Chapter 6
New Evidence Concerning the Structure, Composition, and Maturation of Class I (Polylabdanoid) Resinites 1
Ken B. Anderson
Amoco Oil Company, P.O. Box 3011, Naperville, IL 60566-7011
Pyrolysis-Gas Chromatographic-Mass Spectrometric (Py-GC-MS) analysis of a range of Class I resinites indicates that the macromolecular structures of these resinites are typically derivedfromcopolymers of labdanoid carboxylic acids, alcohols and hydrocarbons. ClassIaandIbresinites are derived from copolymers of regular [1S, 4aR, 5S, 8aR] labdanoid diterpenes, especially communic acid, communol and/or biformenes. Class Ic resinites are based on structures derived from enantio [1S, 4aS, 5R, 8aS] labdanoids, especially ozic acid, ozol and/or enantio biformenes. The monomeric components of Class I resinites are readily determined in PyGC-MS analyses by recognition of characteristic bicyclic products derived from the A/B ring system of the original labdanoid monomers. In addition to bicyclic products derived directlyfromthe labdanoid A/B ring system, products derived by modification of this ring system are also observed in Py-GC-MS analyses of some Class I resinites. These include: bicyclic methyl ethers produced by pyrolytic hydrolysis/O-methylation of succinylated (esterified) communol monomers within the macromolecular structure of Class Ia resinites; and, in samples of moderate maturity, C and C bicyclic products derived from Α-ring defunctionalized labdanoids. The existence of these Α-ring defunctionalized products indicates the existence of a heretofore unrecognized maturation pathway in polylabdanoid resinites. 13
14
N O T E : This chapter is Part V of a series entitled "The Nature and Fate of Natural Resins in the Geosphere." Part IV of this series is by Anderson, Κ. B. Org. Geochem, 1994, 21(2), 209-212. 1Current address: Chemistry Division, Argonne National Laboratory, 9 7 0 0 South Cass Avenue, Argonne, IL 60439
0097-6156/95/0617-0105$13.25/0 © 1995 American Chemical Society
106
AMBER, RESINITE, AND FOSSIL RESINS
Introduction and Background. Amber has long been prized by entomologists and paleontologists for the extraordinary degree of preservation of fossil materials entrapped within it, especially insects and plant matter. Recent studies have shown that this high degree of preservation includes preservation of individual soft tissues and organs (1) and even preservation of molecular characteristics and DNA (2-4). Less well recognized however, is the fact that fossil resins preserve details of their own original molecular structure to a greater degree than perhaps any other form of sedimentary organic matter. The preservation of molecular structure in these materials, and the changes to the original structures which do occur in response to (primarily) thermal stress, have the potential to be of significant value in organic geochemical studies. For this reason, the structural characteristics of resinites are currently receiving considerable attention (5-14). In earlier reports in this series it has been shown that most resinites can be classified into one of five classes based on the nature of their (macro)molecular structure (6-8). It has also been demonstrated that resinites derived from resins based on polymers of labdanoid diterpenes (Class I resinites), which are by far the most common and abundant class of resinite, can be further divided into three subclasses (6,8).
The macromolecular structures of all Class I resinites are based on polymers of labdanoid diterpenes. In many cases they also contain occluded mono-, sesqui-, di-, and/or triterpenoids and in some samples, especially immature samples, these may be more abundant than the polymeric component of the resinite. The composition of these occluded materials varies widely in both recent and fossil resins. The nature of the polylabdanoid macromolecular structure however, is highly conservative across a wide range of modern and especially fossil resins. Hence although low molecular weight non polymeric materials may be highly useful for provenance and paleobotanical studies, their usefulness for the purposes of classification and geochemical studies is limited. Sub-classification of Class I resinites is based on details of the structural characteristics of the labdanoid diterpenes from which the polymeric component of the resinite is derived. The macromolecular component of Class la and lb resinites is based on polymers of labdanoid diterpenes having a regular configuration (ie, [IS, 4aR, 5S, 8aR]) especially communie acid (III) and communol (II). These two subclasses are then further differentiated by the presence of succinic acid, which is incorporated in significant amounts into the macromolecular structure of Class la resinites (presumably as a cross-linking agent between labdanoid alcohols, especially communol). Conversely, Class Ic resinites are based on polymers of labdanoid diterpenes with the enantio [IS, 4aS, 5R, 8aS] configuration such as ozic acid (VI) (6,8).
In Py-GC-MS analyses, differentiation of sub-classes of Class I resinites is achieved by recognition and identification of characteristic bicyclic products derived from the A/B ring system of the original labdanoid monomers. A large percentage of Class I resinites contain significant amounts of polymerized communie acid (III), and in some cases, the macromolecular component of the resinites is derived virtually entirely from this monomer. Pyrolysis of such materials results in generation of four characteristic bicyclic acids (in addition to release of occluded material) which are
6. ANDERSON
New Evidence Concerning Class I (Polylabdanoid) Resinites 107
Ο
c/)
DC ω OK ε Il II II g
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-ο
u
u u u u
i l i l c*i il il oci
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108
AMBER, RESINITE, AND FOSSIL RESINS
Victorian Brown Coal (Yallourn seam) Resinite Unextracted
0T
*V0 CH 2
3
* C0 CH 2
3
' co cn, 2
42 20
44
46
48 JUUJL
30
40
50
60
70
30
40 50 Time (min)
60
70
Extracted
20
Figure 1. Total ion chromatograms obtained by Py-GC-MS analysis of whole (unextracted) and extracted Victorian Brown Coal resinite (Yallourn seam). Expansion of chromatogram showing elution of characteristic bicyclic acids is inset. Products observed in the chromatogram of the extracted sample are derived from the macromolecular component of the resinite.
6. ANDERSON
New Evidence Concerning Class I (Polylabdanoid) Resinites 109
readily identifiedfromthe mass spectral characteristics and chromatographic behavior of their corresponding methyl esters Vila - Xa (6). An example is illustrated in Figure 1. That these characteristic bicyclic products are derived from the macromolecular component of the resinites is readily established by pyrolysis of thoroughly extracted samples, as also illustrated in Figure 1. Pyrolysis of ambers derived from resins based on ozic acid (VI) results in generation of four analogous epimeric bicyclic acids, which are readily differentiated from those derived from communie acid on the basis of the chromatographic behavior of their corresponding methyl esters XIa - XlVa (6). Until recently, the presence of these characteristic bicyclic acids (methyl esters) in the pyrolysate of a resinite was a primary basis for classification of Class I resinites (6). However, in a recent report in this series (8), Py-GC-MS data for an Upper Cretaceous resinite from the Taimyr Peninsula (Siberia) were presented which indicate the presence of series of bicyclic hydrocarbons (VIIc - Xc) and alcohols (Vllb - Xb) in the pyrolysate of this sample. These data, and datafromtwo additional related Middle and Upper Cretaceous samples from the same region are illustrated in Figure 2. The pyrolysates of all of these samples are dominated by bicyclic hydrocarbons (VIIc - Xc) and alcohols (Vllb - Xb) which are clearly analogous to the characteristic bicyclic acids observed in the pyrolysates of samples such as that illustrated in Figure 1. The presence of these characteristic bicyclic alcohols and hydrocarbons are interpreted as indicating that the macromolecular structures of these ambers are derived from a copolymeric structure in which the primary monomers are biformene (I) and communol (II). The absence of compounds Vila - Xa indicates that communie acids are absent. These observations suggest that these samples represent a previously unrecognized form of Class lb resinite. (A third series of compounds, viz, A-H, will be discussed letter in this text). The observation and assignment of compounds Vllb - Xb and VIIc - Xc immediately raises the question of the significance of these products in the pyrolysates of other Class I resinites, and hence the significance of labdanoids other than communie and ozic acids in the macromolecular structures of other Class I resinites. A review of unpublished Py-GC-MS data generated by the author over the past several years suggested that these characteristic alcohols and hydrocarbons were present in a significant number of Class I resinites. Therefore, in order to determine the significance of diterpenoids other than communie and ozic acids in the macromolecular structures of other Class I resinites, Py-GC-MS analyses of a diverse series of samples have been undertaken. All samples were reanalyzed under identical conditions, and as much as possible, series of samples were analyzed "back-to-back" to ensure that both chromatographic and mass spectral data were directly comparable. The primary purpose of this report is to discuss the observation of characteristic products derived from communol and biformene in a wide range of Class la and lb resinites, and to report identification of analogous enantio bicyclic alcohols and hydrocarbons in the pyrolysates of Class Ic resinites. Furthermore, insights gained during identification of these characteristic alcohols and hydrocarbons have subsequently enabled identification of a number of other significant series of compounds. The identification of these compounds, which are described in detail below, has significant implications for the structure, maturation, and analysis of Class I resinites.
110
AMBER, RESINITE, AND FOSSIL RESINS
C
Yantardakh Hill #1
ψ
r
C
)CI
l OH ^
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2
2
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k
2
2
'
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1 20
30
50
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.
20
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L
7
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M:O CH, 2
CH OF 2
i
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d
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60
y
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2
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I OH
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JuJ. m40 50 Time (min.)
^t0 CH,jy 2
WJx—JJ^ 60
^'COjCH, 70
Figure 2. Total ion chromatograms obtained by Py-GC-MS of communol/ biformene copolymeric Class Ib resinites obtained from Middle and Upper Cretaceous sediments in the Taimyr Peninsula, Siberia. See also reference 8.
6. ANDERSON
New Evidence Concerning Class I (Polylabdanoid) Resinites 111
Experimental. Py-GC-MS analyses were conducted using an HP 5890(11) GC directly coupled to an HP 5970 or 5971 mass selective detector. Pyrolyses were carried out using a CDS "Pyroprobe" pyrolysis system. T = 480°C in all cases reported herein. Methylation of acidic components of the pyrolysates was achieved in-situ by co-pyrolysis with tetramethyl ammonium hydroxide (TMAH) as described elsewhere (5). Chromatographic conditions used to obtain the data reported herein are as follows: Column = 60m DB-1701, Temperature program: T j = 40°C (1.5 min), Ramp rate = 4°C/min, T = 280°C. Additional data, (not illustrated) have also been generated using 60m DB-5 and RTx-200 capillary GC-columns. Off-line pyrolysis was carried out as follows. Pre-extracted Victorian Brown Coal resinite (0.75g) was loaded into an N purged pyrex pyrolysis tube. After allowing sufficient time for all air to be purged, the pyrolysis tube was then placed into a furnace preset at 450°C for 2 minutes. (Note: pyrolysis of this sample was found to be significantly exothermic. Direct measurement of the temperature at the sample indicated that after this period of time an actual T of 600°C had been reached). The tube was removed and allowed to cool to ambient temperature. Volatile products evolved during pyrolysis were trapped using a coil type water cooled glass condenser, loosely packed with glass wool. (Note: trapping of volatile products was imperfect, therefore, no attempt was made to calculate pyrolysis yields.) The pyrolysate was then collected in THF (20mL) and reacted with 1.0M LiAlH (10 mL) for 2.5 hours. The product of this reaction was then filtered and concentrated by rotary evaporation. GC MS analysis confirmed that the desired alcohol products had been generated as the primary products of this reaction. A small aliquot of this product (re-dissolved in CH C1 ) was then methylated using (CH ) SiCHN /HBF (75). GC-MS analysis confirmed that the desired methyl ether products had been prepared. The identity of these products with those observed in the pyrolysates of Class la (and lb) resinites was confirmed by co-pyrolysis. Succinylation of Baikura-Neru Resinite: Resinite (132.7 mg) was placed in a small flask together with CH C1 (10 mL). After stirring for 30 min to allow time for the resinite to swell, succinic anhydride (126 mg) was added and the reaction was then stirred overnight at room temperature. CH OH (10 mL) was then added to the reaction. This resulted in complete dissolution of the product. Therefore, the product was diluted with CH OH (70 mL) and H 0 (15mL) to precipitate the product and concentrated by rotary evaporation. The precipitate was then filtered through a 0.45 μηι filter, washed with a few mL of H 0, and dried under vacuum at room temperature. Yield =119 mg. Reductive decarboxylation products were preparedfromthe off- line pyrolysate of extracted Victorian Brown Coal resinite using the method initially described by Barton and co-workers (16). Briefly, the offline pyrolysate of extracted VBC resinite (~0.5mmol) was reacted with (1) oxalyl chloride (2M in Benzene) and DMF (trace) to produce the corresponding acid chlorides. The product of this reaction was then allowed to react with a dry suspension of 2-mercaptopyridine N-oxide sodium salt (250mg) and DMAP (26mg) in benzene (60mL) (N ). After stirring for 30 minutes, freshly distilled t-butyl mercaptan (500 μL) was added and the reaction was then refluxed for 2.5 hours. The product was then extracted with H 0 (2X40mL), dried over MgS0 and chromatographed over silica gel with n-pentane in the usual way. Py
(
nitian
(finaI)
2
max
4
2
2
3
2
3
2
4
2
3
3
2
2
2
2
4
112
AMBER, RESINITE, A N D FOSSIL RESINS
Goodwins /
,,
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.
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40
Q
Reefton
& FK
•
Φ A
E
θΦ
mû
30
35
40
45
50
55
T i m e (min.)
Figure 3. Partial chromatograms obtained by Py-GC-MS analysis of Class lb resinites of increasing rank collected from a series of New Zealand coal fields. Characteristic bicyclic acids, alcohols, and hydrocarbons reflecting the distribution of labdanoid monomers in the original resin are indicated.
6. ANDERSON
New Evidence Concerning Class I (Polylabdanoid) Resinites 113
Results and Discussion. Partial Py-GC-MS total ion chromatograms for four Class lb resinites collected from coalfieldsin New Zealand are illustrated in Figure 3. These data illustrate the distribution of polymer-derived characteristic bicyclic products in the pyrolysates of these samples. (Some data generated from earlier analyses of these samples has been reported previously (5,6)). These samples are all likely to be derived from a common botanical source (Agathis) and hence give an indication of the variability of the composition of related resinites. As illustrated in this Figure, in addition to characteristic bicyclic acids derived from structures related to communie acid, varying amounts of the bicyclic alcohols and hydrocarbons observed in the pyrolysates of the Siberian resinites (see Figure 2) are also observed in the pyrolysates of all of these samples, indicating that the polymeric components of these resinites incorporate communol and biformene in addition to communie acid. (The effects of increasing maturity on the distribution of products observed in the pyrolysates of these four resinites will be discussed latter in this text. (See also reference 6). Communol and biformene derived characteristic bicyclic products are also observed in the pyrolysates of Class la resinites, as illustrated in Figure 4. A number of previous workers have suggested that the macromolecular structure of succinite probably incorporates communol units to allow for cross linking of the structure by succinic acid (/ 7,18). The observation of compounds VIIb-Xb in the data illustrated in Figure 4 provides direct evidence confirming that this monomer is abundant in the macromolecular structure of Class la resinites. The presence of abundant biformene monomers, (indicated by the observation of compounds VIIc -Xc) in the structures of these resinites has not been previously reported. In addition to compounds Vll(a-c) - X(a-c), a fourth series of analogous compounds was also observed in the pyrolysates of these samples. Based on interpretation their mass spectra, these compounds were tentatively assigned as methyl ethers analogous to the bicyclic alcohols derived from communol. (See structures Vlld - Xd.) Mass spectra for these compounds are given in Table I below. In order to confirm the identification of compounds Vlld - Xd and to substantiate the assignment and stereochemistry of compounds VIIb-Xb, mixtures of these compounds were prepared synthetically by sequential reduction (LiAlH ) and methylation ((CH )3SiCHN /HBF4) of the off-line pyrolysate of an extracted Victorian Brown Coal resinite derived virtually entirely from communie acid. (See Figure 1) The chromatographic behavior and mass spectral characteristics of the products from these reactions were identical to those of the products generated in-situ by Py-GC-MS analysis. (Note: Analogous epimeric bicyclic methyl ether products have now also been observed in the pyrolysates of fossil resins derivedfromPseudolarix. (See Part VI of this series, Anderson and Lepage, in this volume.) A search of the literature indicates that although a small number of labdanoid methyl ethers are known (19-21) these compounds are rare and generally occur in low abundance. Furthermore, those which have been characterized lack the side-chain characteristics necessary for polymerization into polylabdanoid structures. This suggested that compounds Vlld - Xd might be secondary products resultingfromthe in-situ methylation procedure used for these analyses. To verify this premise, Py-GCMS analyses were carried out using D TMAH. The results of these experiments 4
3
2
13
114
AMBER, RESINITE, AND FOSSIL RESINS
Anhalt (Saxony) Resinite υπ
G
ι
' ro cH 2
Β
3
a"
φ τ
φ f 'ΐΗ ΟΗ OCHjO
I φ τ
2
1) is inconsistent with the values observed in other immature resinites. And secondly; the distributions of regular and enantio
