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Structural identification of the C25 highly branched isoprenoid pentaene in the marine diatom Rhizosolenia setigera
Organic Geochemistry 30 (1999) 1581±1583
Note
www.elsevier.nl/locate/orggeochem
Structural identi®cation of the C25 highly branched isoprenoid pentaene in the marine diatom Rhizosolenia setigera Jaap S. Sinninghe Damste a,*, Stefan Schouten a, W. Irene C. Rijpstra a, Ellen C. Hopmans a, Harry Peletier a, Winfried W.C. Gieskes b, Jan A.J. Geenevasen c a
Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59, 1790 AB Den Burg, Texel, The Netherlands b Department of Marine Biology, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands c University of Amsterdam, Faculty of Chemistry, Department of Organic Chemistry, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands Received 20 September 1999; accepted 6 October 1999 (Returned to author for revision 28 September 1999)
Abstract 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadeca-2,5E,9E,13-tetraene I possessing a C25 highly branched isoprenoid skeleton has been isolated from the marine diatom Rhizosolenia setigera and identi®ed by 1H and 13C NMR spectroscopy. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Highly branched isoprenoid alkenes; NMR spectroscopy; Rhizosolenia setigera
Highly branched isoprenoid (HBI) alkanes and alkenes are ubiquitous components in recent and ancient sediments [for a review see Rowland and Robson (1990)]. In recent years it has become clear that diatoms are the biological source of the C25 and C30 sedimentary HBI alkenes and detailed studies of HBI alkenes in diatoms are thus relevant for the interpretation of the sedimentary HBI record. Volkman et al. (1994, 1998) identi®ed C25 HBI alkenes with 3-5 double bonds and C30 HBI alkenes with 5±6 double bonds in laboratory cultures of the diatoms Haslea ostrearia and Rhizosolenia setigera, respectively. Belt et al. (1996) and Wraige et al. (1997) have established the double bond positions in a C25:3, a C25:4, two C25:5 and a C25:6 HBI alkene(s) biosynthesised by H. ostrearia Simonsen. Recently, we determined the lipid composition of a North Atlantic strain of the marine diatom R. setigera (Sinninghe Damste et al., 1999), indicating that C25 HBI * Corresponding author. Tel.: +31-222-369550; fax: +31222-319674. E-mail address: [email protected] (J.S. Sinninghe DamsteÂ).
polyenes are not restricted to Haslea species. The R. setigera strain contained an abundant C25 pentaene possessing a HBI (II) skeleton as determined by hydrogenation and mass spectral analysis. To establish the positions and stereochemistry of the ®ve double bonds of this C25 highly branched isoprenoid in R. setigera, it was isolated. To this end, large cell masses of the R. setigera (strain CCMP 1330, isolated from Vineyard Sound, MA, USA) were grown at 12 C in batch cultures under identical conditions previously described (Sinninghe Damste et al., 1999). Cells were harvested by ®ltration on precombusted glass ®bre ®lters and extracted ultrasonically with hexane, yielding 51 mg of total extract. An apolar fraction was obtained from the extract by column chromatography (Al2O3, hexane:dichloromethane 9:1 eluent). Subsequently, I was isolated from this fraction (5.5 mg) by high-performance liquid chromatography using a semi-preparative reversed phase column (Waters Symmetry Prep C18; 7.8150 mm, 7 mm) and a mobile phase of methanol:water (95:5) delivered at 2 ml/min. The separation was monitored by UV detection at 210 nm. A fraction (1.2 mg) consisting of
0146-6380/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(99)00140-0
1582
J.S. Sinninghe Damste et al. / Organic Geochemistry 30 (1999) 1581±1583
>99% (as determined by GC) of I resulted. This is the same HBI isomer previously reported (Sinninghe Damste et al., 1999) with identical RI and MS data. Analysis by high ®eld 1H and 13C NMR of I led to complete assignment of proton and carbon chemical shifts (Table 1). The 1H NMR spectrum revealed the presence of 7 ole®nic H, 2 di-allylic H, 8 allylic H, 6 ole®nic CH3 and 1 aliphatic CH3, 4 aliphatic protons in CH2 groups. Carbon multiplicities were established by an APT spectrum and revealed that I contains 25 carbon atoms with 4 ole®nic C, 5 ole®nic and 2 aliphatic CH, 1 ole®nic and 6 aliphatic CH2 and 7 CH3 units. Homonuclear (COSY; Fig. 1a) and heteronuclear (HMQC, HMBC; Fig. 1b) 2-D NMR spectra were used to assign chemical shifts. These assignments, in combination with the known highly branched isoprenoid carbon skeleton of I established by hydrogenation (Sinninghe Damste et al., 1999), proved that the double bonds are at positions 2, 5, 9, 13 and 23. Allylic and homoallylic long-range couplings observed in the 1H±1H COSY spectrum (Fig. 1a) further established the structure of I. One dimensional NOE experiments
indicated the stereochemistry of the 5 and 9 double bonds to be both trans (5E,9E) by the observed NOE interactions (Fig. 1c), no NOE interactions being observed between H-5 and H-17 or between H-9 and H-18. The stereochemistry of the chiral centres at C-7 and C-22 could not be established. The assignments of NMR signals is generally in good agreement with those of the structurally related HBI hexaene III identi®ed by Wraige et al. (1997) in H. ostrearia, except that the assignment of the chemical shifts of C-4 and C-11 in the 13C NMR spectrum have been interchanged. The positions and stereochemistries of the double bonds of I in R. setigera are identical to those of I tentatively identi®ed in H. ostrearia and to ®ve of the six double bonds of the HBI hexaene III in H. ostrearia (Wraige et al., 1997). This seems to indicate a common biosynthetic pathway in diatoms for HBI alkene biosynthesis. However, in our North Atlantic strain of R. setigera the HBI distribution is dominated by only one isomer and unaected by dierent growth temperatures (Sinninghe Damste et al., 1999), whereas the HBI distribution in H.
Table 1 1 H (400 MHz) and 13C (100 MHz) NMR data of 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadeca-2,5E,9E,13-tetraene I in C6D6 C-number H-shift
C-shift CH2
CH3 1 2 3 4
1.66 (s, 3H) ± 5.27 (m, 1H) 2.80 (quasi t, 2H, J=7 Hz) 5.33 (m, 1H) ± 2.08 (m, 1H) 2.14 (m, 2H) 5.30 (m, 1H) ± 2.11 (m, 2H) 2.20 (m, 2H) 5.27 (m, 1H) ± 1.68 (s, 3H) 1.58 (s, 3H) 1.54 (s, 3H) 1.63 (s, 3H) 1.58 (s, 3H) 1.40 (m, 2H) 1.25 (m, 2H) 2.08 (m, 1H) 5.70 (m, 1H) 4.97 (Ha, dd, 1H, J=10 and 1 Hz) 5.05 (Hb, dd, 1H, J=17 and 1 Hz) 0.98 (d, 3H, J=7 Hz)
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
25 a±d
CH
C
a
26.53
27.95c
125.90b 125.69b
33.57
40.97 28.02c 26.44a 18.42 12.82 17.01 18.42
31.49 35.65
113.47
21.45
Assignments may be interchanged.
50.43 124.77b
124.71b
131.72d
137.58
135.76
131.76d
39.00 145.59
Fig. 1. (a) Connectivities from a COSY experiment (stippled lines indicate long-range allylic and homoallylic couplings); (b) connectivities for methyl groups from an inverse long-range (2J and 3J) 1H±13C correlation (HMBC) experiment; (c) long-distance NOE interactions of selected protons, indicating the stereochemistry of the 5 and 9 double bonds.
J.S. Sinninghe Damste et al. / Organic Geochemistry 30 (1999) 1581±1583
ostrearia is much more variable and aected by growth conditions (Wraige et al., 1997, 1998, 1999). It indicates that the controls on the HBI distribution in diatoms and, thus, in sediments are complicated and still poorly understood.
Acknowledgements We thank an anonymous referee for useful comments. This work was partly supported by the Research Council for Earth and Life Sciences (ALW) from the Netherlands Organisation for Scienti®c Research (NWO). This is NIOZ Contribution 3423. Associate EditorÐA.G. Douglas Appendix
1583
References Belt, S.T., Cooke, D.A., Robert, J.-M., Rowland, S.J., 1996. Structural characterisation of widespread polyunsaturated isoprenoid biomarkers: A C25 triene, tetraene and pentaene from the diatom Haslea ostrearia Simonsen. Tetrahedron Letters 37, 4755±4758. Rowland, S.J., Robson, J.N., 1990. The widespread occurrence of highly branched acyclic C20, C25 and C30 hydrocarbons in recent sediments and biota Ð a review. Marine Environmental Research 30, 191±216. Sinninghe DamsteÂ, J.S., Rijpstra, W.I.C., Schouten, S., Peletier, H., van der Maarel, M.J.E.C., Gieskes, W.W.C., 1999. A C25 highly branched isoprenoid alkene and C25 and C27 n-polyenes in the marine diatom Rhizosolenia setigera. Organic Geochemistry 30, 95±100. Volkman, J.K., Barrett, S.M., Dunstan, G.A., 1994. C25 and C30 highly branched isoprenoid alkenes in laboratory cultures of two marine diatoms. Organic Geochemistry 21, 407±413. Volkman, J.K., Barrett, S.M., Blackburn, S.I., Mansour, M.P., Sikes, E.L., Gelin, F., 1998. Microalgal biomarkers: a review of recent research developments. Organic Geochemistry 29, 1163±1179. Wraige, E.J., Belt, S.T., MasseÂ, G., Robert, J.-M., Rowland, S.J., 1997. Variations in structures and distributions of C25 highly branched isoprenoid (HBI) alkenes in cultures of the diatom, Haslea ostrearia (Simonson). Organic Geochemistry 27, 497±505. Wraige, E.J., Belt, S.T., Lewis, C.A., Cooke, D.A., Robert, J.M., MasseÂ, G., Rowland, S.J., 1998. Variations in distributions of C25 highly branched isoprenoid (HBI) alkenes in the diatom, Haslea ostrearia: in¯uence of salinity. Organic Geochemistry 28, 855±859. Wraige, E.J., Johns, L., Belt, S.T., MasseÂ, G., Robert, J.-M., Rowland, S.J., 1999. Highly branched C25 isoprenoids in axenic cultures of Haslea ostrearia. Phytochemistry 51, 69±73.
Note
www.elsevier.nl/locate/orggeochem
Structural identi®cation of the C25 highly branched isoprenoid pentaene in the marine diatom Rhizosolenia setigera Jaap S. Sinninghe Damste a,*, Stefan Schouten a, W. Irene C. Rijpstra a, Ellen C. Hopmans a, Harry Peletier a, Winfried W.C. Gieskes b, Jan A.J. Geenevasen c a
Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59, 1790 AB Den Burg, Texel, The Netherlands b Department of Marine Biology, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands c University of Amsterdam, Faculty of Chemistry, Department of Organic Chemistry, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands Received 20 September 1999; accepted 6 October 1999 (Returned to author for revision 28 September 1999)
Abstract 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadeca-2,5E,9E,13-tetraene I possessing a C25 highly branched isoprenoid skeleton has been isolated from the marine diatom Rhizosolenia setigera and identi®ed by 1H and 13C NMR spectroscopy. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Highly branched isoprenoid alkenes; NMR spectroscopy; Rhizosolenia setigera
Highly branched isoprenoid (HBI) alkanes and alkenes are ubiquitous components in recent and ancient sediments [for a review see Rowland and Robson (1990)]. In recent years it has become clear that diatoms are the biological source of the C25 and C30 sedimentary HBI alkenes and detailed studies of HBI alkenes in diatoms are thus relevant for the interpretation of the sedimentary HBI record. Volkman et al. (1994, 1998) identi®ed C25 HBI alkenes with 3-5 double bonds and C30 HBI alkenes with 5±6 double bonds in laboratory cultures of the diatoms Haslea ostrearia and Rhizosolenia setigera, respectively. Belt et al. (1996) and Wraige et al. (1997) have established the double bond positions in a C25:3, a C25:4, two C25:5 and a C25:6 HBI alkene(s) biosynthesised by H. ostrearia Simonsen. Recently, we determined the lipid composition of a North Atlantic strain of the marine diatom R. setigera (Sinninghe Damste et al., 1999), indicating that C25 HBI * Corresponding author. Tel.: +31-222-369550; fax: +31222-319674. E-mail address: [email protected] (J.S. Sinninghe DamsteÂ).
polyenes are not restricted to Haslea species. The R. setigera strain contained an abundant C25 pentaene possessing a HBI (II) skeleton as determined by hydrogenation and mass spectral analysis. To establish the positions and stereochemistry of the ®ve double bonds of this C25 highly branched isoprenoid in R. setigera, it was isolated. To this end, large cell masses of the R. setigera (strain CCMP 1330, isolated from Vineyard Sound, MA, USA) were grown at 12 C in batch cultures under identical conditions previously described (Sinninghe Damste et al., 1999). Cells were harvested by ®ltration on precombusted glass ®bre ®lters and extracted ultrasonically with hexane, yielding 51 mg of total extract. An apolar fraction was obtained from the extract by column chromatography (Al2O3, hexane:dichloromethane 9:1 eluent). Subsequently, I was isolated from this fraction (5.5 mg) by high-performance liquid chromatography using a semi-preparative reversed phase column (Waters Symmetry Prep C18; 7.8150 mm, 7 mm) and a mobile phase of methanol:water (95:5) delivered at 2 ml/min. The separation was monitored by UV detection at 210 nm. A fraction (1.2 mg) consisting of
0146-6380/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(99)00140-0
1582
J.S. Sinninghe Damste et al. / Organic Geochemistry 30 (1999) 1581±1583
>99% (as determined by GC) of I resulted. This is the same HBI isomer previously reported (Sinninghe Damste et al., 1999) with identical RI and MS data. Analysis by high ®eld 1H and 13C NMR of I led to complete assignment of proton and carbon chemical shifts (Table 1). The 1H NMR spectrum revealed the presence of 7 ole®nic H, 2 di-allylic H, 8 allylic H, 6 ole®nic CH3 and 1 aliphatic CH3, 4 aliphatic protons in CH2 groups. Carbon multiplicities were established by an APT spectrum and revealed that I contains 25 carbon atoms with 4 ole®nic C, 5 ole®nic and 2 aliphatic CH, 1 ole®nic and 6 aliphatic CH2 and 7 CH3 units. Homonuclear (COSY; Fig. 1a) and heteronuclear (HMQC, HMBC; Fig. 1b) 2-D NMR spectra were used to assign chemical shifts. These assignments, in combination with the known highly branched isoprenoid carbon skeleton of I established by hydrogenation (Sinninghe Damste et al., 1999), proved that the double bonds are at positions 2, 5, 9, 13 and 23. Allylic and homoallylic long-range couplings observed in the 1H±1H COSY spectrum (Fig. 1a) further established the structure of I. One dimensional NOE experiments
indicated the stereochemistry of the 5 and 9 double bonds to be both trans (5E,9E) by the observed NOE interactions (Fig. 1c), no NOE interactions being observed between H-5 and H-17 or between H-9 and H-18. The stereochemistry of the chiral centres at C-7 and C-22 could not be established. The assignments of NMR signals is generally in good agreement with those of the structurally related HBI hexaene III identi®ed by Wraige et al. (1997) in H. ostrearia, except that the assignment of the chemical shifts of C-4 and C-11 in the 13C NMR spectrum have been interchanged. The positions and stereochemistries of the double bonds of I in R. setigera are identical to those of I tentatively identi®ed in H. ostrearia and to ®ve of the six double bonds of the HBI hexaene III in H. ostrearia (Wraige et al., 1997). This seems to indicate a common biosynthetic pathway in diatoms for HBI alkene biosynthesis. However, in our North Atlantic strain of R. setigera the HBI distribution is dominated by only one isomer and unaected by dierent growth temperatures (Sinninghe Damste et al., 1999), whereas the HBI distribution in H.
Table 1 1 H (400 MHz) and 13C (100 MHz) NMR data of 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadeca-2,5E,9E,13-tetraene I in C6D6 C-number H-shift
C-shift CH2
CH3 1 2 3 4
1.66 (s, 3H) ± 5.27 (m, 1H) 2.80 (quasi t, 2H, J=7 Hz) 5.33 (m, 1H) ± 2.08 (m, 1H) 2.14 (m, 2H) 5.30 (m, 1H) ± 2.11 (m, 2H) 2.20 (m, 2H) 5.27 (m, 1H) ± 1.68 (s, 3H) 1.58 (s, 3H) 1.54 (s, 3H) 1.63 (s, 3H) 1.58 (s, 3H) 1.40 (m, 2H) 1.25 (m, 2H) 2.08 (m, 1H) 5.70 (m, 1H) 4.97 (Ha, dd, 1H, J=10 and 1 Hz) 5.05 (Hb, dd, 1H, J=17 and 1 Hz) 0.98 (d, 3H, J=7 Hz)
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
25 a±d
CH
C
a
26.53
27.95c
125.90b 125.69b
33.57
40.97 28.02c 26.44a 18.42 12.82 17.01 18.42
31.49 35.65
113.47
21.45
Assignments may be interchanged.
50.43 124.77b
124.71b
131.72d
137.58
135.76
131.76d
39.00 145.59
Fig. 1. (a) Connectivities from a COSY experiment (stippled lines indicate long-range allylic and homoallylic couplings); (b) connectivities for methyl groups from an inverse long-range (2J and 3J) 1H±13C correlation (HMBC) experiment; (c) long-distance NOE interactions of selected protons, indicating the stereochemistry of the 5 and 9 double bonds.
J.S. Sinninghe Damste et al. / Organic Geochemistry 30 (1999) 1581±1583
ostrearia is much more variable and aected by growth conditions (Wraige et al., 1997, 1998, 1999). It indicates that the controls on the HBI distribution in diatoms and, thus, in sediments are complicated and still poorly understood.
Acknowledgements We thank an anonymous referee for useful comments. This work was partly supported by the Research Council for Earth and Life Sciences (ALW) from the Netherlands Organisation for Scienti®c Research (NWO). This is NIOZ Contribution 3423. Associate EditorÐA.G. Douglas Appendix
1583
References Belt, S.T., Cooke, D.A., Robert, J.-M., Rowland, S.J., 1996. Structural characterisation of widespread polyunsaturated isoprenoid biomarkers: A C25 triene, tetraene and pentaene from the diatom Haslea ostrearia Simonsen. Tetrahedron Letters 37, 4755±4758. Rowland, S.J., Robson, J.N., 1990. The widespread occurrence of highly branched acyclic C20, C25 and C30 hydrocarbons in recent sediments and biota Ð a review. Marine Environmental Research 30, 191±216. Sinninghe DamsteÂ, J.S., Rijpstra, W.I.C., Schouten, S., Peletier, H., van der Maarel, M.J.E.C., Gieskes, W.W.C., 1999. A C25 highly branched isoprenoid alkene and C25 and C27 n-polyenes in the marine diatom Rhizosolenia setigera. Organic Geochemistry 30, 95±100. Volkman, J.K., Barrett, S.M., Dunstan, G.A., 1994. C25 and C30 highly branched isoprenoid alkenes in laboratory cultures of two marine diatoms. Organic Geochemistry 21, 407±413. Volkman, J.K., Barrett, S.M., Blackburn, S.I., Mansour, M.P., Sikes, E.L., Gelin, F., 1998. Microalgal biomarkers: a review of recent research developments. Organic Geochemistry 29, 1163±1179. Wraige, E.J., Belt, S.T., MasseÂ, G., Robert, J.-M., Rowland, S.J., 1997. Variations in structures and distributions of C25 highly branched isoprenoid (HBI) alkenes in cultures of the diatom, Haslea ostrearia (Simonson). Organic Geochemistry 27, 497±505. Wraige, E.J., Belt, S.T., Lewis, C.A., Cooke, D.A., Robert, J.M., MasseÂ, G., Rowland, S.J., 1998. Variations in distributions of C25 highly branched isoprenoid (HBI) alkenes in the diatom, Haslea ostrearia: in¯uence of salinity. Organic Geochemistry 28, 855±859. Wraige, E.J., Johns, L., Belt, S.T., MasseÂ, G., Robert, J.-M., Rowland, S.J., 1999. Highly branched C25 isoprenoids in axenic cultures of Haslea ostrearia. Phytochemistry 51, 69±73.