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Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata (Fucales, Phaeophyta)
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Biochemical Systematics and Ecology 36 (2008) 484e489 www.elsevier.com/locate/biochemsyseco
Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata (Fucales, Phaeophyta) Mohamed El Hattab a, Mohammed Ben Mesaoud b, Mohammed Daoudi b, Annick Ortalo-Magne´ c, Ge´rald Culioli c, Robert Valls d, Louis Piovetti c,* a
Laboratoire des Plantes Aromatiques et Me´dicinales, De´partement de Chimie, Faculte´ des Sciences, Universite´ de Blida, BP 270, Blida, Algeria b Laboratoire de Chimie Organique et Bioorganique, Faculte´ des Sciences Choua€ıb Doukkali, El Jadida, Morocco c Laboratoire des Mate´riaux a` Finalite´s Spe´cifiques (MFS), The´matique «Biofouling & Substances Naturelles Marines», Universite´ du Sud Toulon-Var, Avenue de l’Universite´, BP 20132, F-83957 La Garde Cedex, France d UMR 6180 CNRS «Chirotechnologies: Catalyse et Biocatalyse», Groupe «Se´paration, Identification, Synthe`se», Universite´ Paul Ce´zanne, Aix-Marseille III, Avenue Escadrille Normandie-Niemen, Service 551, F-13397 Marseille Cedex 20, France Received 27 November 2007; accepted 19 December 2007
Keywords: Sargassaceae; Phaeophyceae; Bifurcaria bifurcata; Brown alga; Trihydroxylated linear diterpenes; Chemotaxonomy
1. Subject and source The most prolific species of marine algae concerning the number of metabolites are those belonging to the Rhodomelaceae family for the red algae and those of the Dictyotaceae in the case of brown seaweeds. Important contribution has also been provided by Sargassaceae, in particular with the genus Cystoseira. For these two last families, compounds are different: for the Dictyotacean species, cyclic diterpenes have been isolated while acyclic diterpenoids and meroditerpenoids have been described for the Sargassaceae (Blunt et al., 2007). In the course of our phytochemical study on Mediterranean and Atlantic brown algae (Culioli et al., 2004, and references cited therein; El Hattab et al., 2008; Ortalo-Magne´ et al., 2005; Valls and Piovetti, 1995; Valls et al., 1995), we have re-investigated the polar fraction of the lipid extract from Bifurcaria bifurcata (Velley) Ross collected at Oualidia (Morocco) in September 2002, with the object of studying undescribed trihydroxylated linear diterpenes from this species. A voucher specimen of this species (No. P192.7) was deposited in the herbarium of Dr. Pellegrini, Laboratoire de Biologie Marine Fondamentale et Applique´e, Universite´ de la Me´diterrane´e, Marseille, France. 2. Previous work In the algal family Sargassaceae1 (order Fucales, class Phaeophyceae), Bifurcaria is a relative small genus by contrast to the main genera Sargassum, Cystoseira and Turbinaria. It currently includes only three species: B. bifurcata * Corresponding author. Tel.: þ33 4 94 14 23 46; fax: þ33 4 94 14 21 68. E-mail address: [email protected] (L. Piovetti). 1 The Cystoseiraceae species are now merged into the large Sargassaceae family (Rousseau and De Reviers, 1999; Cho et al., 2006). 0305-1978/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.12.007
M. El Hattab et al. / Biochemical Systematics and Ecology 36 (2008) 484e489
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(Velley) R. Ross, distributed on Atlantic coasts from Morocco (southern limit) to north-western Ireland (northern limit) (Ross, 1961); Bifurcaria brassicaeformis (Ku¨tzing) Barton confined to the western (Stegenga et al., 1997) and Indian Ocean coasts of South Africa (Silva et al., 1996); and Bifurcaria galapegensis (Piccone & Grunow) Womersley (Womersley, 1964) which is endemic to the Galapagos archipelago. Previous studies of the secondary metabolites from lipid extracts of these three species have shown that B. bifurcata contains a rich array of acyclic diterpenes (Biard et al., 1980; Combaut and Piovetti, 1983; Culioli et al., 1999a,b, 2000, 2001, 2004; Daoudi et al., 2001; Hougaard et al., 1991a,b; Ortalo-Magne´ et al., 2005; Semmak et al., 1988; Valls et al., 1993a,b, 1995). By contrast, the main constituent of the lipid extract of B. galapegensis is a meroditerpene (bifurcarenone) (Sun et al., 1980; Mori and Uno, 1989), while B. brassicaeformis shows the total absence of diterpenes in its lipid extract (Daoudi et al., 2001). During our chemotaxonomic studies on B. bifurcata (Combaut and Piovetti, 1983; Culioli et al., 1999a,b, 2000, 2001; Semmak et al., 1988; Valls et al., 1986, 1993a,b, 1995), including the geographical variation of its diterpenoid composition (Valls et al., 1993a,b, 1995), we have investigated the moderately polar fraction of the lipid extract obtained from specimens of the alga collected in different locations off the Moroccan, Spanish, French and Irish Atlantic coasts. The acyclic diterpenes obtained were mono-and dioxygenated geranylgeraniol derivatives in which the second oxygenated function was located at C-12 or C-13 depending on the place where the alga was collected (Valls et al., 1993a,b, 1995). 3. Present work We have recently started a study of the polar fraction of the lipid extracts of the alga in order to isolate trioxygenated acyclic diterpenes and to verify if our previous chemotaxonomic data on this species (Valls and Piovetti, 1995; Valls et al., 1993a,b, 1995) are applicable to these compounds. From this work, two trihydroxylated linear diterpenes derived from 12-hydroxygeranylgeraniol were isolated from a specimen of B. bifurcata collected at Oualidia (Morocco), (Culioli et al., 2004), and five polar linear diterpenes derived from 13-oxo- and 13-hydroxygeranylgeraniol were described from a specimen of B. bifurcata collected off the Atlantic coast of Southern Brittany (Quiberon, France), (Ortalo-Magne´ et al., 2005). The lipid extract of shade-dried B. bifurcata collected near Oualidia was re-investigated with the object of isolating the polar diterpenes undescribed in this extract. The CHCl3eMeOH extract of 83 g (2.63% of dried alga) was partitioned in the mixture MeOHeisooctane (1:1) leading to a MeOH extract which was then dissolved in the mixture MeOHeCHCl3eH2O (4:3:1). About 20.5 g of organic extract obtained from the last extraction was fractionated on silica gel column. The fractions eluted with EtOAceisooctane (1:1 to 3:1) contained sterols, geranylgeraniol, monohydroxylated diterpenes (5.6 g) and the dihydroxylated diterpene bifurcadiol (1) (5.5 g) previously described (Culioli et al., 2001). The fractions eluted with EtOAc and EtOAceMeOH (98:2 and 95:5), respectively, were further purified by HPLC on a C-18 reversed-phase column, eluting with MeCNeH2O (1:1 and/or 2:3). From this separation we obtained the two trihydroxylated linear diterpenes 2 (200 mg) and 3 (55 mg) previously identified in this extract (Culioli et al., 2004) and two new compounds 4 (15 mg) and 5 (19 mg). Diterpene 4 (Fig. 1), C20H34O3 (HRMS), was an optically active oil ([a]20 D ¼ 3.5 (c 0.2, CH2Cl2)) which showed several spectral features in common with geranylgeraniol-derived diterpenes, particularly with bifurcadiol (1) and compound 3. It showed a strong hydroxyl absorption (nOH ¼ 3372 cm1) in its IR spectrum. Inspection of the 1H and 13C NMR data (Tables 1 and 2) compared with those of bifurcadiol (1) and compound 3 showed that the last two isoprenic units (C-9 to C-18) and a part of the first (C-1 to C-3 with C-20) of the three molecules were similar. In contrast, the main differences were present in the second isoprene unit: (i) a tertiary hydroxyl group with a 13C signal at d 72.9 ppm (quaternary sp3 carbon); (ii) a methyl group on the latter sp3 carbon with a 1H signal at d 1.32 ppm (s, H-19); (iii) two ethylenic methine groups with 13C signals at d 125.3 and 138.8 ppm and 1H signals at d 5.57 ppm (d, H-6) and d 5.65 ppm (dt, H-5); (iv) a methylene group with a 1H signal at d 2.77 ppm (d, H-4). The E configuration of the double bond at C-5 was revealed by the value of the coupling constant between H-5 and H-6 (3JHeH ¼ 15 Hz) obtained by irradiating the protons at d 2.77 ppm which led to an AB system at d 5.57e 5.65 ppm (JAB ¼ 15 Hz). The assignment of proton and carbon signals (Tables 1 and 2, respectively) was confirmed by means of 1He1H homonuclear (COSY, NOESY) and 1He13C heteronuclear (HSQC, HMBC) 2D experiments, and by DEPT sequences. In particular, the location of the double bond at C-5 was precisely determined with the 3JHeH correlation between H-4/H-5 and the 4JHeH correlation between H-4/H-6, as well as the location of double bounds at C-2, C-10 and C-14 with the 4JHeH correlations between H-2/H-20, H-10/H-18 and H14/H-16, H-14/H-17, respectively. The NOE correlations between H-2/H-4 and H-1/H-20 on one hand and between H-10/H-12 and H-9/H-18 on
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OH 16
14 15 17
10 13
12
11
6
8 9
5
7
18
4
19
2 3
OH 1
20
1 OH
OH
OH
2 OH OH OH 3 OH OH OH 4 OH OH OH 5
OH
6 Fig. 1.
the other hand, clearly showed the E configuration of double bonds at C-2 and C-10, respectively. On the basis of biosynthetic considerations, we have attributed an R configuration to the secondary alcohol C-12, like for compounds 1 and 3 present in the same extract, whereas the configuration at C-7 remains to be ascertained (Culioli et al., 2004). The second new compound 5 (Fig. 1) had the molecular formula C20H36O3 (HRMS). It was isolated as an optically 1 active oil ([a]20 D ¼ 11.0 (c 0.2, CH2Cl2)). Like 4, it showed a strong hydroxyl absorption (nOH ¼ 3362 cm ) in its 1 13 IR spectrum. Inspection of the H and C NMR data (Tables 1 and 2) compared with those of 3 showed that the two first isoprenic units of the two molecules were similar. The only one difference was the upfield shift of C-7 (Dd ¼ 10 ppm) for compound 5. Moreover, the further inspection of spectra showed that the last isoprenic unit was similar to the one of compound 6 previously described for this extract (Culioli et al., 2001). In contrast, the main
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Table 1 1 H NMR data for compounds 1, 3e6a, b H
1c CDCl3, 360 MHz
3d CDCl3, 400 MHz
4 CDCl3, 250 MHz
5 CDCl3, 400 MHz
6e CDCl3, 200 MHz
1 2 4 5 6 8
4.09 5.35 1.99 2.07 5.07 1.99
4.12 d (6.9) 5.38 t (7.0) 1.99 t (7.0) 1.40e1.48 m 1.38e1.44 m 1.45e1.50 m
4.19 5.46 2.77 5.65 5.57 1.58
4.15 d (7.0) 5.42 t (7.0) 2.00e2.20 m 2.00e2.20 m 5.15 t (7.0) 2.75 d (7.0)
9
2.07 m
2.05 m
2.08 m
10 12 13 14 16 17 18 19 20
5.32 3.92 2.20 5.04 1.68 1.60 1.57 1.56 1.63
5.37 t (7.0) 3.94 t (6.7) 2.20e2.28 m 5.05 t (7.2) 1.69 s 1.60 s 1.60 s 1.14 s 1.64 s
5.43 4.00 2.21 5.11 1.75 1.66 1.64 1.32 1.70
4.15 d (6.8) 5.41 t (7.0) 2.02 d (6.5) 1.45e1.50 m 1.45e1.50 m 1.68 m 1.80 m 1.78 m 1.91 m 4.25 t (7.0) 5.41 t (7.0) 2.71 t (7.0) 5.10 t (7.0) 1.68 s 1.62 s 1.62 s 1.22 s 1.67 s
a b c d e
d (6.8) t (6.8) m m t (6.8) m
t (6.8) t (6.7) dd (6.7; 7.0) t (7.0) s s s s s
d (6.8) t (6.8) d (5.0) dt (15.0; 5.0) d (15.0) m
t (6.6) t (7.0) m t (7.0) s s s s s
5.51 dt (15.0; 7.0) 6.05 5.35 2.81 5.11 1.68 1.64 1.75 1.59 1.68
d (15.0) t (7.0) t (7.0) t (7.0) s s s s s
d in ppm (TMS as int. standard), coupling constants (J in parentheses) are given in Hz. Assignments were confirmed by decoupling and 2D NMR experiments (COSY 1He1H, 1H J-resolved, HMQC and HMBC). 1 H NMR data of Semmak et al. (1988) (added for comparison). 1 H NMR data of Culioli et al. (2004) (added for comparison). 1 H NMR data of Culioli et al. (2001) (added for comparison).
Table 2 13 C NMR data for compounds 1, 3e6a C
1b CDCl3, 90 MHz
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
59.2 123.5 139.1 39.4 26.7 124.0 134.9 39.2 26.0 125.8 136.7 77.1 34.2 120.2 134.2 25.8 17.9 11.6 15.9 16.2
a
c
e
4 CDCl3, 62.5 MHz
5 CDCl3, 100 MHz
6e CDCl3, 50 MHz
59.2 123.6 139.4 39.8 21.9 41.4 72.8 41.2 22.1 126.1 136.9 77.1 34.2 120.1 134.7 25.9 18.0 11.7 26.7 16.1
59.4 124.2 138.4 42.3 125.3 138.8 72.9 42.2 22.5 126.1 137.0 77.1 34.2 120.1 134.9 25.9 18.0 11.7 28.2 16.4
59.4 123.3 139.9 39.9 22.6 41.0 82.8 37.2 30.8 84.5 134.9 125.4 26.8 122.8 131.5 25.6 17.7 11.4 26.7 16.1
59.4 123.4 139.6 39.5 26.4 124.5 134.6 43.1 125.6 135.9 131.8 129.4 27.2 122.5 133.3 25.7 17.7 12.5 16.1 16.2
t d s t t t s t t d s d t d s q q q q q
d in ppm (TMS as int. standard). H NMR data of Semmak et al. (1988) (added for comparison). Multiplicities were obtained with DEPT sequences. 1 H NMR data of Culioli et al. (2004) (added for comparison). 1 H NMR data of Culioli et al. (2001) (added for comparison).
b 1
d
tc d s t t d s t t d s d t d s q q q q q
3d CDCl3, 100 MHz
t d s t d d s t t d s t t d s q q q q q
t d s t t t s t t d s d t d s q q q q q
t d s t t d s t d d s d t d s q q q q q
488
M. El Hattab et al. / Biochemical Systematics and Ecology 36 (2008) 484e489
differences with 3 and 6 occurred in the third isoprenic unit: (i) a secondary hydroxyl group with a 13C signal at d 84.5 ppm (instead of d 77.1 ppm for the C-12 of 3) and a 1H signal at d 4.25 ppm (t, H-10); (ii) a methylene group, in b position from a secondary alcohol function, with a 13C signal at d 30.8 ppm; (iii) a trisubstituted double bond with the 13C signals at d 125.4 and d 134.9 ppm, and the 1H signal at d 5.41 ppm (t, H-12); (iv) a methyl group fixed on a E double bond with a 13C signal at d 11.4 ppm and a 1H signal at d 1.62 ppm (s, H-18). These results show that compound 5 is a new trihydroxylated linear diterpene in which the secondary alcohol function is not attached to C-12 or C-13 as that is habitually the case for diterpenes isolated from B. bifurcata (Valls and Piovetti, 1995), but at C-10. The assignment of proton and carbon signals (Tables 1 and 2, respectively) was confirmed by means of 2D NMR experiments and by DEPT sequences. With the help of the 1He1H COSY spectrum, locations of the double bonds at C-2, C-11 and C-14 were precisely determined with the 4JHeH correlations between H-2/H-20, H12/H-18 and H-14/H-16, H-14/H-17, respectively. The spatial correlations between H-1/H-20 and H-2/H-4, and between H-12/H-10 and H-13/H-18, clearly showed the E configuration of double bonds at C-2 and C-11. Moreover, the location of the secondary hydroxyl group at C-10 was precisely confirmed by the 2J and 3JCeH correlations between C-11/H-10, C-18/H-10, C-10/H12, C-9/H-10 and C-8/H-10 in its HMBC spectrum, as well as the location of the tertiary hydroxyl group at C-7 with the correlations between C-7/H-19, C-8/H-19 and C-6/H-19. As for C-7 in compound 4, the configurations of the stereogenic centers at C-7 and C-10 have not been determined. 4. Chemotaxonomic significance Our previous studies of the geographical variations in the diterpenoid composition of B. bifurcata (Valls et al., 1993a,b, 1995; Culioli et al., 1999a,b, 2000, 2001) had revealed that the moderately polar fraction of the lipid extract of this species collected off the Moroccan coast, in the Oualidia zone, was clearly distinguishable from the extracts obtained from other zones of collection. In this case, all the difunctionalized oxygenated linear diterpenes were derived from the 12-hydroxygeranylgeraniol (1), instead of 13-hydroxygeranylgeraniol like those of all the lipid extracts obtained from the other zones of collection (Culioli et al., 1999a,b). Moreover, our recent studies of the polar fraction of the crude extract obtained from two specimens of the species collected at Oualidia (Morocco) and Quiberon (Atlantic coast of southern Brittany), respectively, confirm this chemical characteristic (Culioli et al., 2004; OrtaloMagne´ et al., 2005). By contrast, present work shows the presence, for the first time in B. bifurcata, of a linear diterpene in which an oxygenated function is located at C-10 instead of C-12 or C-13, as that is habitually the case for this species (Valls and Piovetti, 1995). Now, it would be of further interest to study the trihydroxylated linear diterpenes of B. bifurcata collected at different other locations, with the aim to verify if the hydroxylated diterpenes at C-10 are present in the whole of this species or only in the Oualidia specimens. Acknowledgements This work has been realized as a part of our project 06 MDU 680 from the C.M.E.P. (Comite´ Mixte d’Evaluation et de Prospective de Cooperation Interuniversitaire Franco-Alge´rienne). The authors wish to thank the French embassy in Algeria and the C.M.E.P. for partial financial support. References Biard, J.F., Verbist, J.F., Floch, R., Letourneux, Y., 1980. Tetrahedron Lett. 21, 1849. Blunt, J.W., Copp, B.R., Hu, W.-P., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2007. Nat. Prod. Rep. 24, 31, and previous reports in the series. Cho, G.Y., Rousseau, F., De Reviers, B., Boo, S.M., 2006. Phycologia 45, 512. Combaut, G., Piovetti, L., 1983. Phytochemistry 22, 1787. Culioli, G., Mesguiche, V., Piovetti, L., Valls, R., 1999a. Biochem. Syst. Ecol. 27, 665. Culioli, G., Daoudi, M., Mesguiche, V., Valls, R., Piovetti, L., 1999b. Phytochemistry 52, 1447. Culioli, G., Di Guardia, S., Valls, R., Piovetti, L., 2000. Biochem. Syst. Ecol. 28, 185. Culioli, G., Daoudi, M., Ortalo-Magne´, A., Valls, R., Piovetti, L., 2001. Phytochemistry 57, 529. Culioli, G., Ortalo-Magne´, A., Daoudi, M., Thomas-Guyon, H., Valls, R., Piovetti, L., 2004. Phytochemistry 65, 2063. Daoudi, M., Bakkas, S., Culioli, G., Ortalo-Magne´, A., Piovetti, L., Guiry, M.D., 2001. Biochem. Syst. Ecol. 29, 973. El Hattab, M., Culioli, G., Valls, R., Richou, M., Piovetti, L., 2008. Biochem. Syst. Ecol. 36, 447.
M. El Hattab et al. / Biochemical Systematics and Ecology 36 (2008) 484e489 Hougaard, L., Anthoni, U., Christophersen, C., Nielsen, P.H., 1991a. Phytochemistry 30, 3049. Hougaard, L., Anthoni, U., Christophersen, C., Nielsen, P.H., 1991b. Tetrahedron Lett. 32, 3577. Mori, K., Uno, T., 1989. Tetrahedron 45, 1945. Ortalo-Magne´, A., Culioli, G., Valls, R., Pucci, B., Piovetti, L., 2005. Phytochemistry 66, 2316. Ross, R., 1961. J. Linn. Soc. Lond. (Bot.) 56, 512. Rousseau, F., De Reviers, B., 1999. Eur. J. Phycol. 34, 53. Semmak, L., Zerzouf, A., Valls, R., Banaigs, B., Jeanty, G., Francisco, C., 1988. Phytochemistry 27, 2347. Silva, P.C., Basson, P.W., Moe, R.L., 1996. Univ. Calif. Publ. Bot. 79, 1259. Stegenga, H., Bolton, J.J., Anderson, R.J., 1997. Contrib. Bolus Herb. 18, 1. Sun, H.H., Ferrara, N.M., McConnell, O.J., Fenical, W., 1980. Tetrahedron Lett. 21, 3123. Valls, R., Banaigs, B., Francisco, C., Codomier, L., Cave, A., 1986. Phytochemistry 25, 751. Valls, R., Banaigs, B., Piovetti, L., Archavlis, A., Artaud, J., 1993a. Phytochemistry 34, 1585. Valls, R., Banaigs, B., Piovetti, L., Zerzouf, A., 1993b. Ann. Inst. Oce´anogr. 63, 215. Valls, R., Piovetti, L., Banaigs, B., Archavlis, A., Pellegrini, M., 1995. Phytochemistry 39, 145. Valls, R., Piovetti, L., 1995. Biochem. Syst. Ecol. 23, 723. Womersley, H.B.S., 1964. Aust. J. Bot. 12, 53.
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Biochemical Systematics and Ecology 36 (2008) 484e489 www.elsevier.com/locate/biochemsyseco
Trihydroxylated linear diterpenes from the brown alga Bifurcaria bifurcata (Fucales, Phaeophyta) Mohamed El Hattab a, Mohammed Ben Mesaoud b, Mohammed Daoudi b, Annick Ortalo-Magne´ c, Ge´rald Culioli c, Robert Valls d, Louis Piovetti c,* a
Laboratoire des Plantes Aromatiques et Me´dicinales, De´partement de Chimie, Faculte´ des Sciences, Universite´ de Blida, BP 270, Blida, Algeria b Laboratoire de Chimie Organique et Bioorganique, Faculte´ des Sciences Choua€ıb Doukkali, El Jadida, Morocco c Laboratoire des Mate´riaux a` Finalite´s Spe´cifiques (MFS), The´matique «Biofouling & Substances Naturelles Marines», Universite´ du Sud Toulon-Var, Avenue de l’Universite´, BP 20132, F-83957 La Garde Cedex, France d UMR 6180 CNRS «Chirotechnologies: Catalyse et Biocatalyse», Groupe «Se´paration, Identification, Synthe`se», Universite´ Paul Ce´zanne, Aix-Marseille III, Avenue Escadrille Normandie-Niemen, Service 551, F-13397 Marseille Cedex 20, France Received 27 November 2007; accepted 19 December 2007
Keywords: Sargassaceae; Phaeophyceae; Bifurcaria bifurcata; Brown alga; Trihydroxylated linear diterpenes; Chemotaxonomy
1. Subject and source The most prolific species of marine algae concerning the number of metabolites are those belonging to the Rhodomelaceae family for the red algae and those of the Dictyotaceae in the case of brown seaweeds. Important contribution has also been provided by Sargassaceae, in particular with the genus Cystoseira. For these two last families, compounds are different: for the Dictyotacean species, cyclic diterpenes have been isolated while acyclic diterpenoids and meroditerpenoids have been described for the Sargassaceae (Blunt et al., 2007). In the course of our phytochemical study on Mediterranean and Atlantic brown algae (Culioli et al., 2004, and references cited therein; El Hattab et al., 2008; Ortalo-Magne´ et al., 2005; Valls and Piovetti, 1995; Valls et al., 1995), we have re-investigated the polar fraction of the lipid extract from Bifurcaria bifurcata (Velley) Ross collected at Oualidia (Morocco) in September 2002, with the object of studying undescribed trihydroxylated linear diterpenes from this species. A voucher specimen of this species (No. P192.7) was deposited in the herbarium of Dr. Pellegrini, Laboratoire de Biologie Marine Fondamentale et Applique´e, Universite´ de la Me´diterrane´e, Marseille, France. 2. Previous work In the algal family Sargassaceae1 (order Fucales, class Phaeophyceae), Bifurcaria is a relative small genus by contrast to the main genera Sargassum, Cystoseira and Turbinaria. It currently includes only three species: B. bifurcata * Corresponding author. Tel.: þ33 4 94 14 23 46; fax: þ33 4 94 14 21 68. E-mail address: [email protected] (L. Piovetti). 1 The Cystoseiraceae species are now merged into the large Sargassaceae family (Rousseau and De Reviers, 1999; Cho et al., 2006). 0305-1978/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.12.007
M. El Hattab et al. / Biochemical Systematics and Ecology 36 (2008) 484e489
485
(Velley) R. Ross, distributed on Atlantic coasts from Morocco (southern limit) to north-western Ireland (northern limit) (Ross, 1961); Bifurcaria brassicaeformis (Ku¨tzing) Barton confined to the western (Stegenga et al., 1997) and Indian Ocean coasts of South Africa (Silva et al., 1996); and Bifurcaria galapegensis (Piccone & Grunow) Womersley (Womersley, 1964) which is endemic to the Galapagos archipelago. Previous studies of the secondary metabolites from lipid extracts of these three species have shown that B. bifurcata contains a rich array of acyclic diterpenes (Biard et al., 1980; Combaut and Piovetti, 1983; Culioli et al., 1999a,b, 2000, 2001, 2004; Daoudi et al., 2001; Hougaard et al., 1991a,b; Ortalo-Magne´ et al., 2005; Semmak et al., 1988; Valls et al., 1993a,b, 1995). By contrast, the main constituent of the lipid extract of B. galapegensis is a meroditerpene (bifurcarenone) (Sun et al., 1980; Mori and Uno, 1989), while B. brassicaeformis shows the total absence of diterpenes in its lipid extract (Daoudi et al., 2001). During our chemotaxonomic studies on B. bifurcata (Combaut and Piovetti, 1983; Culioli et al., 1999a,b, 2000, 2001; Semmak et al., 1988; Valls et al., 1986, 1993a,b, 1995), including the geographical variation of its diterpenoid composition (Valls et al., 1993a,b, 1995), we have investigated the moderately polar fraction of the lipid extract obtained from specimens of the alga collected in different locations off the Moroccan, Spanish, French and Irish Atlantic coasts. The acyclic diterpenes obtained were mono-and dioxygenated geranylgeraniol derivatives in which the second oxygenated function was located at C-12 or C-13 depending on the place where the alga was collected (Valls et al., 1993a,b, 1995). 3. Present work We have recently started a study of the polar fraction of the lipid extracts of the alga in order to isolate trioxygenated acyclic diterpenes and to verify if our previous chemotaxonomic data on this species (Valls and Piovetti, 1995; Valls et al., 1993a,b, 1995) are applicable to these compounds. From this work, two trihydroxylated linear diterpenes derived from 12-hydroxygeranylgeraniol were isolated from a specimen of B. bifurcata collected at Oualidia (Morocco), (Culioli et al., 2004), and five polar linear diterpenes derived from 13-oxo- and 13-hydroxygeranylgeraniol were described from a specimen of B. bifurcata collected off the Atlantic coast of Southern Brittany (Quiberon, France), (Ortalo-Magne´ et al., 2005). The lipid extract of shade-dried B. bifurcata collected near Oualidia was re-investigated with the object of isolating the polar diterpenes undescribed in this extract. The CHCl3eMeOH extract of 83 g (2.63% of dried alga) was partitioned in the mixture MeOHeisooctane (1:1) leading to a MeOH extract which was then dissolved in the mixture MeOHeCHCl3eH2O (4:3:1). About 20.5 g of organic extract obtained from the last extraction was fractionated on silica gel column. The fractions eluted with EtOAceisooctane (1:1 to 3:1) contained sterols, geranylgeraniol, monohydroxylated diterpenes (5.6 g) and the dihydroxylated diterpene bifurcadiol (1) (5.5 g) previously described (Culioli et al., 2001). The fractions eluted with EtOAc and EtOAceMeOH (98:2 and 95:5), respectively, were further purified by HPLC on a C-18 reversed-phase column, eluting with MeCNeH2O (1:1 and/or 2:3). From this separation we obtained the two trihydroxylated linear diterpenes 2 (200 mg) and 3 (55 mg) previously identified in this extract (Culioli et al., 2004) and two new compounds 4 (15 mg) and 5 (19 mg). Diterpene 4 (Fig. 1), C20H34O3 (HRMS), was an optically active oil ([a]20 D ¼ 3.5 (c 0.2, CH2Cl2)) which showed several spectral features in common with geranylgeraniol-derived diterpenes, particularly with bifurcadiol (1) and compound 3. It showed a strong hydroxyl absorption (nOH ¼ 3372 cm1) in its IR spectrum. Inspection of the 1H and 13C NMR data (Tables 1 and 2) compared with those of bifurcadiol (1) and compound 3 showed that the last two isoprenic units (C-9 to C-18) and a part of the first (C-1 to C-3 with C-20) of the three molecules were similar. In contrast, the main differences were present in the second isoprene unit: (i) a tertiary hydroxyl group with a 13C signal at d 72.9 ppm (quaternary sp3 carbon); (ii) a methyl group on the latter sp3 carbon with a 1H signal at d 1.32 ppm (s, H-19); (iii) two ethylenic methine groups with 13C signals at d 125.3 and 138.8 ppm and 1H signals at d 5.57 ppm (d, H-6) and d 5.65 ppm (dt, H-5); (iv) a methylene group with a 1H signal at d 2.77 ppm (d, H-4). The E configuration of the double bond at C-5 was revealed by the value of the coupling constant between H-5 and H-6 (3JHeH ¼ 15 Hz) obtained by irradiating the protons at d 2.77 ppm which led to an AB system at d 5.57e 5.65 ppm (JAB ¼ 15 Hz). The assignment of proton and carbon signals (Tables 1 and 2, respectively) was confirmed by means of 1He1H homonuclear (COSY, NOESY) and 1He13C heteronuclear (HSQC, HMBC) 2D experiments, and by DEPT sequences. In particular, the location of the double bond at C-5 was precisely determined with the 3JHeH correlation between H-4/H-5 and the 4JHeH correlation between H-4/H-6, as well as the location of double bounds at C-2, C-10 and C-14 with the 4JHeH correlations between H-2/H-20, H-10/H-18 and H14/H-16, H-14/H-17, respectively. The NOE correlations between H-2/H-4 and H-1/H-20 on one hand and between H-10/H-12 and H-9/H-18 on
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M. El Hattab et al. / Biochemical Systematics and Ecology 36 (2008) 484e489
OH 16
14 15 17
10 13
12
11
6
8 9
5
7
18
4
19
2 3
OH 1
20
1 OH
OH
OH
2 OH OH OH 3 OH OH OH 4 OH OH OH 5
OH
6 Fig. 1.
the other hand, clearly showed the E configuration of double bonds at C-2 and C-10, respectively. On the basis of biosynthetic considerations, we have attributed an R configuration to the secondary alcohol C-12, like for compounds 1 and 3 present in the same extract, whereas the configuration at C-7 remains to be ascertained (Culioli et al., 2004). The second new compound 5 (Fig. 1) had the molecular formula C20H36O3 (HRMS). It was isolated as an optically 1 active oil ([a]20 D ¼ 11.0 (c 0.2, CH2Cl2)). Like 4, it showed a strong hydroxyl absorption (nOH ¼ 3362 cm ) in its 1 13 IR spectrum. Inspection of the H and C NMR data (Tables 1 and 2) compared with those of 3 showed that the two first isoprenic units of the two molecules were similar. The only one difference was the upfield shift of C-7 (Dd ¼ 10 ppm) for compound 5. Moreover, the further inspection of spectra showed that the last isoprenic unit was similar to the one of compound 6 previously described for this extract (Culioli et al., 2001). In contrast, the main
M. El Hattab et al. / Biochemical Systematics and Ecology 36 (2008) 484e489
487
Table 1 1 H NMR data for compounds 1, 3e6a, b H
1c CDCl3, 360 MHz
3d CDCl3, 400 MHz
4 CDCl3, 250 MHz
5 CDCl3, 400 MHz
6e CDCl3, 200 MHz
1 2 4 5 6 8
4.09 5.35 1.99 2.07 5.07 1.99
4.12 d (6.9) 5.38 t (7.0) 1.99 t (7.0) 1.40e1.48 m 1.38e1.44 m 1.45e1.50 m
4.19 5.46 2.77 5.65 5.57 1.58
4.15 d (7.0) 5.42 t (7.0) 2.00e2.20 m 2.00e2.20 m 5.15 t (7.0) 2.75 d (7.0)
9
2.07 m
2.05 m
2.08 m
10 12 13 14 16 17 18 19 20
5.32 3.92 2.20 5.04 1.68 1.60 1.57 1.56 1.63
5.37 t (7.0) 3.94 t (6.7) 2.20e2.28 m 5.05 t (7.2) 1.69 s 1.60 s 1.60 s 1.14 s 1.64 s
5.43 4.00 2.21 5.11 1.75 1.66 1.64 1.32 1.70
4.15 d (6.8) 5.41 t (7.0) 2.02 d (6.5) 1.45e1.50 m 1.45e1.50 m 1.68 m 1.80 m 1.78 m 1.91 m 4.25 t (7.0) 5.41 t (7.0) 2.71 t (7.0) 5.10 t (7.0) 1.68 s 1.62 s 1.62 s 1.22 s 1.67 s
a b c d e
d (6.8) t (6.8) m m t (6.8) m
t (6.8) t (6.7) dd (6.7; 7.0) t (7.0) s s s s s
d (6.8) t (6.8) d (5.0) dt (15.0; 5.0) d (15.0) m
t (6.6) t (7.0) m t (7.0) s s s s s
5.51 dt (15.0; 7.0) 6.05 5.35 2.81 5.11 1.68 1.64 1.75 1.59 1.68
d (15.0) t (7.0) t (7.0) t (7.0) s s s s s
d in ppm (TMS as int. standard), coupling constants (J in parentheses) are given in Hz. Assignments were confirmed by decoupling and 2D NMR experiments (COSY 1He1H, 1H J-resolved, HMQC and HMBC). 1 H NMR data of Semmak et al. (1988) (added for comparison). 1 H NMR data of Culioli et al. (2004) (added for comparison). 1 H NMR data of Culioli et al. (2001) (added for comparison).
Table 2 13 C NMR data for compounds 1, 3e6a C
1b CDCl3, 90 MHz
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
59.2 123.5 139.1 39.4 26.7 124.0 134.9 39.2 26.0 125.8 136.7 77.1 34.2 120.2 134.2 25.8 17.9 11.6 15.9 16.2
a
c
e
4 CDCl3, 62.5 MHz
5 CDCl3, 100 MHz
6e CDCl3, 50 MHz
59.2 123.6 139.4 39.8 21.9 41.4 72.8 41.2 22.1 126.1 136.9 77.1 34.2 120.1 134.7 25.9 18.0 11.7 26.7 16.1
59.4 124.2 138.4 42.3 125.3 138.8 72.9 42.2 22.5 126.1 137.0 77.1 34.2 120.1 134.9 25.9 18.0 11.7 28.2 16.4
59.4 123.3 139.9 39.9 22.6 41.0 82.8 37.2 30.8 84.5 134.9 125.4 26.8 122.8 131.5 25.6 17.7 11.4 26.7 16.1
59.4 123.4 139.6 39.5 26.4 124.5 134.6 43.1 125.6 135.9 131.8 129.4 27.2 122.5 133.3 25.7 17.7 12.5 16.1 16.2
t d s t t t s t t d s d t d s q q q q q
d in ppm (TMS as int. standard). H NMR data of Semmak et al. (1988) (added for comparison). Multiplicities were obtained with DEPT sequences. 1 H NMR data of Culioli et al. (2004) (added for comparison). 1 H NMR data of Culioli et al. (2001) (added for comparison).
b 1
d
tc d s t t d s t t d s d t d s q q q q q
3d CDCl3, 100 MHz
t d s t d d s t t d s t t d s q q q q q
t d s t t t s t t d s d t d s q q q q q
t d s t t d s t d d s d t d s q q q q q
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M. El Hattab et al. / Biochemical Systematics and Ecology 36 (2008) 484e489
differences with 3 and 6 occurred in the third isoprenic unit: (i) a secondary hydroxyl group with a 13C signal at d 84.5 ppm (instead of d 77.1 ppm for the C-12 of 3) and a 1H signal at d 4.25 ppm (t, H-10); (ii) a methylene group, in b position from a secondary alcohol function, with a 13C signal at d 30.8 ppm; (iii) a trisubstituted double bond with the 13C signals at d 125.4 and d 134.9 ppm, and the 1H signal at d 5.41 ppm (t, H-12); (iv) a methyl group fixed on a E double bond with a 13C signal at d 11.4 ppm and a 1H signal at d 1.62 ppm (s, H-18). These results show that compound 5 is a new trihydroxylated linear diterpene in which the secondary alcohol function is not attached to C-12 or C-13 as that is habitually the case for diterpenes isolated from B. bifurcata (Valls and Piovetti, 1995), but at C-10. The assignment of proton and carbon signals (Tables 1 and 2, respectively) was confirmed by means of 2D NMR experiments and by DEPT sequences. With the help of the 1He1H COSY spectrum, locations of the double bonds at C-2, C-11 and C-14 were precisely determined with the 4JHeH correlations between H-2/H-20, H12/H-18 and H-14/H-16, H-14/H-17, respectively. The spatial correlations between H-1/H-20 and H-2/H-4, and between H-12/H-10 and H-13/H-18, clearly showed the E configuration of double bonds at C-2 and C-11. Moreover, the location of the secondary hydroxyl group at C-10 was precisely confirmed by the 2J and 3JCeH correlations between C-11/H-10, C-18/H-10, C-10/H12, C-9/H-10 and C-8/H-10 in its HMBC spectrum, as well as the location of the tertiary hydroxyl group at C-7 with the correlations between C-7/H-19, C-8/H-19 and C-6/H-19. As for C-7 in compound 4, the configurations of the stereogenic centers at C-7 and C-10 have not been determined. 4. Chemotaxonomic significance Our previous studies of the geographical variations in the diterpenoid composition of B. bifurcata (Valls et al., 1993a,b, 1995; Culioli et al., 1999a,b, 2000, 2001) had revealed that the moderately polar fraction of the lipid extract of this species collected off the Moroccan coast, in the Oualidia zone, was clearly distinguishable from the extracts obtained from other zones of collection. In this case, all the difunctionalized oxygenated linear diterpenes were derived from the 12-hydroxygeranylgeraniol (1), instead of 13-hydroxygeranylgeraniol like those of all the lipid extracts obtained from the other zones of collection (Culioli et al., 1999a,b). Moreover, our recent studies of the polar fraction of the crude extract obtained from two specimens of the species collected at Oualidia (Morocco) and Quiberon (Atlantic coast of southern Brittany), respectively, confirm this chemical characteristic (Culioli et al., 2004; OrtaloMagne´ et al., 2005). By contrast, present work shows the presence, for the first time in B. bifurcata, of a linear diterpene in which an oxygenated function is located at C-10 instead of C-12 or C-13, as that is habitually the case for this species (Valls and Piovetti, 1995). Now, it would be of further interest to study the trihydroxylated linear diterpenes of B. bifurcata collected at different other locations, with the aim to verify if the hydroxylated diterpenes at C-10 are present in the whole of this species or only in the Oualidia specimens. Acknowledgements This work has been realized as a part of our project 06 MDU 680 from the C.M.E.P. (Comite´ Mixte d’Evaluation et de Prospective de Cooperation Interuniversitaire Franco-Alge´rienne). The authors wish to thank the French embassy in Algeria and the C.M.E.P. for partial financial support. References Biard, J.F., Verbist, J.F., Floch, R., Letourneux, Y., 1980. Tetrahedron Lett. 21, 1849. Blunt, J.W., Copp, B.R., Hu, W.-P., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2007. Nat. Prod. Rep. 24, 31, and previous reports in the series. Cho, G.Y., Rousseau, F., De Reviers, B., Boo, S.M., 2006. Phycologia 45, 512. Combaut, G., Piovetti, L., 1983. Phytochemistry 22, 1787. Culioli, G., Mesguiche, V., Piovetti, L., Valls, R., 1999a. Biochem. Syst. Ecol. 27, 665. Culioli, G., Daoudi, M., Mesguiche, V., Valls, R., Piovetti, L., 1999b. Phytochemistry 52, 1447. Culioli, G., Di Guardia, S., Valls, R., Piovetti, L., 2000. Biochem. Syst. Ecol. 28, 185. Culioli, G., Daoudi, M., Ortalo-Magne´, A., Valls, R., Piovetti, L., 2001. Phytochemistry 57, 529. Culioli, G., Ortalo-Magne´, A., Daoudi, M., Thomas-Guyon, H., Valls, R., Piovetti, L., 2004. Phytochemistry 65, 2063. Daoudi, M., Bakkas, S., Culioli, G., Ortalo-Magne´, A., Piovetti, L., Guiry, M.D., 2001. Biochem. Syst. Ecol. 29, 973. El Hattab, M., Culioli, G., Valls, R., Richou, M., Piovetti, L., 2008. Biochem. Syst. Ecol. 36, 447.
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