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Biomarkers of Holocene buried conifer logs from Bella Coola and north Vancouver, British Columbia, Canada
Organic Geochemistry 33 (2002) 1241–1251 www.elsevier.com/locate/orggeochem
Biomarkers of Holocene buried conifer logs from Bella Coola and north Vancouver, British Columbia, Canada Angelika Otto*, Bernd R.T. Simoneit Environmental and Petroleum Geochemistry Group, College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331, USA Received 5 April 2002; accepted 13 August 2002 (returned to author for revision 18 June 2002)
Abstract The biomarker compositions of a Holocene seep oil (liquefied resin) originating from a buried log of Douglas fir (Pseudotsuga menziesii) from Bella Coola, BC, Canada, and a Holocene pine wood (Pinus sp.) sample from north Vancouver, BC, Canada, were analyzed using gas chromatography–mass spectrometry (GC–MS). The total extract of the Bella Coola sample contains the sesquiterpenoid derivative calamenene and diterpenoids of the labdane, abietane, pimarane, and phyllocladane classes. A novel series of successively degraded labdane derivatives (labdan-15-oic acid, norlabdanoic acids, drimenoic acid) was detected in the polar fraction and a diagenetic pathway is proposed. Aromatic abietanes are the major components in the neutral fraction. The neutral fraction of the pine wood contains two sesquiterpenoids (calamenene, cadalene), diterpenoids of the abietane, pimarane, isopimarane, and kaurane classes, and a series of steroids. 18-Norabieta-8,11,13-triene (dehydroabietin) is the major component among the predominantly aromatic abietanes. Based on the compositions of the terpenoids it is concluded that the diagenesis of the pine and Douglas fir logs probably occurred under anaerobic conditions leading to reductive reactions such as decarboxylation of resin acids, with concurrent oxidation reactions to form the aromatic derivatives. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction The aromatic diterpenoid 18-norabieta-8,11,13-triene (4b-dehydroabietin) was reported as the major compound in the acetone extracts (bitumen) of a Holocene pine wood log (Pinus sp.) excavated from the talus of a land slide in north Vancouver, British Columbia, and a Holocene seep oil (liquefied resin) probably originating from the decomposition of a buried trunk of Douglas fir (Pseudotsuga menziesii) in Bella Coola, British Columbia, Canada (Swan, 1965, 1968). Additionally, further aromatic abietanes (19-norabietatriene, dehydroabietane) and a series of aromatic sesquiterpenoids
(calamenene, 5,6,7,8-tetrahydrocadalene, cadalene) were identified in the Bella Coola seep oil (Simoneit et al., 1986). The diagenesis of the pine and Douglas fir wood probably occurred under alkaline anaerobic conditions leading to reductive reactions such as decarboxylation (Swan, 1965, 1968). The previous analyses documented only the hydrocarbons of the extracts; polar compounds were not included. Here we report the composition of the polar compounds with the hydrocarbons in these conifer remains which were fossilized under unusual conditions.
2. Samples and methods * Corresponding author at present address: Institut fu¨r Mineralogie—Umweltanalytik, J. W. Goethe-Universita¨t Frankfurt/Main, Georg-Voigt-Strasse 14, D-60054 Frankfurt/ Main, Germany. Fax: +49-69-798-28-702. E-mail address: [email protected] (A. Otto).
2.1. Samples Soil with a high proportion of dark brown seep oil (liquefied resin) of a wood origin was sampled near
0146-6380/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(02)00139-0
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Bella Coola, British Columbia. The seep oil originated from the decomposition of a buried log of a conifer, possibly Douglas Fir (Pseudotsuga menziesii), of Holocene age (Swan, 1965). The exact age of the sample could not be determined. The sample of ancient pine wood originated from pine logs (Pinus sp.) buried in a mud land slide near north Vancouver, British Columbia (Swan, 1968). The wood was partly silicified, but had a fibrous character. The age of the fossil pine wood was determined to be 32,200 3300 years by radiocarbon dating (Swan, 1968). 2.2. Extraction and fractionation The soil from Bella Coola was extracted with acetone. The total extract was treated with benzene on a silicic acid column, and then eluted with petroleum ether on an alumina column to separate the neutral fraction and the polar fraction (Swan, 1965). An aliquot of the polar fraction was treated with diazomethane in diethyl ether to generate methyl esters of acids. Aliquots of the total extract, the methylated polar fraction and the neutral fraction were analyzed by gas chromatography–mass spectrometry (GC–MS). The total extract was also analyzed after derivatization with BSTFA. The pine wood was ground to a meal and extracted with acetone:hydrochloric acid (1:1; v:v) (Swan, 1968). The total extract was washed with dilute sodium bicarbonate to remove the acids and then with dilute sodium hydroxide to remove the phenols (Swan, 1968). The remaining neutral fraction was analyzed non-derivatized and derivatized with BSTFA by GC–MS. 2.3. Gas Chromatography–Mass Spectrometry Prior to GC-MS analysis, aliquots of the total extract of the Bella Coola sample and the neutral fraction of the pine wood extract were converted to trimethylsilyl derivatives by reaction with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine for 3 h at 70 C. GC-MS analyses of the non-derivatized and the derivatized total extract and separated fractions were performed on a Hewlett-Packard model 6890 GC coupled to a Hewlett Packard 5973 MSD. Separation was achieved on a fused silica capillary column coated with DB5 (30 m0.25 mm i.d., film thickness 0.25 mm). The GC operating conditions were as follows: temperature hold at 65 C for 2 min, increase from 65 to 300 C at a rate of 6 C min 1 with final isothermal hold at 300 C for 20 min. Helium was used as carrier gas. The sample was injected splitless at an injector temperature of 300 C. The mass spectrometer was operated in the electron impact mode at 70 eV and scanned from 50 to 650 Da. Data were acquired and processed with the Chemstation software. Individual compounds were identified by comparison of mass spectra with literature
and library data, comparison with authentic standards and interpretation of mass spectrometric fragmentation patterns.
3. Results and discussion 3.1. Seep oil, Bella Coola The total extract of the Bella Coola seep oil contains one cadalane type sesquiterpenoid and diterpenoids of the pimarane, abietane, phyllocladane and labdane classes (Fig. 1a, Table 1). Mass spectrometric data of novel compounds are listed in Table 2. Calamenene (s1; the chemical structures of terpenoids cited in the text are shown in the Appendix) is the only sesquiterpenoid present in the Bella Coola seep oil. The diterpenoids can be assigned to the following classes: labdanes, abietanes, pimaranes, isopimaranes, and phyllocladanes. Major components in the non-derivatized total extract are 18-norabieta-8,11,13-triene (a5) and pimarane (p4; Fig. 1a), while silylated diterpenoid alcohols and acids, e.g., tetrahydro(-epi-)manool (L8; co-elutes with pimarane), labdan-15-oic acid (L10), and dehydroabietic acid (a13), are predominant in the derivatized extract (Fig. 1b). A series of diterpenoids was identified as successively degraded novel labdane derivatives, i.e., 13,14,15,16,17pentanorlabdan-12-oic acid (L3), 14,15,16,17-tetranorlabdan-13-oic acid (L4), 14,15-bisnorlabdan-13-one (L5), 14,15,16-trisnorlabdan-13-oic acid (L6), 14,15-bisnorlabdan-16-oic acid (L7), ethyl labdan-15-oate (L9), and labdan-15-oic acid (L10). The drimane derivatives 11-nordriman-9-one (L1) and drim-7-en-11-oic acid (L2) were also interpreted as diagenetic products of labdane type precursors. The abietanes are composed of 18-norabieta-4(19),8,11,13-tetraene (a4), 18-norabieta8,11,13-triene (a5), 18-norabieta-8,11,13-trien-7-one (a9), abieta-8,11,13-trien-7-one (a10), and the following abietanoic acids: abieta-6,8,11,13-tetraenoic acid (a11), 13b(H)-abietan-18-oic acid (a12), dehydroabietic acid (a13), 7-oxodehydroabietic acid (a14), 7-oxoabieta5,8,11,13-tetraenoic acid (a15), and 3-oxodehydroabietic acid (a16). Isopimarane (p3) and pimarane (p4) are the only diterpenoids of the pimarane/isopimarane class present in the extract. Minor amounts of phyllocladanes, i.e., isophyllocladene (k3) and phyllocladene (k4), were also detected. The polar fraction (Fig. 2a) contains the series of successively degraded labdanoic acids (L3-L10) with labdan-15-oic acid (L10) as the major compound. 13b(H)-Abietan-18-oic acid (a12) is the predominant abietanoic acid accompanied by 7- and 3-oxydehydroabietic acids. The composition of the neutral fraction (Fig. 2b) confirms the results observed by Swan (1965). The fraction is mainly composed of 18-norabieta-
A. Otto, B.R.T. Simoneit / Organic Geochemistry 33 (2002) 1241–1251
8,11,13-triene (a5) and 18-norabieta-8,11,13-trien-7-one (a9). The 18-norabieta-8,11,13-trien-7-one (a9) may also be an oxidation product of 18-norabieta-8,11,13-triene (a5) generated in the neutral fraction during the extensive storage time.
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The composition of the terpenoids in the Bella Coola seep oil is in accordance with its presumed origin from a species of the pine family (Pinaceae). Diterpenoids of the labdane, abietane and pimarane/isopimarane classes are the major components of extant species of the
Fig. 1. GC–MS (TIC) traces of (a) the total extract and (b) the silylated total extract (TMS derivatives) of the Holocene Bella Coola seep oil (liquefied resin). Peak annotation see Table 1. u=unknown compounds, *=contamination.
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Table 1 Compounds identified in the total extract of the Bella Coola seep oil and the neutral fraction of the pine wood extract No.
Compound namea
MW
Composition
Relative abundanceb Bella Coola
Pine wood
IDc
s1 s2
SESQUITERPENOIDS Calamenene Cadalene
202 198
C15H22 C15H18
0.5 –
4.6 3.2
P S
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10
DITERPENOIDS Labdanes 11-Nordriman-9-one Drim-7-en-11-oic acid 13,14,15,16,17-Pentanorlabdan-12-oic acid 14,15,16,17-Tetranorlabdan-13-oic acid 14,15-Bisnorlabdan-13-one 14,15,16-Trisnorlabdan-13-oic acid 14,15-Bisnorlabdan-16-oic acid Tetrahydro(-epi-)manool Ethyl labdan-15-oate Labdan-15-oic acid
208 236 238 252 264 266 280 294 336 308
C14H24O C15H24O2 C15H26O2 C16H28O2 C18H32O C17H30O2 C18H32O2 C20H38O C22H40O2 C20H36O2
2.3 0.6 7.1 0.5 1.8 9.9 0.6 100.0 9.3 58.2
– – – – – – – – – –
I I I I I I I I I P, I
a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 a11 a12 a13 a14 a15 a16
Abietanes 18-Norabieta-7,13-diene 16,17-Bisnordehydroabietane Fichtelite 18-Norabieta-4(19),8,11,13-tetraene 18-Norabieta-8,11,13-triene Dehydroabietane 1,2,3,4-Tetrahydroretene Retene 18-Norabieta-8,11,13-trien-7-one Abieta-8,11,13-trien-7-one Abieta-6,8,11,13-tetraenoic acid 13b(H)-Abietan-18-oic acid Dehydroabietic acid 7-Oxodehydroabietic acid 7-Oxoabieta-5,8,11,13-tetraenoic acid 3-Oxodehydroabietic acid
258 242 262 254 256 270 238 234 270 284 298 306 300 314 312 314
C19H30 C18H26 C19H34 C19H26 C19H28 C20H30 C18H22 C18H18 C19H26O C20H28O C20H26O2 C20H34O2 C20H28O2 C20H26O3 C20H24O3 C20H26O3
– – – 0.2 23.0 – – – 7.4 0.2 1.2 11.9 38.3 17.7 0.9 1.1
0.9 0.7 2.9 100.0 3.9 22.5 2.7 3.6 – 3.3 – 6.3 5.1 – –
I I S S P S P S S I P S S S I I
p1 p2 p3 p4 p5
Pimaranes and Isopimaranes Isopimara-8,15-diene Pimara-8(14),15-diene Isopimarane Pimarane Pimaric acid
272 272 276 276 302
C20H32 C20H32 C20H36 C20H36 C20H30O2
– – 5.1 17.2 –
1.6 1.4 – – 4.7
I I P S S
k1 k2 k3 k4
Kauranes and Phyllocladanes 18-Norkaurene ent-18-Norkaurane Isophyllocladene Phyllocladene
258 260 272 272
C19H30 C19H32 C20H32 C20H32
– – 6.5 1.4
0.7 1.4 – –
I I P L
st1 st2 st3 st4 st5 st6 st7 st8 st9 st10 st11 st12 st13 st14 st15
STEROIDS 24-Methylcholest-2-ene 24-Methylcholest-4-ene Stigmast-2-ene Stigmast-4-ene Stigmasta-4,6,22-triene Stigmasta-3,5-diene 5b(H)-Campestanol Campesterol 5b(H)-Stigmastanol 5a(H)-Campestanol 24-Methylcholesta-3,5-dien-7-one 5a-Stigmastan-3-one b-Sitosterol 5a(H)-Stigmastanol Stigmasta-3,5-dien-7-one
384 384 398 398 394 396 402 400 416 402 396 414 414 416 410
C28H48 C28H48 C29H50 C29H50 C29H46 C29H48 C28H50O C28H48O C29H52O C28H50O C28H44O C29H50O C29H50O C29H52O C29H46O
– – – – – – – – – – – – – – –
1.4 0.6 1.6 0.6 1.4 0.9 21.3 34.8 38.5 16.3 2.0 2.2 58.2 53.0 9.4
I I I P I P I S S I I L P L I
a
Polar compounds analyzed as trimethylsilyl derivatives. Relative abundance normalized to major peak=100. Identification: S=standard, P=published mass spectrum (Kitadani et al., 1970; Zinkel et al., 1971; Simoneit, 1977; Simoneit and Mazurek, 1982; Philp, 1985; Noble et al., 1986; Li et al., 1990), L=Wiley MS library, I=interpretation of MS fragmentation pattern. b c
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Table 2 Mass spectrometric data of novel and derivatized (silylated or methylated) terpenoids No.
Compound name
MW
Characteristic fragments m/z (%)
L1
11-Nordriman-9-one
208
L2
Drim-7-en-11-oic acid
236
L3
13,14,15,16,17-Pentanorlabdan-12-oic acid 13,14,15,16,17-Pentanorlabdan-12-oic acid-TMS Methyl 13,14,15,16,17pentanorlabdan-12-oate
238 310 252
L4
Methyl 14,15,16,17-tetranorlabdan-13-oate
266
L5
14,15-Bisnorlabdan-13-one
264
L6
14,15,16-Trisnorlabdan-13-oic acid 14,15,16Trisnorlabdan-13-oic acid-TMS Methyl 14,15,16-trisnorlabdan-13-oate
266 338 280
L7
Methyl 14,15-bisnorlabdan-16-oate
294
L8
Tetrahydro(-epi-)manool-TMS
364
L9
Ethyl labdan-15-oate
336
L10
Labdan-15-oic acid Labdan-15-oic acid-TMS
308 380
a1
18-Norabieta-7,13-diene
258
a2 a4
16,17-Bisnordehydroabietane 18-Norabieta-4(19),8,11,13-tetraene
242 254
a9
18-Norabieta-8,11,13-trien-7-one
270
a10
Abieta-8,11,13-trien-7-one
284
a14
7-Oxodehydroabietic acid-TMS Methyl 7-oxodehydroabietate
386 328
a15
Methyl 7-oxoabieta-5,8,11,13-tetraenoate
326
a16
3-Oxodehydroabietic acid-TMS Methyl 3-oxodehydroabietate
386 328
p1
Isopimara-8,15-diene
272
p2
Pimara-8(14),15-diene
272
k1
18-Norkaurene
258
k2
ent-18-Norkaurane
260
k3
Isophyllocladene
272
123 (100), 71 (55), 84 (52), 95 (43), 69 (41), 175 (39), 81 (34), 109 (31), 208 (9) 123 (100), 192 (59), 55 (52), 69 (52), 177 (52), 81 (49), 95 (46), 109 (37), 236 (3) 223 (100), 178 (92), 81 (56), 55 (54), 163 (54), 95 (51), 69 (50), 107 (50), 238 (20) 117 (100), 295 (56), 132 (45), 73 (40), 75 (40), 133 (24), 55 (22), 178 (17), 310 (12) 178 (100), 237 (57), 81 (42), 107 (39), 55 (38), 95 (35), 163 (33), 93 (32), 252 (32) 178 (100), 251 (39), 88 (37), 81 (36), 107 (34), 67 (31), 266 (32), 163 (29) 123 (100), 109 (44), 95 (44), 81 (43), 69 (42), 55 (39), 67 (33), 191 (26), 264 (12) 123 (100), 69 (39), 251 (37), 55 (35), 95 (34), 81 (34), 109 (33), 67 (27), 266 (15) 123 (100), 323 (100), 73 (63), 75 (55), 69 (45), 55 (44), 117 (40), 95 (38), 338 (38) 123 (100), 265 (40), 55 (37), 69 (35), 81 (35), 95 (35), 109 (34), 67 (30), 280 (19) 123 (100), 279 (43), 69 (37), 55 (35), 95 (35), 81 (34), 109 (34), 67 (27), 294 (22) 145 (100), 73 (57), 146 (53), 75 (44), 191 (30), 69 (28), 337 (27), 95 (22), 109 (22) 123 (100), 69 (48), 321 (40), 55 (39), 95 (36), 109 (36), 81 (34), 336 (28), 67 (26) 123 (100 ), 293 (35), 55 (33), 69 (33), 95 (33), 109 (33), 81 (32), 124 (23), 308 (18) 123 (100), 365 (85), 73 (52), 69 (40), 75 (40), 109 (37), 95 (35), 380 (34), 81 (32) 243 (100), 105 (54), 161 (45), 91 (43), 258 (37), 187 (33), 119 (32), 55 (30) 227 (100), 242 (64), 157 (36), 145 (24), 131 (22) 197 (100), 254 (85), 141 (77), 183 (45), 239 (45), 155 (39), 169 (37), 153 (28) 255 (100), 173 (67), 270 (43), 199 (29), 256 (20), 128 (18), 129 (17), 115 (14) 269 (100), 284 (48), 187 (40), 199 (38), 201 (32), 227 (26), 128 (21), 185 (20) 253 (100), 268 (78), 73 (49), 327 (33), 269 (32), 187 (30), 386 (25), 254 (22) 253 (100), 328 (35), 254 (21), 187 (17), 269 (11), 211 (10), 213 (10), 128 (9) 251 (100), 326 (42), 252 (21), 185 (17), 211 (12), 115 (10), 128 (10), 197 (9) 268 (100), 73 (98), 327 (82), 253 (62), 75 (33), 187 (28), 342 (27), 269 (26), 386 (7) 253 (100), 328 (52), 187 (49), 268 (33), 213 (28), 269 (25), 313 (20), 115 (18) 257 (100), 145 (72), 105 (64), 227 (62), 91 (61), 131 (57), 161 (50), 272 (37) 137 (100), 257 (62), 91 (61), 79 (42), 105 (41), 81 (40), 272 (36), 55 (35), 69 (35) 91 (100), 123 (82), 243 (70), 81 (65), 79 (55), 93 (54), 55 (50), 121 (47), 258 (36) 123 (100), 217 (87), 245 (57), 81 (55), 107 (38), 136 (37), 91 (35), 260 (32) 120 (100), 272 (98), 106 (72), 91 (58), 105 (49), 107 (48), 119 (45), 93 (38), 133 (33)
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Pinaceae (Hegnauer, 1962, 1986; Thomas, 1970; Otto and Wilde, 2001). Diterpenoid acids such as abietic and isopimaric acids were reported from the resin of extant Douglas Fir (Pseudotsuga menziesii; Erdtman et al., 1968; Otto and Wilde, 2001). A diagenetic pathway of the degradation of labdane derivatives is suggested from the presence of labdan-15oic acid (L10) and a series of labdane type diagenetic
products (Fig. 3). The degradation of natural product precursors, e.g. manoyl oxide and manool, leads to the generation of tetrahydromanool (L8) and labdan-15-oic acid (L10). The successive decarboxylation and demethylation of the labdane acid generates 14,15-bisnorlabdan-16-oic acid (L7), 14,15,16-trisnorlabdan-13-oic acid (L6), 14,15,16,17-tetranorlabdan-13-oic acid (L4), and 13,14,15,16,17-pentanorlabdan-12-oic acid (L3). In an
Fig. 2. GC–MS (TIC) traces of (a) the methylated polar fraction and (b) the neutral fraction of the Holocene Bella Coola seep oil (liquefied resin). For peak annotation see Table 1. u=unknown compound, *=contamination.
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alternative pathway, the degradation of 14,15,16-trisnorlabdan-13-oic acid (L6) can result in the formation of drim-7-en-11-oic acid (L2) and 11-nordriman-9-one (L1). The bisnorlabdanone (L5) is probably generated from the oxidation of manool. Similar series of acids and ketones were observed as the major intermediates in the aerobic bacterial degradation of dehydroabietic acid (Biellmann and Branlant, 1973a,b). Thus, the diagenesis of the labdanes (inferred precursors: manoyl oxide, labdadienol) in the Holocene conifer log from Bella Coola is probably caused by aerobic bacterial degradation of the liquified resin. The formation of 13b(H)-abietan-18-oic acid (a12), isopimarane (p3) and pimarane (p4) in the Bella Coola sample suggests a first step of aerobic decarboxylation of the parent acids, with concomitant anaerobic alteration to the saturated derivatives under strong reducing conditions.
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Thus, both oxidative and reductive reactions occurred simultaneously. The process of disproportionation suggested by Skrigan (1964) and Frenkel and Heller-Kallai (1977) may also be the cause for the generation of both saturated and aromatic derivatives, but not all abietane degradation products observed in the Bella Coola seep oil could have formed by disproportionation. 3.2. Ancient pine wood, North Vancouver The neutral fraction of the pine wood extract contains sesquiterpenoids, diterpenoids of the abietane, pimarane, isopimarane and kaurane classes and a group of steroids (Fig. 4). Aromatic abietanes are predominant in the nonderivatized (Fig. 4a) and in the BSTFA-derivatized neutral fraction (Fig. 4b). Calamenene (s1) and cadalene (s2) are the only sesquiterpenoids identified. The abietanes are
Fig. 3. Proposed diagenetic degradation scheme of the labdane type diterpenoids from the Holocene Bella Coola seep oil (liquefied resin). Compounds in boxes represent natural product precursors.
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composed of 18-norabieta-7,13-diene (a1), 16,17-bisnordehydroabietane (a2), fichtelite (a3), 18-norabieta-8,11,13triene (a5), dehydroabietane (a6), 1,2,3,4-tetrahydroretene (a7), retene (a8), 18-norabieta-8,11,13-trien-7-one (a9), abieta-6,8,11,13-tetraenoic acid (a11), dehydroabietic acid (a13), and 7-oxodehydroabietic acid (a14). 18-Norabieta-8,11,13-triene (a5, relative abundance 100%) is
the major compound in the neutral fraction as Swan (1968) reported. The pimaranes/isopimaranes are represented by isopimara-8,15-diene (p1), pimara-8(14),15diene (p2), and pimaric acid (p5). The kauranes are 18norkaurene (k1) and ent-18-norkaurane (k2). The steroids consist of the hydrocarbons 24-methylcholest-2-ene (st1), 24-methylcholest-4-ene (st2), stigmast-
Fig. 4. GC–MS (TIC) traces of (a) the total extract and (b) the silylated total extract (TMS derivatives) of the Holocene pine wood from north Vancouver. Peak annotation see Table 1. v=n-alkanol, *=contamination.
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2-ene (st3), stigmast-4-ene (st4), stigmasta-4,6,22-triene (st5), and stigmasta-3,5-diene (st6), and the polars 5b(H)-campestanol (st7), campesterol (st8), 5b(H)-stigmastanol (st9), 5a(H)-campestanol (st10), 24-methylcholesta-3,5-dien-7-one (st11), 5a-stigmastan-3-one (st12), b-sitosterol (st13), 5a(H)-stigmastanol (st14), and stigmasta-3,5-dien-7-one (st15). b-Sitosterol was also identified in the pine wood by Swan (1968). The composition of the terpenoids in the Holocene wood is in accordance with its botanical assignment to
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pine (Pinus sp.), because abietic acid associated with pimarane and isopimarane type diterpenoids are the major constituents of the resins of contemporary pine species (Hegnauer, 1962, 1986; Otto and Wilde, 2001). Swan (1968) suggested the diagenesis of abietane type diterpenoids in the pine wood under alkaline anaerobic conditions with major reductive processes, i.e. decarboxylation and hydrogenation of resin acids. Abietic acid and dehydroabietane are probably the biological precursors of the saturated and aromatic abietanes
Fig. 5. Proposed diagenetic degradation scheme of the abietane and pimarane type diterpenoids from the Holocene pine wood. Compounds in boxes represent natural product precursors.
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(Fig. 5). Dehydroabietane (a6), pimaradiene (p2) or isopimaradiene (p3) are the presumed precursors of 16,17-bisnordehydroabietane (a2). The decarboxylation of the natural product abietic acid followed by reduction leads to the saturated fichtelite (a3) as a minor product in the pine wood extract. The dominant alteration pathway is the oxidation of abietic acid to dehydroabietic acid (a13) and its decarboxylation which generates the major diagenetic product 18-norabieta8,11,13-triene (a5). Further aromatization of the B and A rings leads to 1,2,3,4-tetrahydroretene (a7) and finally to retene (a8). The generation of 1,2,3,4-tetrahydroretene from 18-norabieta-8,11,13-trien-7-one is yet unclear.
4. Conclusions The extracts of two Holocene conifer remains contain sesqui- and diterpenoids similar to the terpenoid patterns observed in their related contemporary species.
Appendix
The major compounds in the Bella Coola seep oil are series of successively degraded labdanes while abietanes predominate in the pine wood from north Vancouver. These samples represent the degradation of resin compounds by an unusual fossilization process under semiaerobic conditions in soil or land slides. The presence of both saturated and aromatic products in the samples suggests the occurrence of concomitant oxidative and reductive processes during the degradation of the diterpenoids.
Acknowledgements We thank Dr. E.P. Swan, Canadian Forestry Service, for the seep oil (liquefied resin) and extract fractions, and the Max Kade-Foundation, New York, for financial support. This paper benefitted from the reviews by Dr. G.A. Logan and Dr. H.P. Nytoft. Associate Editor—G. Abbott
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References Biellmann, J.F., Branlant, G., 1973a. De´gradation bacte´rienne de l’acide de´hydroabie´tique par Flavobacterium resinovorum. Tetrahedron 29, 1227–1236. Biellmann, J.F., Branlant, G., 1973b. De´gradation becte´rienne de l’acide de´hydroabie´tique par un Pseudomonas et un Alcaligenes. Tetrahedron 29, 1237–1241. Erdtman, H., Kimland, B., Norin, T., Daniels, P. J. L., 1968. The constituents of the ‘‘pocket resin’’ from Douglas fir Pseudotsuga menziesii (MIRB.) FRANCO. Acta Chemica Scandinavica 22, 938–942. Frenkel, M., Heller-Kallai, L., 1977. Aromatization of limonene—a geochemical model. Organic Geochemistry 1, 3–5. Hegnauer, R., 1962. Chemotaxonomie der Pflanzen. Vol. 1. Thallophyten, Bryophyten, Pteridophyten und Gymnospermen. Basel, Birkha¨user. Hegnauer, R., 1986. Chemotaxonomie der Pflanzen. Vol. 7. Nachtra¨ge zu Band 1 und Band 2. Basel, Birkha¨user. Kitadani, M., Yoshikoshi, A., Kitahara, Y., de Paiva Campello, J., McChesney, J.D., Watts, D.J., Wenkert, E., 1970. Natural ar-abietatriene. Chemical and Pharmaceutical Bulletin 18, 402–405. Li, M., Johns, R.B., Mei, B., 1990. A study in early diagenesis: biomarker composition of a suite of immature coals and coaly shales. Organic Geochemistry 16, 1067–1075. Noble, R.A., Alexander, R., Kagi, R.I., Knox, J., 1986. Identification of some diterpenoid hydrocarbons in petroleum. Organic Geochemistry 10, 825–829.
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Otto, A., Wilde, V., 2001. Sesqui-, di-, and triterpenoids as chemosystematic markers in extant conifers- A review. Botanical Review 67, 141–238. Philp, R.P., 1985. Fossil Fuel Biomarkers. Applications and Spectra. Methods in Geochemistry and Geophysics, 23. Elsevier, Amsterdam. Simoneit, B.R.T., 1977. Diterpenoid compounds and other lipids in deep-sea sediments and their geochemical significance. Geochimica et Cosmochimica Acta 41, 463–476. Simoneit, B.R.T., Mazurek, M.A., 1982. Organic matter of the troposphere—II. Natural background of biogenic lipid matter in aerosols over the rural western United States. Atmospheric Environment 16, 2139–2159. Simoneit, B.R.T., Grimalt, J.O., Wang, T.G., Cox, R.E., Hatcher, P.G., Nissenbaum, A., 1986. Cyclic terpenoids of contemporary resinous plant detritus and of fossil woods, ambers and coals. Organic Geochemistry 10, 877–889. Skrigan, A.I., 1964. Preparation and utilization of fichtelite and retene. Tr. Vses. Nauchno-Tekhn. Soveshch. Gorki 1963, 108–115. Chemical Abstracts 62, 10664a. Swan, E.P., 1965. Identity of a hydrocarbon found in a forest soil. Forest Products Journal 15, 272. Swan, E.P., 1968. Extractives of an Ancient Pine. Bi-Monthly Research Notes. Canadian Forestry Service 24, 8. Thomas, B.R., 1970. Modern and fossil plant resins. In: Harborne, J.B. (Ed.), Phytochemical Phylogeny. Academic Press, London, pp. 59–79. Zinkel, D.F., Zank, L.C., Wesolowski, M.F., 1971. Diterpene resin acids, mass spectra of diterpene resin acid methyl esters. Journal of the American Oil Chemists’ Society 48, 455–461.
Biomarkers of Holocene buried conifer logs from Bella Coola and north Vancouver, British Columbia, Canada Angelika Otto*, Bernd R.T. Simoneit Environmental and Petroleum Geochemistry Group, College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331, USA Received 5 April 2002; accepted 13 August 2002 (returned to author for revision 18 June 2002)
Abstract The biomarker compositions of a Holocene seep oil (liquefied resin) originating from a buried log of Douglas fir (Pseudotsuga menziesii) from Bella Coola, BC, Canada, and a Holocene pine wood (Pinus sp.) sample from north Vancouver, BC, Canada, were analyzed using gas chromatography–mass spectrometry (GC–MS). The total extract of the Bella Coola sample contains the sesquiterpenoid derivative calamenene and diterpenoids of the labdane, abietane, pimarane, and phyllocladane classes. A novel series of successively degraded labdane derivatives (labdan-15-oic acid, norlabdanoic acids, drimenoic acid) was detected in the polar fraction and a diagenetic pathway is proposed. Aromatic abietanes are the major components in the neutral fraction. The neutral fraction of the pine wood contains two sesquiterpenoids (calamenene, cadalene), diterpenoids of the abietane, pimarane, isopimarane, and kaurane classes, and a series of steroids. 18-Norabieta-8,11,13-triene (dehydroabietin) is the major component among the predominantly aromatic abietanes. Based on the compositions of the terpenoids it is concluded that the diagenesis of the pine and Douglas fir logs probably occurred under anaerobic conditions leading to reductive reactions such as decarboxylation of resin acids, with concurrent oxidation reactions to form the aromatic derivatives. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction The aromatic diterpenoid 18-norabieta-8,11,13-triene (4b-dehydroabietin) was reported as the major compound in the acetone extracts (bitumen) of a Holocene pine wood log (Pinus sp.) excavated from the talus of a land slide in north Vancouver, British Columbia, and a Holocene seep oil (liquefied resin) probably originating from the decomposition of a buried trunk of Douglas fir (Pseudotsuga menziesii) in Bella Coola, British Columbia, Canada (Swan, 1965, 1968). Additionally, further aromatic abietanes (19-norabietatriene, dehydroabietane) and a series of aromatic sesquiterpenoids
(calamenene, 5,6,7,8-tetrahydrocadalene, cadalene) were identified in the Bella Coola seep oil (Simoneit et al., 1986). The diagenesis of the pine and Douglas fir wood probably occurred under alkaline anaerobic conditions leading to reductive reactions such as decarboxylation (Swan, 1965, 1968). The previous analyses documented only the hydrocarbons of the extracts; polar compounds were not included. Here we report the composition of the polar compounds with the hydrocarbons in these conifer remains which were fossilized under unusual conditions.
2. Samples and methods * Corresponding author at present address: Institut fu¨r Mineralogie—Umweltanalytik, J. W. Goethe-Universita¨t Frankfurt/Main, Georg-Voigt-Strasse 14, D-60054 Frankfurt/ Main, Germany. Fax: +49-69-798-28-702. E-mail address: [email protected] (A. Otto).
2.1. Samples Soil with a high proportion of dark brown seep oil (liquefied resin) of a wood origin was sampled near
0146-6380/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(02)00139-0
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Bella Coola, British Columbia. The seep oil originated from the decomposition of a buried log of a conifer, possibly Douglas Fir (Pseudotsuga menziesii), of Holocene age (Swan, 1965). The exact age of the sample could not be determined. The sample of ancient pine wood originated from pine logs (Pinus sp.) buried in a mud land slide near north Vancouver, British Columbia (Swan, 1968). The wood was partly silicified, but had a fibrous character. The age of the fossil pine wood was determined to be 32,200 3300 years by radiocarbon dating (Swan, 1968). 2.2. Extraction and fractionation The soil from Bella Coola was extracted with acetone. The total extract was treated with benzene on a silicic acid column, and then eluted with petroleum ether on an alumina column to separate the neutral fraction and the polar fraction (Swan, 1965). An aliquot of the polar fraction was treated with diazomethane in diethyl ether to generate methyl esters of acids. Aliquots of the total extract, the methylated polar fraction and the neutral fraction were analyzed by gas chromatography–mass spectrometry (GC–MS). The total extract was also analyzed after derivatization with BSTFA. The pine wood was ground to a meal and extracted with acetone:hydrochloric acid (1:1; v:v) (Swan, 1968). The total extract was washed with dilute sodium bicarbonate to remove the acids and then with dilute sodium hydroxide to remove the phenols (Swan, 1968). The remaining neutral fraction was analyzed non-derivatized and derivatized with BSTFA by GC–MS. 2.3. Gas Chromatography–Mass Spectrometry Prior to GC-MS analysis, aliquots of the total extract of the Bella Coola sample and the neutral fraction of the pine wood extract were converted to trimethylsilyl derivatives by reaction with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and pyridine for 3 h at 70 C. GC-MS analyses of the non-derivatized and the derivatized total extract and separated fractions were performed on a Hewlett-Packard model 6890 GC coupled to a Hewlett Packard 5973 MSD. Separation was achieved on a fused silica capillary column coated with DB5 (30 m0.25 mm i.d., film thickness 0.25 mm). The GC operating conditions were as follows: temperature hold at 65 C for 2 min, increase from 65 to 300 C at a rate of 6 C min 1 with final isothermal hold at 300 C for 20 min. Helium was used as carrier gas. The sample was injected splitless at an injector temperature of 300 C. The mass spectrometer was operated in the electron impact mode at 70 eV and scanned from 50 to 650 Da. Data were acquired and processed with the Chemstation software. Individual compounds were identified by comparison of mass spectra with literature
and library data, comparison with authentic standards and interpretation of mass spectrometric fragmentation patterns.
3. Results and discussion 3.1. Seep oil, Bella Coola The total extract of the Bella Coola seep oil contains one cadalane type sesquiterpenoid and diterpenoids of the pimarane, abietane, phyllocladane and labdane classes (Fig. 1a, Table 1). Mass spectrometric data of novel compounds are listed in Table 2. Calamenene (s1; the chemical structures of terpenoids cited in the text are shown in the Appendix) is the only sesquiterpenoid present in the Bella Coola seep oil. The diterpenoids can be assigned to the following classes: labdanes, abietanes, pimaranes, isopimaranes, and phyllocladanes. Major components in the non-derivatized total extract are 18-norabieta-8,11,13-triene (a5) and pimarane (p4; Fig. 1a), while silylated diterpenoid alcohols and acids, e.g., tetrahydro(-epi-)manool (L8; co-elutes with pimarane), labdan-15-oic acid (L10), and dehydroabietic acid (a13), are predominant in the derivatized extract (Fig. 1b). A series of diterpenoids was identified as successively degraded novel labdane derivatives, i.e., 13,14,15,16,17pentanorlabdan-12-oic acid (L3), 14,15,16,17-tetranorlabdan-13-oic acid (L4), 14,15-bisnorlabdan-13-one (L5), 14,15,16-trisnorlabdan-13-oic acid (L6), 14,15-bisnorlabdan-16-oic acid (L7), ethyl labdan-15-oate (L9), and labdan-15-oic acid (L10). The drimane derivatives 11-nordriman-9-one (L1) and drim-7-en-11-oic acid (L2) were also interpreted as diagenetic products of labdane type precursors. The abietanes are composed of 18-norabieta-4(19),8,11,13-tetraene (a4), 18-norabieta8,11,13-triene (a5), 18-norabieta-8,11,13-trien-7-one (a9), abieta-8,11,13-trien-7-one (a10), and the following abietanoic acids: abieta-6,8,11,13-tetraenoic acid (a11), 13b(H)-abietan-18-oic acid (a12), dehydroabietic acid (a13), 7-oxodehydroabietic acid (a14), 7-oxoabieta5,8,11,13-tetraenoic acid (a15), and 3-oxodehydroabietic acid (a16). Isopimarane (p3) and pimarane (p4) are the only diterpenoids of the pimarane/isopimarane class present in the extract. Minor amounts of phyllocladanes, i.e., isophyllocladene (k3) and phyllocladene (k4), were also detected. The polar fraction (Fig. 2a) contains the series of successively degraded labdanoic acids (L3-L10) with labdan-15-oic acid (L10) as the major compound. 13b(H)-Abietan-18-oic acid (a12) is the predominant abietanoic acid accompanied by 7- and 3-oxydehydroabietic acids. The composition of the neutral fraction (Fig. 2b) confirms the results observed by Swan (1965). The fraction is mainly composed of 18-norabieta-
A. Otto, B.R.T. Simoneit / Organic Geochemistry 33 (2002) 1241–1251
8,11,13-triene (a5) and 18-norabieta-8,11,13-trien-7-one (a9). The 18-norabieta-8,11,13-trien-7-one (a9) may also be an oxidation product of 18-norabieta-8,11,13-triene (a5) generated in the neutral fraction during the extensive storage time.
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The composition of the terpenoids in the Bella Coola seep oil is in accordance with its presumed origin from a species of the pine family (Pinaceae). Diterpenoids of the labdane, abietane and pimarane/isopimarane classes are the major components of extant species of the
Fig. 1. GC–MS (TIC) traces of (a) the total extract and (b) the silylated total extract (TMS derivatives) of the Holocene Bella Coola seep oil (liquefied resin). Peak annotation see Table 1. u=unknown compounds, *=contamination.
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Table 1 Compounds identified in the total extract of the Bella Coola seep oil and the neutral fraction of the pine wood extract No.
Compound namea
MW
Composition
Relative abundanceb Bella Coola
Pine wood
IDc
s1 s2
SESQUITERPENOIDS Calamenene Cadalene
202 198
C15H22 C15H18
0.5 –
4.6 3.2
P S
L1 L2 L3 L4 L5 L6 L7 L8 L9 L10
DITERPENOIDS Labdanes 11-Nordriman-9-one Drim-7-en-11-oic acid 13,14,15,16,17-Pentanorlabdan-12-oic acid 14,15,16,17-Tetranorlabdan-13-oic acid 14,15-Bisnorlabdan-13-one 14,15,16-Trisnorlabdan-13-oic acid 14,15-Bisnorlabdan-16-oic acid Tetrahydro(-epi-)manool Ethyl labdan-15-oate Labdan-15-oic acid
208 236 238 252 264 266 280 294 336 308
C14H24O C15H24O2 C15H26O2 C16H28O2 C18H32O C17H30O2 C18H32O2 C20H38O C22H40O2 C20H36O2
2.3 0.6 7.1 0.5 1.8 9.9 0.6 100.0 9.3 58.2
– – – – – – – – – –
I I I I I I I I I P, I
a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 a11 a12 a13 a14 a15 a16
Abietanes 18-Norabieta-7,13-diene 16,17-Bisnordehydroabietane Fichtelite 18-Norabieta-4(19),8,11,13-tetraene 18-Norabieta-8,11,13-triene Dehydroabietane 1,2,3,4-Tetrahydroretene Retene 18-Norabieta-8,11,13-trien-7-one Abieta-8,11,13-trien-7-one Abieta-6,8,11,13-tetraenoic acid 13b(H)-Abietan-18-oic acid Dehydroabietic acid 7-Oxodehydroabietic acid 7-Oxoabieta-5,8,11,13-tetraenoic acid 3-Oxodehydroabietic acid
258 242 262 254 256 270 238 234 270 284 298 306 300 314 312 314
C19H30 C18H26 C19H34 C19H26 C19H28 C20H30 C18H22 C18H18 C19H26O C20H28O C20H26O2 C20H34O2 C20H28O2 C20H26O3 C20H24O3 C20H26O3
– – – 0.2 23.0 – – – 7.4 0.2 1.2 11.9 38.3 17.7 0.9 1.1
0.9 0.7 2.9 100.0 3.9 22.5 2.7 3.6 – 3.3 – 6.3 5.1 – –
I I S S P S P S S I P S S S I I
p1 p2 p3 p4 p5
Pimaranes and Isopimaranes Isopimara-8,15-diene Pimara-8(14),15-diene Isopimarane Pimarane Pimaric acid
272 272 276 276 302
C20H32 C20H32 C20H36 C20H36 C20H30O2
– – 5.1 17.2 –
1.6 1.4 – – 4.7
I I P S S
k1 k2 k3 k4
Kauranes and Phyllocladanes 18-Norkaurene ent-18-Norkaurane Isophyllocladene Phyllocladene
258 260 272 272
C19H30 C19H32 C20H32 C20H32
– – 6.5 1.4
0.7 1.4 – –
I I P L
st1 st2 st3 st4 st5 st6 st7 st8 st9 st10 st11 st12 st13 st14 st15
STEROIDS 24-Methylcholest-2-ene 24-Methylcholest-4-ene Stigmast-2-ene Stigmast-4-ene Stigmasta-4,6,22-triene Stigmasta-3,5-diene 5b(H)-Campestanol Campesterol 5b(H)-Stigmastanol 5a(H)-Campestanol 24-Methylcholesta-3,5-dien-7-one 5a-Stigmastan-3-one b-Sitosterol 5a(H)-Stigmastanol Stigmasta-3,5-dien-7-one
384 384 398 398 394 396 402 400 416 402 396 414 414 416 410
C28H48 C28H48 C29H50 C29H50 C29H46 C29H48 C28H50O C28H48O C29H52O C28H50O C28H44O C29H50O C29H50O C29H52O C29H46O
– – – – – – – – – – – – – – –
1.4 0.6 1.6 0.6 1.4 0.9 21.3 34.8 38.5 16.3 2.0 2.2 58.2 53.0 9.4
I I I P I P I S S I I L P L I
a
Polar compounds analyzed as trimethylsilyl derivatives. Relative abundance normalized to major peak=100. Identification: S=standard, P=published mass spectrum (Kitadani et al., 1970; Zinkel et al., 1971; Simoneit, 1977; Simoneit and Mazurek, 1982; Philp, 1985; Noble et al., 1986; Li et al., 1990), L=Wiley MS library, I=interpretation of MS fragmentation pattern. b c
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Table 2 Mass spectrometric data of novel and derivatized (silylated or methylated) terpenoids No.
Compound name
MW
Characteristic fragments m/z (%)
L1
11-Nordriman-9-one
208
L2
Drim-7-en-11-oic acid
236
L3
13,14,15,16,17-Pentanorlabdan-12-oic acid 13,14,15,16,17-Pentanorlabdan-12-oic acid-TMS Methyl 13,14,15,16,17pentanorlabdan-12-oate
238 310 252
L4
Methyl 14,15,16,17-tetranorlabdan-13-oate
266
L5
14,15-Bisnorlabdan-13-one
264
L6
14,15,16-Trisnorlabdan-13-oic acid 14,15,16Trisnorlabdan-13-oic acid-TMS Methyl 14,15,16-trisnorlabdan-13-oate
266 338 280
L7
Methyl 14,15-bisnorlabdan-16-oate
294
L8
Tetrahydro(-epi-)manool-TMS
364
L9
Ethyl labdan-15-oate
336
L10
Labdan-15-oic acid Labdan-15-oic acid-TMS
308 380
a1
18-Norabieta-7,13-diene
258
a2 a4
16,17-Bisnordehydroabietane 18-Norabieta-4(19),8,11,13-tetraene
242 254
a9
18-Norabieta-8,11,13-trien-7-one
270
a10
Abieta-8,11,13-trien-7-one
284
a14
7-Oxodehydroabietic acid-TMS Methyl 7-oxodehydroabietate
386 328
a15
Methyl 7-oxoabieta-5,8,11,13-tetraenoate
326
a16
3-Oxodehydroabietic acid-TMS Methyl 3-oxodehydroabietate
386 328
p1
Isopimara-8,15-diene
272
p2
Pimara-8(14),15-diene
272
k1
18-Norkaurene
258
k2
ent-18-Norkaurane
260
k3
Isophyllocladene
272
123 (100), 71 (55), 84 (52), 95 (43), 69 (41), 175 (39), 81 (34), 109 (31), 208 (9) 123 (100), 192 (59), 55 (52), 69 (52), 177 (52), 81 (49), 95 (46), 109 (37), 236 (3) 223 (100), 178 (92), 81 (56), 55 (54), 163 (54), 95 (51), 69 (50), 107 (50), 238 (20) 117 (100), 295 (56), 132 (45), 73 (40), 75 (40), 133 (24), 55 (22), 178 (17), 310 (12) 178 (100), 237 (57), 81 (42), 107 (39), 55 (38), 95 (35), 163 (33), 93 (32), 252 (32) 178 (100), 251 (39), 88 (37), 81 (36), 107 (34), 67 (31), 266 (32), 163 (29) 123 (100), 109 (44), 95 (44), 81 (43), 69 (42), 55 (39), 67 (33), 191 (26), 264 (12) 123 (100), 69 (39), 251 (37), 55 (35), 95 (34), 81 (34), 109 (33), 67 (27), 266 (15) 123 (100), 323 (100), 73 (63), 75 (55), 69 (45), 55 (44), 117 (40), 95 (38), 338 (38) 123 (100), 265 (40), 55 (37), 69 (35), 81 (35), 95 (35), 109 (34), 67 (30), 280 (19) 123 (100), 279 (43), 69 (37), 55 (35), 95 (35), 81 (34), 109 (34), 67 (27), 294 (22) 145 (100), 73 (57), 146 (53), 75 (44), 191 (30), 69 (28), 337 (27), 95 (22), 109 (22) 123 (100), 69 (48), 321 (40), 55 (39), 95 (36), 109 (36), 81 (34), 336 (28), 67 (26) 123 (100 ), 293 (35), 55 (33), 69 (33), 95 (33), 109 (33), 81 (32), 124 (23), 308 (18) 123 (100), 365 (85), 73 (52), 69 (40), 75 (40), 109 (37), 95 (35), 380 (34), 81 (32) 243 (100), 105 (54), 161 (45), 91 (43), 258 (37), 187 (33), 119 (32), 55 (30) 227 (100), 242 (64), 157 (36), 145 (24), 131 (22) 197 (100), 254 (85), 141 (77), 183 (45), 239 (45), 155 (39), 169 (37), 153 (28) 255 (100), 173 (67), 270 (43), 199 (29), 256 (20), 128 (18), 129 (17), 115 (14) 269 (100), 284 (48), 187 (40), 199 (38), 201 (32), 227 (26), 128 (21), 185 (20) 253 (100), 268 (78), 73 (49), 327 (33), 269 (32), 187 (30), 386 (25), 254 (22) 253 (100), 328 (35), 254 (21), 187 (17), 269 (11), 211 (10), 213 (10), 128 (9) 251 (100), 326 (42), 252 (21), 185 (17), 211 (12), 115 (10), 128 (10), 197 (9) 268 (100), 73 (98), 327 (82), 253 (62), 75 (33), 187 (28), 342 (27), 269 (26), 386 (7) 253 (100), 328 (52), 187 (49), 268 (33), 213 (28), 269 (25), 313 (20), 115 (18) 257 (100), 145 (72), 105 (64), 227 (62), 91 (61), 131 (57), 161 (50), 272 (37) 137 (100), 257 (62), 91 (61), 79 (42), 105 (41), 81 (40), 272 (36), 55 (35), 69 (35) 91 (100), 123 (82), 243 (70), 81 (65), 79 (55), 93 (54), 55 (50), 121 (47), 258 (36) 123 (100), 217 (87), 245 (57), 81 (55), 107 (38), 136 (37), 91 (35), 260 (32) 120 (100), 272 (98), 106 (72), 91 (58), 105 (49), 107 (48), 119 (45), 93 (38), 133 (33)
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Pinaceae (Hegnauer, 1962, 1986; Thomas, 1970; Otto and Wilde, 2001). Diterpenoid acids such as abietic and isopimaric acids were reported from the resin of extant Douglas Fir (Pseudotsuga menziesii; Erdtman et al., 1968; Otto and Wilde, 2001). A diagenetic pathway of the degradation of labdane derivatives is suggested from the presence of labdan-15oic acid (L10) and a series of labdane type diagenetic
products (Fig. 3). The degradation of natural product precursors, e.g. manoyl oxide and manool, leads to the generation of tetrahydromanool (L8) and labdan-15-oic acid (L10). The successive decarboxylation and demethylation of the labdane acid generates 14,15-bisnorlabdan-16-oic acid (L7), 14,15,16-trisnorlabdan-13-oic acid (L6), 14,15,16,17-tetranorlabdan-13-oic acid (L4), and 13,14,15,16,17-pentanorlabdan-12-oic acid (L3). In an
Fig. 2. GC–MS (TIC) traces of (a) the methylated polar fraction and (b) the neutral fraction of the Holocene Bella Coola seep oil (liquefied resin). For peak annotation see Table 1. u=unknown compound, *=contamination.
A. Otto, B.R.T. Simoneit / Organic Geochemistry 33 (2002) 1241–1251
alternative pathway, the degradation of 14,15,16-trisnorlabdan-13-oic acid (L6) can result in the formation of drim-7-en-11-oic acid (L2) and 11-nordriman-9-one (L1). The bisnorlabdanone (L5) is probably generated from the oxidation of manool. Similar series of acids and ketones were observed as the major intermediates in the aerobic bacterial degradation of dehydroabietic acid (Biellmann and Branlant, 1973a,b). Thus, the diagenesis of the labdanes (inferred precursors: manoyl oxide, labdadienol) in the Holocene conifer log from Bella Coola is probably caused by aerobic bacterial degradation of the liquified resin. The formation of 13b(H)-abietan-18-oic acid (a12), isopimarane (p3) and pimarane (p4) in the Bella Coola sample suggests a first step of aerobic decarboxylation of the parent acids, with concomitant anaerobic alteration to the saturated derivatives under strong reducing conditions.
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Thus, both oxidative and reductive reactions occurred simultaneously. The process of disproportionation suggested by Skrigan (1964) and Frenkel and Heller-Kallai (1977) may also be the cause for the generation of both saturated and aromatic derivatives, but not all abietane degradation products observed in the Bella Coola seep oil could have formed by disproportionation. 3.2. Ancient pine wood, North Vancouver The neutral fraction of the pine wood extract contains sesquiterpenoids, diterpenoids of the abietane, pimarane, isopimarane and kaurane classes and a group of steroids (Fig. 4). Aromatic abietanes are predominant in the nonderivatized (Fig. 4a) and in the BSTFA-derivatized neutral fraction (Fig. 4b). Calamenene (s1) and cadalene (s2) are the only sesquiterpenoids identified. The abietanes are
Fig. 3. Proposed diagenetic degradation scheme of the labdane type diterpenoids from the Holocene Bella Coola seep oil (liquefied resin). Compounds in boxes represent natural product precursors.
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composed of 18-norabieta-7,13-diene (a1), 16,17-bisnordehydroabietane (a2), fichtelite (a3), 18-norabieta-8,11,13triene (a5), dehydroabietane (a6), 1,2,3,4-tetrahydroretene (a7), retene (a8), 18-norabieta-8,11,13-trien-7-one (a9), abieta-6,8,11,13-tetraenoic acid (a11), dehydroabietic acid (a13), and 7-oxodehydroabietic acid (a14). 18-Norabieta-8,11,13-triene (a5, relative abundance 100%) is
the major compound in the neutral fraction as Swan (1968) reported. The pimaranes/isopimaranes are represented by isopimara-8,15-diene (p1), pimara-8(14),15diene (p2), and pimaric acid (p5). The kauranes are 18norkaurene (k1) and ent-18-norkaurane (k2). The steroids consist of the hydrocarbons 24-methylcholest-2-ene (st1), 24-methylcholest-4-ene (st2), stigmast-
Fig. 4. GC–MS (TIC) traces of (a) the total extract and (b) the silylated total extract (TMS derivatives) of the Holocene pine wood from north Vancouver. Peak annotation see Table 1. v=n-alkanol, *=contamination.
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2-ene (st3), stigmast-4-ene (st4), stigmasta-4,6,22-triene (st5), and stigmasta-3,5-diene (st6), and the polars 5b(H)-campestanol (st7), campesterol (st8), 5b(H)-stigmastanol (st9), 5a(H)-campestanol (st10), 24-methylcholesta-3,5-dien-7-one (st11), 5a-stigmastan-3-one (st12), b-sitosterol (st13), 5a(H)-stigmastanol (st14), and stigmasta-3,5-dien-7-one (st15). b-Sitosterol was also identified in the pine wood by Swan (1968). The composition of the terpenoids in the Holocene wood is in accordance with its botanical assignment to
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pine (Pinus sp.), because abietic acid associated with pimarane and isopimarane type diterpenoids are the major constituents of the resins of contemporary pine species (Hegnauer, 1962, 1986; Otto and Wilde, 2001). Swan (1968) suggested the diagenesis of abietane type diterpenoids in the pine wood under alkaline anaerobic conditions with major reductive processes, i.e. decarboxylation and hydrogenation of resin acids. Abietic acid and dehydroabietane are probably the biological precursors of the saturated and aromatic abietanes
Fig. 5. Proposed diagenetic degradation scheme of the abietane and pimarane type diterpenoids from the Holocene pine wood. Compounds in boxes represent natural product precursors.
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(Fig. 5). Dehydroabietane (a6), pimaradiene (p2) or isopimaradiene (p3) are the presumed precursors of 16,17-bisnordehydroabietane (a2). The decarboxylation of the natural product abietic acid followed by reduction leads to the saturated fichtelite (a3) as a minor product in the pine wood extract. The dominant alteration pathway is the oxidation of abietic acid to dehydroabietic acid (a13) and its decarboxylation which generates the major diagenetic product 18-norabieta8,11,13-triene (a5). Further aromatization of the B and A rings leads to 1,2,3,4-tetrahydroretene (a7) and finally to retene (a8). The generation of 1,2,3,4-tetrahydroretene from 18-norabieta-8,11,13-trien-7-one is yet unclear.
4. Conclusions The extracts of two Holocene conifer remains contain sesqui- and diterpenoids similar to the terpenoid patterns observed in their related contemporary species.
Appendix
The major compounds in the Bella Coola seep oil are series of successively degraded labdanes while abietanes predominate in the pine wood from north Vancouver. These samples represent the degradation of resin compounds by an unusual fossilization process under semiaerobic conditions in soil or land slides. The presence of both saturated and aromatic products in the samples suggests the occurrence of concomitant oxidative and reductive processes during the degradation of the diterpenoids.
Acknowledgements We thank Dr. E.P. Swan, Canadian Forestry Service, for the seep oil (liquefied resin) and extract fractions, and the Max Kade-Foundation, New York, for financial support. This paper benefitted from the reviews by Dr. G.A. Logan and Dr. H.P. Nytoft. Associate Editor—G. Abbott
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References Biellmann, J.F., Branlant, G., 1973a. De´gradation bacte´rienne de l’acide de´hydroabie´tique par Flavobacterium resinovorum. Tetrahedron 29, 1227–1236. Biellmann, J.F., Branlant, G., 1973b. De´gradation becte´rienne de l’acide de´hydroabie´tique par un Pseudomonas et un Alcaligenes. Tetrahedron 29, 1237–1241. Erdtman, H., Kimland, B., Norin, T., Daniels, P. J. L., 1968. The constituents of the ‘‘pocket resin’’ from Douglas fir Pseudotsuga menziesii (MIRB.) FRANCO. Acta Chemica Scandinavica 22, 938–942. Frenkel, M., Heller-Kallai, L., 1977. Aromatization of limonene—a geochemical model. Organic Geochemistry 1, 3–5. Hegnauer, R., 1962. Chemotaxonomie der Pflanzen. Vol. 1. Thallophyten, Bryophyten, Pteridophyten und Gymnospermen. Basel, Birkha¨user. Hegnauer, R., 1986. Chemotaxonomie der Pflanzen. Vol. 7. Nachtra¨ge zu Band 1 und Band 2. Basel, Birkha¨user. Kitadani, M., Yoshikoshi, A., Kitahara, Y., de Paiva Campello, J., McChesney, J.D., Watts, D.J., Wenkert, E., 1970. Natural ar-abietatriene. Chemical and Pharmaceutical Bulletin 18, 402–405. Li, M., Johns, R.B., Mei, B., 1990. A study in early diagenesis: biomarker composition of a suite of immature coals and coaly shales. Organic Geochemistry 16, 1067–1075. Noble, R.A., Alexander, R., Kagi, R.I., Knox, J., 1986. Identification of some diterpenoid hydrocarbons in petroleum. Organic Geochemistry 10, 825–829.
1251
Otto, A., Wilde, V., 2001. Sesqui-, di-, and triterpenoids as chemosystematic markers in extant conifers- A review. Botanical Review 67, 141–238. Philp, R.P., 1985. Fossil Fuel Biomarkers. Applications and Spectra. Methods in Geochemistry and Geophysics, 23. Elsevier, Amsterdam. Simoneit, B.R.T., 1977. Diterpenoid compounds and other lipids in deep-sea sediments and their geochemical significance. Geochimica et Cosmochimica Acta 41, 463–476. Simoneit, B.R.T., Mazurek, M.A., 1982. Organic matter of the troposphere—II. Natural background of biogenic lipid matter in aerosols over the rural western United States. Atmospheric Environment 16, 2139–2159. Simoneit, B.R.T., Grimalt, J.O., Wang, T.G., Cox, R.E., Hatcher, P.G., Nissenbaum, A., 1986. Cyclic terpenoids of contemporary resinous plant detritus and of fossil woods, ambers and coals. Organic Geochemistry 10, 877–889. Skrigan, A.I., 1964. Preparation and utilization of fichtelite and retene. Tr. Vses. Nauchno-Tekhn. Soveshch. Gorki 1963, 108–115. Chemical Abstracts 62, 10664a. Swan, E.P., 1965. Identity of a hydrocarbon found in a forest soil. Forest Products Journal 15, 272. Swan, E.P., 1968. Extractives of an Ancient Pine. Bi-Monthly Research Notes. Canadian Forestry Service 24, 8. Thomas, B.R., 1970. Modern and fossil plant resins. In: Harborne, J.B. (Ed.), Phytochemical Phylogeny. Academic Press, London, pp. 59–79. Zinkel, D.F., Zank, L.C., Wesolowski, M.F., 1971. Diterpene resin acids, mass spectra of diterpene resin acid methyl esters. Journal of the American Oil Chemists’ Society 48, 455–461.