of the pure compound [14] and by GC/MS in El and CI modes. Component D has a MS identical to that of 1 and we propose it to be theZ-isomer. In fact, a GC trace of pure E-isomer 1, left under daylight for 1 h in methylene chloride solution, showed the accompanying preceding peak that, by UV and NMR changes, can be attributed to the Z-form. Peaks B and C exhibited identical M S and a molecular ion at m / z 330 containing one bromine and one chlorine was found from GC/CIMS, although it was almost absent in the El mode. Component B has been identified as obtusenyne (2) by GC co-injection with an authentic sample

[8] in isothermal mode (see later) and by comparison of their MS. Component C is probably the E-isomer of 2. Cl

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HALOGENATED METABOLITES OF LAURENCIA OBTUSA

243

and a GC co-injection with 2 in isothermal mode (see later) ruled out this assignment. In fact, the MS did not exhibit a molecular ion at m/z 330 and other mass spectral features were different. Thus the major component B from Brucoli remains a still unknown halogenated metabolite. Recently we isolated it, and from NMR and UV data it does not contain the enyne functionality characteristic of compounds 1 and 2. Its structural elucidation is in progress. To the component C we assign the structure of obtusadiol (3), by both GC co-injection of authentic sample [9] and GC/MS data. The sample from Portopalo shows obtusenyne 2 (peak B) and its probable E-isomer (peak C), but no evidence for compound 1 was found. The most interesting fingerprint is shown by L. obtusa from Capo Murro di Porco, where only obtusin 4 [10] was identified with the usual criteria as the component D.

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Peaks B and C (two probable isomers from the identity of MS) show,, at first glance, retention times very similar to that of 1 and its probable isomer identified in the sample from Castelluccio, but also in this case GC/MS data ruled out this possibility, indicating still unknown metabolites with a molecular ion at m/z 436 having a characteristic monobromomonochloro cluster. These GC tracings from unsorted collections from the different locations are reproducible, casting out doubts about inhomogeneous collections. Besides the compounds previously discussed, n-heptadecane has been identified in peak A from all sites, while peak F from Castelluccio, D from Portopalo, E from Capo Murro di Porco and D from Brucoli have been identified as cholesterol. Both compounds are common constituents of most Rhodophyta. Moreover, a careful examination of the GC/MS scanning of the unlabelled peaks in

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246

SALVATORECACCAMESE, ROBERTOAZZOLtNA, ROSA MARIA TOSCANO AND KENNETH L RINEHART, JR

and it was identical for all examined samples. An algal sample (10 g, wet wt) was homogenized for 10 min in a Waring blender in 50 ml of toluene-MeOH (1:3), then centrifuged to remove insoluble material. To the remaining extract 20 ml of 1 N NaNO 3 solution was added for partition of the lipophylic and hydrophylic components and to desalt the organic layer which was separated by centrifuging, evaporated under nitrogen and weighed. The lipid weight was usually 1.2+ 1.5% of the weight of the wet alga. The extracts prepared following this procedure were stored at - 2 0 ° and redissolved in an opportune amount of methylene chloride before GC analyses. Not significant differences in the GC traces of specimens after prolonged storage were detected. GC and MS. GC analyses were performed on a Varian Model 1700 gas chromatograph and on a Carlo Erba Model 2530 gas chromatograph, both equipped with a hydrogen flame ionization detector using helium as the carrier gas. GC/ ElMS and GC/CIMS were performed on a Varian MAT mass spectrometer Model 311 A, equipped with a combination EICI (chemical ionization, isobutane as reactant gas) ion source, a Varian Model 2700 gas chromatograph and a Varian M S data system Model SS 100. The GC conditions were as follows, unless otherwise specified: 3% OV 17 on 100-120 mesh GCQ, 6 ft × 2 mm i.d glass column; flow 35 ml/min; attenuation 1 x 10 10, 10 mV; temp. programme10 °/rain (150 290 ° )

Acknowledgements-The authors thank Drs. G. Furnari and M. Cormaci (Institute of Botany, University of Catania) for identification of plant material. We wish to thank D r B Howard for authentic samples of obtusin and obtusadiol and Dr. A. Oztunc for an authentic sample of obtusenyne. This work was supported in part by a NATO Research Grant (SRG 16, to S. C. and K. L. R.)andin part byNIH Research GrantsAI 04769 and GM 27029 (to K L.R.) and National Research Council Grant CT 79.01676.03 (to S C )

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