“Dinoflagellate Sterols” in marine diatoms

Phytochemistry 72 (2011) 1896–1901

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‘‘Dinoflagellate Sterols’’ in marine diatoms José-Luis Giner a,⇑, Gary H. Wikfors b a b

Department of Chemistry, SUNY-ESF, Syracuse, NY 13210, USA Northeast Fisheries Science Center, NMFS, NOAA, Milford, CT 06460, USA

a r t i c l e

i n f o

Article history: Received 30 September 2010 Received in revised form 7 January 2011 Available online 31 May 2011 Keywords: 23-Methyl sterols 27-Norsterols Chemotaxonomy Diatoms Dinoflagellates Marine algae Marine lipids Marine sterols

a b s t r a c t Sterol compositions for three diatom species, recently shown to contain sterols with side chains typically found in dinoflagellates, were determined by HPLC and 1H NMR spectroscopic analyses. The centric diatom Triceratium dubium (= Biddulphia sp., CCMP 147) contained the highest percentage of 23-methylated sterols (37.2% (24R)-23-methylergosta-5,22-dienol), whereas the pennate diatom Delphineis sp. (CCMP 1095) contained the cyclopropyl sterol gorgosterol, as well as the 27-norsterol occelasterol. The sterol composition of Ditylum brightwellii (CCMP 358) was the most complex, containing D0- and D7-sterols, in addition to the predominant D5-sterols. A pair of previously unknown sterols, stigmasta-5,24,28-trienol and stigmasta-24,28-dienol, were detected in D. brightwellii and their structures were determined by NMR spectroscopic analysis and by synthesis of the former sterol from saringosterol. Also detected in D. brightwellii was the previously unknown 23-methylcholesta-7,22-dienol. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Recently, sterols containing a methyl group at the 23-position of the side chain were shown by Dutch geochemists to be widely distributed among diatom algae (Rampen et al., 2009a,b,c, 2010). Prior to this, 23-methylated sterols such as dinosterol (10D, Fig. 1) (Shimizu et al., 1976) and gorgosterol (12A) (Finer et al., 1978) were considered to be typical of dinoflagellates (Withers, 1983; Giner, 1993), despite a few reports of their presence as minor sterols in other algae (Volkman, 2003). Recently, however, 27norsterols, also previously believed to be typical of dinoflagellates (Giner, 1993), were encountered in silicoflagellate algae (Giner et al., 2008). The identification of the diatom sterols was based entirely upon GC–MS data, with one exception for which NMR spectroscopic data were given (Rampen et al., 2009b). Because of the uncertainties of GC–MS structural assignments, we have used HPLC and NMR spectroscopy to analyze the sterols of three species: the pennate diatom Delphineis sp. (CCMP 1095), and two centric diatoms, Ditylum brightwellii (CCMP 358) and Triceratium dubium (= Biddulphia sp., CCMP 147), respectively.

2. Results The same strains analyzed by the Dutch group (Rampen et al., 2009a,b,c, 2010) were investigated here by RP-HPLC purification ⇑ Corresponding author. Tel.: +1 315 470 6895; fax: +1 315 470 6856. E-mail address: [email protected] (J.-L. Giner). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.05.002

and 600 MHz 1H NMR spectroscopic analysis. The sterol compositions of the three diatoms were in good agreement with the published GC–MS analyses (Table 1) (Rampen et al., 2010). The diatoms contained a fairly complex mixture of sterols, mainly ordinary phytosterols. Thus the free sterols of Delphineis sp. contained 46.6% brassicasterol (4A) and 10.0% of its C-24 epimer, epibrassicasterol (5A). The major sterols of T. dubium were 24methylenecholesterol (2A, 22.4%), brassicasterol (4A, 16.2%) and epibrassicasterol (5A, 18.7%). Small amounts of cholesterol (1A) were detected in these two species, but this was the major sterol of D. brightwellii (23.2%), which also contained 24-ethyl sterols, fucosterol (6A, 15.3%) and isofucosterol (7A, 7.8%). Except for a trace of the latter sterol (0.9% in T. dubium), no 24-ethyl sterols were detected in the other two species. In contrast to the other species, D. brightwellii exclusively had the b-configuration at C24. This species also contained minor amounts of D0 and D7 sterols, whereas the other two species contained only D5-sterols. All three diatoms contained the 23-methyl sterol, 4-demethyl5-dehydrodinosterol (10A) (Kobayashi et al., 1979; Withers et al., 1982), the largest percentage of which was found in T. dubium (37.2%). The 23-methyl cyclopropyl sterol, gorgosterol (12A), was found in Delphineis sp. (17.4%). Small amounts of 23-methyl sterols lacking the usual 24-methyl group, i.e., 23-methyl-22-dehydrocholestanol (11C, 2.3%) and the analogous D5- and D7-sterols, 23methyl-22-dehydrocholesterol (11A, 1.0%) and the previously unreported 23-methylcholesta-7,22-dienol (11B, 0.3%), were found in D. brightwellii, along with the D0- and D7-analogs of 4-demethyl5-dehydrodinosterol (10C, 16.3% and 10B, 5.5%). The order of prevalence of the 23-methyl sterols in D. brightwellii was

J.-L. Giner, G.H. Wikfors / Phytochemistry 72 (2011) 1896–1901

24

22 R

R

R

R

R

R

R

HO

3

23 1

25 27

R

4

R

8

R

11

R

14

R

17

R

20

5

R

7

A

2

5

9

12

15

18

21

HO

R

R

R

R

R

R

R

3

(E ) = 6 (Z ) = 7

10

13

16

19

22

B

1897

A small peak in the HPLC chromatogram from D. brightwellii was attributable to a sterol that proved to be relatively unstable, largely decomposing during purification. An analysis of a second culture yielded the same results and provided more of this substance. The 1H NMR spectrum displayed signals indicative of a vinyl group (6.723 (dd, J = 17.3, 11.0, 1H), 5.105 (d, J = 17.3, 1H), 4.947 (d, J = 11.0, 1H)), and the downfield shifts suggested conjugation, although no other olefinic signals were evident. The HPLC retention time was similar to 24-methylenecholesterol (2A), which suggested the presence of an extra carbon to counteract the chromatographic effect of an extra double bond. A structure consistent with these observations was proposed (15A), and, because it had never been reported, was confirmed by synthesis by means of phosphorus oxychloride dehydration (Giner et al., 1989b) of saringosterol (23, Scheme 1). The D0 analog of this new sterol (15C) was also detected. The esterified sterols of Delphineis sp., D. brightwellii, and T. dubium were analyzed separately from the free sterols. The amount of sterol esters varied from 30% of the total sterols in T. dubium, to 20% in Delphineis sp., and 5% in D. brightwellii. The composition of the ester sterols in T. dubium showed much more 24-methylenecholesterol (2A) than the free sterols, and much less 4-demethyl-5-dehydrodinosterol (10A). The most noticeable difference in the sterols of Delphineis sp. was that the esters had proportionally more brassicasterol (4A) than the free sterols. The amount of ester sterols was very small for D. brightwellii, but no 23-methyl sterols could be detected there. The trend among all three species seemed to be that the ordinary sterols were enriched in the ester sterols compared to the 23-methyl sterols. Traces of alternative double bond isomers of 4-demethyl-5dehydrodinosterol (10A) were searched for in the NMR spectra using authentic samples for comparison. Neither of the C-23 stereoisomers of 23-methyl-24-methylenecholesterol (16A) (Giner et al., 1989a), or (24R)-24-methyl-23-methylenecholesterol (17A) (Giner et al., 1989b) (none of which are yet known in nature), however, could be detected in the NMR spectra of any of the diatoms. Traces of alternative isomers of 23-methyl-22-dehydrocholesterol (11A) were also sought. No traces of (22Z)-23-methyl-22-dehydrocholesterol (Z-11A) (Li and Djerassi, 1982), (23E)-23-methyl-23dehydrocholesterol (18A) (Li and Djerassi, 1982), both (22E)- and (22Z)-22-methyl-22-dehydrocholesterol (19A) (Li and Djerassi, 1982), 23-methylenecholesterol (20A) (Li et al., 1983), or 22methylenecholesterol (21A) (Zielinski et al., 1981) were detected. Only the last of these has been found in nature, in a marine sponge (Zielinski et al., 1981). Although 22-dehydrocholesterol (22A) is the expected biosynthetic precursor of 23-methyl-22-dehydrocholesterol (11A) (Kokke et al., 1979), it was not detected in any of the species in this study.

3. Discussion

HO

C

HO

D

Fig. 1. Structures of sterols.

D0 > D5 > D7; however, the other sterols in this species predominantly had the D5-nucleus. The total 23-methyl sterol contents of T. dubium, Delphineis sp., and D. brightwellii were 37.2%, 31.6%, and 28.5%, respectively. The 27-norsterol, occelasterol (13A), which was recently found as the predominant sterol of two silicoflagellates (Giner et al., 2008), was found in Delphineis sp. (7.9%).

Dinoflagellates were the first known algal sources of 23-methyl sterols (Shimizu et al., 1976; Finer et al., 1978), and the presence of 23-methyl sterols was used chemotaxonomically to help in classifying algae as dinoflagellates (Nichols et al., 1983; Jones et al., 1983). There have long been reports, however, of the presence of 4-demethyl-5-dehydrodinosterol (10A) (3.3–12.5%) in non-dinoflagellate algae such as the prymnesiophyte Pleurochrysis carterae (= Hymenomonas carterae) (Volkman et al., 1981; Neal et al., 1986; Gladu et al., 1990; Ghosh et al., 1998), and in the diatoms Biddulphia sinensis (0.9–2.9%) (Volkman et al., 1980), Navicula sp. (2.0–4.6%) (Volkman et al., 1993), Fragilaria pinnata (18.0%) (Barrett et al., 1995), and Haslea ostrearia (Véron et al., 1998). Dinosterol (10D) itself was detected in the diatom Navicula sp. in trace amounts (0.3–0.6%) (Volkman et al., 1993), however, the presence

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Table 1 Sterol composition of diatom species. RRTa

Sterols

Percentage of total sterols Delphineis sp. CCMP 1095

a b c d

RRT = HPLC retention time Literature values (Rampen Literature values (Rampen Literature values (Rampen

0.67 0.79 0.86 0.86 0.86 0.86 0.88 0.90 0.94 0.94 0.94 0.96 1.00 1.00 1.00 1.00 1.00 1.02 1.05 1.05 1.09 1.12 1.12 1.12 1.13 1.17 1.53

Free sterols (80%)

Sterol esters (20%)

7.9 0.7

7.9 1

10.0 46.6

7.4 69.6

1.6

8.7

14.2

1.6

Lit.b

6.4

62

10

Free sterols (95%)

12.1 0.3 1.0 0.3 1.6 3.5 0.3 0.3 2.3 23.2 15.3 7.8 0.9 0.3 5.5 3.1 1.1 0.8 2.5 1.0 0.5

Sterol esters (5%)

21

Lit.c

5

Lit.d

Free sterols (70%)

Sterol esters (30%)

0.6

1.7

22.4

56.0

37

18.7 16.2

16.1 16.2

32

0.3

0.9

0.9

0.6

37.2

4.9

25

3.7

3.6

7

4

2

43 7 10

3 35 26 13 1 4

19

2

1 16.3

17.4

Triceratium dubium CCMP 147

10

13

relative to cholesterol. et al., 2010). In addition, 10% (22E)-22-dehydrocholesterol and 3% ergosterol were reported. et al., 2010). In addition, a trace of (22E)-22-dehydrocholesterol, 1% 23-methylenecholesterol, and 5% 23-methyl-24-methylenecholesterol were reported. et al., 2010). In addition, a trace of 24-ethylcholesterol was reported.

J.-L. Giner, G.H. Wikfors / Phytochemistry 72 (2011) 1896–1901

Ergosta-5,22,24(28)-trienol (14A) (24S)-27-Norergosta-5,22-dienol (13A) (occelasterol) Ergosta-5,24(28)-dienol (2A) (24-methylenecholesterol) Ergosta-7,24(28)-dienol (2B) 23-Methylcholesta-5,22-dienol (11A) 23-Methylcholesta-7,22-dienol (11B) Stigmasta-5,24,28-trienol (15A) (24S)-Ergosta-5,22-dienol (5A) (24-epibrassicasterol) (24R)-Ergosta-5,22-dienol (4A) (brassicasterol) (24R)-Ergosta-7,22-dienol (4B) (24S)-Stigmasta-5,25-dienol (8A) 23-Methylcholest-22-enol (11C) Cholest-5-enol (1A) (cholesterol) (24E)-Stigmasta-5,24(28)-dienol (6A) (fucosterol) (24Z)-Stigmasta-5,24(28)-dienol (7A) (isofucosterol) Stigmasta-24,28-dienol (15C) Cholest-7-enol (1B) (24R)-23-Methylergosta-7,22-dienol (10B) (24R)-23-Methylergosta-5,22-dienol (10A) (24R)-Ergost-22-enol (4C) (24R)-Stigmasta-5,22-dienol (9A) Cholestanol (1C) (24E)-Stigmast-24(28)-enol (6C) (24Z)-Stigmast-24(28)-enol (7C) (24S)-Ergost-5-enol (3A) (24R)-23-Methylergost-22-enol (10C) Gorgost-5-enol (12A) (gorgosterol)

D. brightwellii CCMP 358

J.-L. Giner, G.H. Wikfors / Phytochemistry 72 (2011) 1896–1901

1899

OH a,b,c

HO

23

HO

15A

Scheme 1. Synthesis of stigmasta-5,24,28-trienol. (a) Ac2O/pyr.; (b) POCl3/pyr.; (c) NaOH.

of the 4a-methyl-D0 nucleus in the major sterols of some prymnesiophytes (Volkman et al., 1990) suggests that more abundant nondinoflagellate sources of dinosterol may yet be found. We were previously skeptical of some of these claims. The generally low amounts of 23-methyl sterols suggested the possibility of culture contamination, which was not discussed in any of the papers, and the algal strains were generally not from major culture collections, making replication difficult. Another difficulty was that the sterols were identified solely by GC–MS, which provides considerably less information than NMR spectroscopic analyses. Furthermore, many sterols display identical molecular ions and similar fragmentation patterns. Because of the great variety of sterols, many also have indistinguishable retention times, although the use of multiple GC columns can help to address this problem (Gerst et al., 1997). The presence of stereoisomers has routinely been ignored, although many are separable (Thompson et al., 1981). The use of GC retention times for identification requires that authentic standards be used; however, the sources and identities of these have seldom been given. These objections have been addressed in part by the recent publications of NIOZ group, which included some strain information, and NMR spectroscopic data for 4-demethyl-5-dehydrocholestanol (10C) (Rampen et al., 2009a,b,c, 2010). The stereochemical configuration of each sterol was determined in this study. The C-24 epimers of ergosta-5,22-dienol (4A, 5A) were well separated by HPLC, but this is not typical of 24-methyl and 24-ethyl sterols. The E and Z isomers of 24-ethylidenecholesterol (6A, 7A) were not separated; however, these, and indeed all sterols, can be distinguished by their 1H NMR spectra. The ratio of brassicasterol/epibrassicasterol (4A, 5A) was ca. 5:1 for Delphineis sp. and ca. 1:1 for T. dubium. Only brassicasterol (4A) was detected in D. brightwellii. The configuration at C-24 is likely to provide chemotaxonomic information (Gladu et al., 1990). All 23,24-dimethyl sterols had exclusively the 24b-configuration – the 24a-configuration is not yet known in nature. In contrast, occelasterol (13A), the 27-norsterol isolated from Delphineis sp. had the 24a-configuration. Occelasterol (13A) was recently isolated for the first time from an algal source, two silicoflagellates in the genus Pseudochattonella, in which it represents the predominant sterol (Giner et al., 2008). The 24b-configuration is found in 27norsterols from dinoflagellates (Goad and Withers, 1982; Giner et al., 2003), but in contrast the 24a-configuration was found in the diatom and the silicoflagellates. The detection of a 27-norsterol in a diatom shows that both of the sterol side chain modifications previously thought to be limited to dinoflagellates are also present in diatoms. Although there was generally very good agreement between our NMR spectroscopic analysis and the reported GC–MS analysis, there were a few discrepancies (Table 1). We suggest that the sterol identified as 23-methyl-24-methylenecholesterol (16A) from D. brightwellii (5% of the total sterols) (Rampen et al., 2010) is actually 4-demethyl-7-dehydrodinosterol (10B), which we detected in a similar quantity (5.5%). We suggest that the sterol reported from Delphineis sp. as 10% of the total sterols and identified as (22E)22-dehydrocholesterol (22A) (Rampen et al., 2010) is actually

occelasterol (13A), which we detected in a similar quantity (7.9%). We found no trace of 23-methylenecholesterol (20A) which was reported to be 1% of the total sterols of D. brightwellii, (Rampen et al., 2010), but has otherwise never been detected in nature. Perhaps the isomeric sterols ergosta-7,24(28)-dienol (2B), (24R)-ergosta-7,22-dienol (4B), and 23-methylcholesta-7,22-dienol (11B) might account instead for this. Although most of the sterols detected here are well known, there were a few rarities. Although the side chain of 22-dehydro24-methylenecholesterol (14A) is well known from fungi as the D5,7-sterol, the D5-sterol has only been reported once (Jarzebski, 1991). There have been only a few reports of either 4-demethyldinosterol (10C) (Withers et al., 1982), or its D7-analog (10B) (Kanazawa et al., 1980; Stonik et al., 1998). Likewise, although there are very few reports of 23-methyl-22-dehydrocholesterol (11A) (Kobayashi et al., 1979; Withers et al., 1982), there are even fewer of the D0-analog (11C) (Withers et al., 1982), and hitherto none of the D7-analog (11B). A new sterol with a conjugated diene in the side chain (15A) was detected and its structure determined by synthesis (Scheme 1). This was also detected as a D0-sterol (15C). This side chain is likely to result from a desaturase acting on the fucosterol (6) or isofucosterol (7) side chain. The presence of 23-methyl sterols, cyclopropyl sterols and 27norsterols is thought to provide an adaptive benefit to marine algae (Giner et al., 2003), possibly by interfering with the sterol metabolism of grazers. Accordingly, the sterols can be divided into two groups, the normal sterols that function as membrane lipids, and the unusual, possibly toxic, sterols. Diatoms are known to have multiple lines of defense, including siliceous shells, neurotoxins and fatty aldehydes (Koski et al., 2008; Ianora and Miralto, 2010). The relatively small amounts of unusual sterols in the diatoms, and the relatively large amounts of sterols beneficial to marine invertebrates (e.g., 23.2% cholesterol (1A) in D. brightwellii) suggests that the defense function for algae is not merely that unusual sterols are non-nutritious to grazers, but that they actively interfere with grazer sterol metabolism. For example, the cyclopropyl functionality of gorgosterol (12A, 17.4% in Delphineis sp.) potentially mediates the mechanism-based inhibition of enzymes involved in phytosterol dealkylation and ecdysteroid biosynthesis (Giner et al., 2003). There are hints that the unusual sterols are in a physiologically different class from the normal sterols. The presence of smaller amounts of 23-methyl sterols in the sterol esters suggests that it is the free sterols that provide the defensive function, while the sterol esters function as reserves for membrane sterols. The preferential modification of the sterol nucleus of 23methyl sterols in D. brightwellii also suggests a different, presumably defensive, role for the unusual sterols, and, furthermore, that the modifications of the sterol nucleus augment the function of the unusual sterol side chains. It is possible that the pathways for 23-methylation and 27demethylation in dinoflagellates originated through horizontal gene transfer from diatoms. Dinoflagellates are known to form symbioses readily (Saldarriaga et al., 2001; Ishida and Green, 2002; Yoon et al., 2005), and a diatom endosymbiont is found in

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J.-L. Giner, G.H. Wikfors / Phytochemistry 72 (2011) 1896–1901

Kryptoperidinium foliaceum (Inagaki et al., 2000; Figueroa et al., 2009). Gene transfer through endosymbiosis has, however, also occurred in the evolution of diatoms (Moustafa et al., 2009); therefore, it is not certain that the ultimate origins of these pathways do not lie elsewhere.

was separated, and the solvents evaporated with a stream of nitrogen. Most of the pigments could be removed by passing the residue through a column of Florisil (elution with hexane/EtOAc 19:1, then 4:1). The pooled fractions were concentrated to dryness with a stream of N2.

4. Conclusions

5.4. Sterol analysis

This study confirmed by NMR spectroscopic analyses the existence of 23-methyl sterols in diatoms and showed that 27-norsterols are also present. This suggests that the dinoflagellate sterols are potentially derived from diatoms via horizontal gene transfer or shared ancestry. The existence of these pathways in ‘‘ordinary’’ algae is anticipated to aid in biosynthetic investigations (Giner and Djerassi, 1991) as dinoflagellates possess an unusual molecular biology (John et al., 2004).

The algal lipids were separated by preparative TLC (hexane/ EtOAc 9:1, then 4:1). The fractions containing the sterol esters, and those containing the free sterols, which were contaminated with fatty acyl diglycerides, were treated separately with 10% NaOH/ CH3OH at reflux under N2 for 22 h. The saponification reactions were extracted with H2O and hexane/EtOAc 1:1, and the organic layers were filtered through neutral alumina. After purification by preparative TLC as described above (free sterols: CCMP 147 – 0.38 mg; CCMP 358 – 0.28 mg; CCMP 1095 – 0.11 mg; ester sterols: CCMP 147 – 0.16 mg; CCMP 358 – 0.01 mg; CCMP 1095 – 0.03 mg), the individual sterols were isolated by reversed phase HPLC. The HPLC solvent was evaporated with a stream of N2, and the sterols characterized by 1 H NMR spectroscopic analyses. The relative proportions of the sterols were determined from the integrals of the HPLC traces. The structures of sterols were assigned by comparison of the NMR spectroscopic data with authentic standards and literature values. 23-Methylcholesta-7,22-dienol (11B). 1H NMR (600 MHz) 5.156 (m, 1H), 4.894 (d, J = 9.8, 1H), 3.596 (m, 1H), 1.563 (s, 3H), 0.951 (d, J = 6.8, 3H), 0.841 (d, J = 6.4, 3H), 0.822 (d, J = 6.4, 3H), 0.805 (s, 3H), 0.570 (s, 3H). Ergosta-5,22,24(28)-trienol (14A). 1H NMR (600 MHz) 5.935 (d, J = 15.8, 1H), 5.588 (dd, J = 15.8, 8.8, 1H), 5.351 (m, 1H), 4.851 (s, 1H), 4.816 (s, 1H), 3.524 (m, 1H), 1.080 (d, J = 6.9, 3H), 1.067 (d, J = 6.8, 3H), 1.061 (d, J = 6.6, 3H), 1.015 (s, 3H), 0.717 (s, 3H). Stigmasta-5,24,28-trienol (15A). 1H NMR (600 MHz) 6.723 (dd, J = 17.4, 10.9, 1H), 5.352 (m, 1H), 5.104 (d, J = 17.4, 1H), 4.947 (d, J = 11.1, 1H), 3.524 (m, 1H), 1.803 (s, 3H), 1.775 (s, 3H), 1.013 (s, 3H), 1.023 (d, J = 6.6, 3H), 0.695 (s, 3H). (See below for further characterization.) Stigmasta-24,28-dienol (15C). 1H NMR (600 MHz) 6.719 (dd, J = 17.8, 11.2, 1H), 5.099 (d, J = 17.8, 1H), 4.943 (d, J = 11.2, 1H), 3.588 (m, 1H), 1.803 (s, 3H), 1.775 (s, 3H), 1.005 (d, J = 6.6, 3H), 0.806 (s, 3H), 0.663 (s, 3H).

5. Experimental 5.1. General methods NMR spectra were acquired using a Bruker Avance-600 instrument with a TXI probe at 30 °C and CDCl3 as the solvent. Calibration was by the residual solvent signal (1H: 7.26 ppm; 13C: 77.0 ppm). Mass spectra were obtained using a Thermo Finnigan MAT 95 XP instrument at 70 eV, UV–vis using an Agilent 8453E. HPLC was carried out with a Waters 6000A pump, Waters 410 differential refractometer, and two Altex Ultrasphere ODS 5 lm 10  250 mm columns in series using a flow rate of 3 ml/min MeOH or, in selected cases, MeCN/MeOH/EtOAc 11:4:4. Preparative TLC was performed on glass backed plates (10 cm in length) coated with a 0.25 mm layer of silica gel 60 F254. All extraction and purification steps were carried out under low light conditions to minimize autoxidation. 5.2. Cultures Microalgal strains were obtained from CCMP (Bigelow Laboratory, W. Boothbay Harbor, Maine) and cultured in 2800-ml Fernbach flasks containing 1250 ml of f/2 + Si enriched Milford Harbor seawater. Flask cultures were incubated in a lighted bioincubator at 18 °C with 200 lmole photons m 2 s 1 illumination from cool-white fluorescent bulbs on a 12:12-h light:dark cycle. Cultures were harvested by centrifugation (1000g, 15 min) after 2–3 weeks when populations had reached stationary phase (CCMP 147 – 6.1  105 cells ml 1; CCMP 358 – 1.8  106 cells ml 1; CCMP 1095 – 1.4  105 cells ml 1). Cell counts (n = 4) of each culture were done by light microscopy on an Improved Neuebauer hemocytometer; cultures were examined for contaminating particles during the counting process. Subsequently, each strain was analyzed by flow-cytometry (Accuri C-6, Ann Arbor, MI) to ascertain that only one chlorophyll-containing population was present. The cell pellets (CCMP 147 – 2.2 g fresh weight (fw); CCMP 358 – 2.4 g fw; CCMP 1095 – 1.6 g fw) were frozen and shipped to Syracuse for analysis. 5.3. Extraction of lipids Solvent extractions were carried out by adding one vol. acetone (ca. 2 ml) to the algal pellets in polypropylene microfuge tubes (1.5 ml) and shaking vigorously with a Mini-Beadbeater at room temperature for 5 min. After microfuge centrifugation, the supernatants were removed, and the extraction of the pellets was continued with another volume of acetone, and then three more times using EtOAc. The organic extracts were combined, the H2O

5.5. Sterol synthesis Stigmasta-5,24,28-trienol (15A) was prepared from saringosterol (23) (26.3 mg) (Scheme 1, Catalan et al., 1983). Treatment with Ac2O/pyridine (r.t., overnight), followed by evaporation of the reagents, gave the 3-acetate (28.8 mg, quant.). Dehydration with POCl3 (176 mg) in dry pyridine (1.0 ml) at 50 °C, 24 h, followed by extraction with H2O and hexane/EtOAc 4:1, and filtration of the organic layer through silica gel, gave the desired sterol as the 3-acetate (12.0 mg, 43%). Deacetylation with 10% KOH/MeOH (50 °C, 1.5 h), followed by extraction with H2O and hexane/EtOAc 2:1, and filtration of the organic layer through silica gel, gave the free sterol 15A, which was purified by preparative TLC and HPLC (5.3 mg, 49%). This sterol was fairly unstable, presumably due to autoxidation and Diels–Alder reactions. 1H NMR (600 MHz) 6.723 (dd, J = 17.3, 11.0, 1H), 5.350 (m, 1H), 5.105 (d, J = 17.3, 1H), 4.947 (d, J = 11.0, 1H), 3.524 (m, 1H), 1.803 (s, 3H), 1.775 (s, 3H), 1.014 (s, 3H), 1.023 (d, J = 6.9, 3H), 0.696 (s, 3H); 13C NMR (151 MHz) 140.8 (C-5), 134.7 (C-28), 132.1 (C-24), 131.1 (C-24), 121.7 (C-6), 110.5 (C-29), 71.8 (C-3), 56.8 (C-14), 55.8 (C-17), 50.1 (C-9), 42.4 (C-4), 42.3 (C-13), 39.8 (C-12), 37.3 (C-1), 36.55 (C-10 or C-20), 36.53 (C-10 or C-20), 34.9 (C-21), 31.9 (C-7 and C-8), 31.7 (C-2), 28.3 (C-16), 24.6 (C-23), 24.4 (C-15), 21.3 (C-27), 21.1 (C-11), 20.2 (C-26), 19.4 (C-19), 18.8 (C-21), 11.8 (C-18); UV kmax C6H12

J.-L. Giner, G.H. Wikfors / Phytochemistry 72 (2011) 1896–1901

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