Biochemical Systematics and Ecology 38 (2010) 861–863
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3b, 7b-Dihydroxy-5a-cholestan-11-one: A new oxidation pattern of cholestane skeleton from Laurencia papillosa Sultan S. Al-lihaibi a, Seif-Eldin N. Ayyad b, Essam Al-wessaby a, Walied M. Alarif a, * a b
Department of Marine Chemistry, Faculty of Marine Sciences, King Abdulaziz University, PO. Box 80207, Jeddah 21589, Saudi Arabia Department of Chemistry, Faculty of Science, King Abdulaziz University, PO. Box 80207, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o Article history: Received 27 May 2010 Accepted 8 August 2010 Keywords: 3b, 7b-Dihydroxy-5a-cholestan-11-one Laurencia papillosa Red Sea
1. Subject and source The red alga Laurencia papillosa was collected in June 2008, off the Saudi Arabia Red Sea Coast at Jeddah. Voucher sample (JAD 03050) is deposited at the Marine Chemistry Department, King Abdulaziz University, Jeddah, Saudi Arabia. 2. Previous study Although L. papillosa (Ceramiales, Rhodomelaceae) belongs to a genus that is a rich source of secondary metabolites (Blunt et al., 2003; Kladi et al., 2007), only a few studies have focused on the identification of steroids using GC/MS (Lisboa et al., 1982; Zornista et al., 2006). 3. Present study The dried material of the red alga L. papillosa (dry weight 380 g) was extracted with a mixture of chloroform and methanol (1:1) at room temperature. The residue was partitioned between chloroform and water, the organic layer (9 g) was chromatographed on silica gel (Merck, 60G) column chromatography using benzene–ethylacetate mixtures with increasing polarity to give a steroidal compound (1) (benzene: ethylacetate ¼ 5:2) in addition to cholesterol, cholestane and 24methylene cholesterol (n-hexane:ethylacetate ¼ 8:2). Compound 1 was further purified by PTLC using benzene–ethylacetate (6:4) and visualized upon spraying with ethanol/sulfuric acid reagent to give an orange spot color after heating. 3b,7b-Dihydroxy-5a-cholestan-11-one (1): White solid (4 mg, 0.001%) m.p. 149 C, [a]20 D ¼ þ40 (CHCl3; c ¼ 0.1), Rf 0.43 (benzene/EtOAc,6:4). IR (neat) nmax 3450, 2951, 1708 cm1. EI-MS m/z: 418 [M, C27H46O3]þ, 400 [M-H2O]þ, 385 [M-CH3H2O]þ, 318 (100) [M-C7H16]þ. 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3): Table 1. The molecular formula was deduced to be C27H46O3 on the basis of the EI-MS, in conjunction with the 1H, 13C, and DEPT NMR data, which indicated 5
* Corresponding author. Tel.: þ966 2 6952383 (work), þ966 56 0352034 (mob.); fax: þ966 2 6401747. E-mail address:
[email protected] (W.M. Alarif). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.08.009
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S.S. Al-lihaibi et al. / Biochemical Systematics and Ecology 38 (2010) 861–863
Table 1 1 H, 13C NMR, 1H-1H COSY and HMBC (d ppm) of compound 1 in CDCl3. No.
H(m, J Hz)
1
(1a) 1.52 m (1b) 1.3 m (2a) 1.75 m (2b) 1.32 m 3.64 (tt, 13.2, 4, 1.8) 2.07 m, 1.7 m 1.47 (dt, 12.6, 3.2) 1.91 m 3.61 (ddd, 11.6, 4, 1.4) 1.87 (ddd, 13.2, 4, 3.9) 2.15 (d, 13.2) – – (b) 2.17 (d, 12.6) (a) 2.19 (d, 12.6) – 1.3 m 1.66 m, 1.22 m 1.76 (2H, m) 1.23 m 0.67 (3H, s) 0.85 (3H, s) 1.36 0.92 (3H, d, 6.5) 1.32 m, 0.98 m 1.33 m, 1.13 m 1.12 (2H, m) 1.39 m 0.89 (3H, d, 6.5) 0.91 (3H, d, 6.5)
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
13
C
COSY
HMBC
29.83
1b, 2a, 2b 1a, 2a, 2b 1a, 1b, 2b 1a, 1b, 2a, 3 2a, 2b, 4a, 4b 3, 5 4a, 4b, 6a, 6b 5, 7 6, 8 7, 9, 14 8 – – –
2, 9, 10, 19
2, 4, 5 2, 5, 6, 10 4, 6, 10 5, 7 5, 6, 8 7, 9, 14 8, 10, 11, 14, 19 – – 11, 13, 17
– 8, 15 14, 16 15, 17 16, 20 – – 17, 21, 22 20 20, 23 22, 24 23, 25 24, 26, 27 25 25
– 8, 13, 15, 18 14, 16 15, 17 13, 16, 20 12, 13, 14, 17 1, 5, 9, 10 17, 21, 22 17, 20, 22 20, 23, 24 22, 24 26,27 26, 27 24, 25, 27 24, 25, 26
27.58 70.18 36.05 42.19 34.31 73.92 41.70 59.33 40.0 213.89 42.91 43.32 53.12 24.07 27.31 56.41 12.38 11.95 37.06 22.46 34.82 20.82 39.56 28.15 22.95 23.15
1, 3, 4
degrees of unsaturation, deduced one degree of unsaturation at d 213.89 as carbonyl group with the absence of any olefinic proton or any double bond signal. The IR spectrum indicated that no bands due to n(CaC) and n(C–H)Olefinic (w1600 and 3000–3300 cm1) but only n(C–H)Aliphatic vibration band was observed at 2951 cm1. Moreover, the spectrum displayed two strong bands at 3450 and 1708 cm1 assigned to n(OH) and a six membered ring n(CaO) (Anderson and Haslewood, 1967), respectively. The DEPT spectrum showed that compound 1 contains five methyl, fourteen methylene, five methine and three quaternary carbons. The 1H and 13C NMR (Table 1) revealed the presence of two hydroxyl groups from signals resonating at d 3.64 (tt, J ¼ 13.2,4, 1.8 Hz; dc 70.18) and 3.61 (ddd, J ¼ 11.6, 4, 1.4 Hz; dc 73.92). The previous data showed that compound 1 should be tetra cyclic with a keto group and two hydroxyl functions. 1H NMR data were typical of sterols it gave two singlets at d 0.67 and d 0.85 corresponding to two angular methyl groups C-18 and C-19, respectively, and three doublets at d 0.92, d 0.89 and d 0.91 corresponding to C-21, C-26 and C-27, respectively. Hence, the compound is a C-27 steroid with a cholestan side chain. Positions of two hydroxyl groups can be determined by examining 1H NMR, 1H-1H COSY and HSQC spectra, a proton at d 3.64 appears as tt with coupling constants (J) 13.2, 4 and 1.8 Hz should located at position 3 with axial orientation i.e. hydroxyl group at C-3 occupies b-position which can also supported from the shape of the signal. The carbinol proton at d 3.61 (1H, ddd, J ¼ 11.6, 4, 1.6 Hz, dc 73.92), i.e. axial orientation, showed cross peaks with signals at d 1.91(2H, m, dc 34.31) and d 1.87 (1H, ddd, J ¼ 13.2, 4, 3.9 Hz, dc 41.70). According to structural connectivities we can locate the –OH group at position 7 with b-orientation. The presence of two geminal protons at 2.17 (d, J ¼ 12.6 Hz) and 2.19 (d, J ¼ 12,6 Hz) which were located at carbon atom at dc 42.91 from HSQC spectrum, HMBC correlation showed that this methylene group is flanked by keto group and a quaternary carbon. These conclusions imply that the carbonyl group should be either at C-2 or C-11. But, the presence of a proton at 1H NMR d 2.15 (d, J ¼ 13.2 Hz; dc 59.33) means that this proton is attached to keto group and to a methine group, this system is only available when keto group is located at position 11 (Fig. 1). The stereochemistry of proton at position 5 was found to be a, owing to its 1H NMR at d 1.47 (dt, J ¼ 12.6, 3.2 Hz) which implies axial orientation for H-5. The chemical structure was further elucidated by following connectivity from 1H-1H COSY, HSQC and HMBC. So compound 1 could be assigned as 3b,7b-dihydroxy-5a-cholestan-11-one. 4. Chemotaxonomic significance L. papillosa is widespread in the coastal region of Jeddah, KSA. Many steroids were identified from L. papillosa in which cholestane and oxygenated cholestane were isolated in considerable yield. Moreover, the identification of the chemical nature
S.S. Al-lihaibi et al. / Biochemical Systematics and Ecology 38 (2010) 861–863
21 18 O 11 19
9
1 3
5
HO
H
12
17
20 25
O : 1H-1 H COSY
8 7
863
OH
HO
OH
: HMBC
(1) Fig. 1. Selacted 1H-1H COSY and HMBC correlations of 1.
of the cholestane steroids of L. papillosa is of chemotaxonomic significance in view of their exploitative potential as biomedical (Brad, 1996) and biogenic antifouling activity (Punyasloke and Phillip, 2004). Polyoxygenated sterols have been occasionally isolated from red and brown algae (D’Auria et al., 1993). Of these steroids, few examples derived from cholestane were identified, such as 5a-cholestane-3,6-dione from some red algae species (Wahidulla et al., 1987) and 11a-hydroxy-5a-cholestane-3,6-dione from Acanthophora spicifera (Prakash et al., 1989). Here, we report a new polyoxygenated cholestane derivative, 3b,7b-dihydroxy-5a-cholestan-11-one, that could be a chemotaxonomic marker for the red alga L. papillosa. Acknowledgement This research work was supported by grants from the Deanship of Scientific Research (DSR) No. 429/003-8, King Abdulaziz university (KAU). Dr. Nessim, I. Rady assistant prof. of algal flora, faculty of science, Umm Al-Qura University and Dr. Fattoun, A. Al-Saegh assistant prof. of applied algae, Faculty of science, King Abdulaziz University, are acknowledged for algae identification. References Anderson, I.G., Haslewood, A.D., 1967. Biochem. J. 104, 1061. Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., Prinsep, M.R., 2003. Nat. Prod. Rep. 20, 1. Brad, K.C., 1996. Biomedical Potential of Marine Natural Products. University of California Press, American Institute of Biological Sciences, pp. 271–286. D’Auria, M.V., Minale, L., Riccio, R., 1993. Chem. Rev. 93, 1839. Kladi, M., Vagias, C., Papazafiri, P., Furnari, G., Serio, D., Roussis, V., 2007. Tetrahedron 63, 7606. Lisboa, B.P., Ganshow, L.I., Halket, J.M., Pinheiro-Joventio, F., 1982. Comp. Biochem. Physiol. 73B, 257. Prakash, O., Ray, R., Bhakuni, R., Wahidulla, S., Kamat, S.Y., 1989. J. Nat. Prod. 52, 686. Punyasloke, B., Phillip, C.W., 2004. Planta J. 219, 561. Wahidulla, S., D’Souza, L., Patel, J., 1987. Phytochem. 26, 2864. Zornista, K., Kamen, S., Rosalina, S., Stefka, D.-K., Simon, P., 2006. Nat. Prod. Res. 20, 113.
