- Open Access
The nature and fate of natural resins in the geosphere. Part X.† Structural characteristics of the macromolecular constituents of modern Dammar resin and Class II ambers
© The Royal Society of Chemistry and the Division of Geochemistry of the American Chemical Society 2000
- Received: 11 January 2000
- Accepted: 25 January 2000
- Published: 2 March 2000
As part of a larger study of the structure and behavior of polyterpenoids in sedimentary systems, the structural characteristics of the macromolecular constituents of Dammar resin and a related Class II amber have been reinvestigated. The conclusions drawn from these analyses are inconsistent with the current widely held "polycadinene" model for the macromolecular structure of these materials. Double bond characteristics observed by one and two dimensional NMR spectroscopy do not match those in the proposed "polycadinene" structure. Based on these observations it is suggested that the proposed "polycadinene" structure for these materials is inadequate and requires revision. Elemental and NMR data also suggest a significant contribution from functionalized monomers.
- Olefinic Proton
- Olefinic Carbon
- Source Rock Extract
- Macromolecular Constituent
- Cosy Data
Ambers of this class have been shown to be composed of both a low MW fraction and a high MW macromolecular phase. [11–15] Extensive analyses of the low MW fraction have identified a large number of functionalized sesqui- and triterpenoids, primarily of the cadinane, oleanane and dammarane skeletal types.[12, 16–18] It is believed that the bicadinanes observed in source rock extracts and crude oils are derived from the HMW fraction of the amber. The current prevailing view of the nature of the HMW fraction, proposed by van Aarssen and coworkers, [3, 12–15] is that this is composed predominantly of a cadinene polymer, loosely referred to as "polycadinene" (Fig. 1, III). Although it is difficult to rationalize the formation of III under natural conditions from biologically reasonable precursors without invoking enzymatic processes  the structure proposed by these authors preserves the "methylene bridged" character observed in the bicadinanes, and readily rationalizes the observed formation of bicadinanes on thermolysis of the macromolecular phase.
It has been shown that the distribution of bicadinane isomers in source rock extracts varies as a direct function of maturity,  which is consistent with the view that these products, and related unassigned "dimers" and higher homologues, are derived by thermal disruption of the HMW component of these ambers. This view also implies that the structural characteristics of these products are directly related to the structure of the HMW material.[2, 3] However, in order to establishthis, it is necessary to have detailed knowledge of the structural characteristics of this HMW material.
In the present study, the structural characteristics of the macromolecular constituents of modern and fossil Dammar have been reinvestigated using high resolution one and two dimensional (1 and 2D) NMR techniques. Effort has focused primarily on investigation of the characteristics of olefinic structures, with the intent of refining details of the overall structural characteristics of these polymers.
Modern "undewaxed" commercial Dammar was purchased from Sigma Chemical Co. Miocene Dammar from the Merit Pila Coal mine was obtained from a commercial amber dealer.
Dammar resin (10 g) was dissolved in CH2C12 (50 mL) with sonication. This solution was then filtered to remove extraneous materials, and the filtrate concentrated to a small volume by rotary evaporation. To this concentrate, CH3OH (100 mL) was then added in small portions to give a white precipitate, which was then recovered by filtration. The precipitate was then resuspended in CH3OH and sonicated to ensure complete removal of CH3OH soluble materials. The product was recovered by filtration and dried. Yield = 9.5 g.
Attempts to recover fossil Dammar polymer by the procedure used for the modern Dammar were unsuccessful due to the presence of substantial amounts of CH2C12 insoluble materials which made filtration impossible. Therefore, amber (9.9 g) was placed in a Gregar extractor and extracted with CH2Cl2 (150 mL) for 24 h. The extract was then worked up as described above. Yield = 6.4 g.
Products from both the modern and fossil Dammar were subjected to analytical pyrolysis at 300°C, in the presence of tetramethyl ammonium hydroxide (TMAH),  to check for the presence of volatile impurities. No products of significant abundance were observed.
All NMR experiments were performed on a Bruker model DMX 500 NMR spectrometer (11.7 T). With the use of the nitrogen pre-cooler, heater coil, and the variable temperature controller, the temperature was stable at 294.3 ± 0.1 K. The 13C NMR spectra and the DEPT (distortionless enhancement by polarization transfer)  spectra were recorded with a two channel 10 mm broadband, direct detection, variable temperature probe with proton decoupling and 2H lock at 76.773 MHz. Inverse gated proton decoupling was accomplished via the WALTZ 16 sequence. [21–23]13C spectra were recorded with 22,000 acquisitions, 10 s recycle delay, 9 μs pulse width (70°) and 33 kHz sweep width. DEPT spectra were recorded with 12,000 acquisitions, 10 s recycle delay, and 42 kHz sweep width.
For the 1H, COSY, HMQC, and HMBC experiments, (see below) a three channel 5 mm inverse detection, three axis gradient, variable temperature probe with 2H lock at 76.773 MHz was used. 1H spectra were recorded with 1000 acquisitions, 4 s recycle delay, 4 μs pulse width (70°) and 6 kHz sweep width. COSY (correlated spectroscopy) [24, 25] spectra were recorded with gradient phase cycling, 200 acquisitions and 1 K data over 3.2 kHz in the F2 dimension, and 4 s recycle delay. The F1 dimension covered 3.2 kHz sweep width with 256 increments. Fourier transformed data were zero filled to 1 K by 1 K points. The 1H 90° pulse was 6.75 μs.
HMQC (heteronuclear multiple quantum correlation) [26, 27] and HMBC (heteronuclear multiple bond correlation) [27, 28] spectra were recorded with gradient phase cycling, 260 acquisitions and 2 K data over 3.8 kHz in the F2 (1H) dimension, and 1.5 s recycle delay. The F1 (13C) dimension covered 33 kHz sweep width with 128 increments. Fourier transformed data were zero filled to 2 K by 1 K points. The 13C and 1H 90° pulses were 12.25 and 6.75 μs respectively. Proton decoupling was accomplished via the GARP composite sequence  and a coupling constant of 145 Hz was used in the HMQC experiment. A delay corresponding to a 20 Hz coupling was used to select long range couplings in the HMBC experiment.
The data illustrated in Fig. 2 and 3 are similar to data previously reported for comparable samples.  Integration data for the modern Dammar polymer show Holefin:Htotal other = 1:27.1, slightly less than the value (ca. 1:23) reported by van Aarssen and coworkers. The Colefin:Ctotalother ratio (1:9.5) is also slightly less than the 1:7.5 required by the prevailing polycadinene model (III).
Elemental analyses a
O+S (wt.%) b
Nominal formula (based on C15)
Modern Dammar polymer
C15H23(O + S)0.75
Fossil Dammar polymer
C15H22(O + S)0.72
Polycadinene (III c )
1D NMR spectra provide excellent information regarding the distribution of carbon and proton types within a sample, but they do not directly provide evidence of either C-C or C-H connectivity. In simple molecules, connectivity can usually be inferred from chemical shift arguments and in many cases structures can be unambiguously assigned from these data alone. In the case of more complex systems, such as those with which the present study is concerned, chemical shift arguments are less definitive. In such cases it is necessary to use 2D analyses in order to derive unambiguous structural assignments.
2D NMR analyses depend on transfer of magnetization from one nuclei to adjacent or more remote nuclei. Those used in this study all rely on transfer of magnetization through covalent bonds and hence provide direct evidence of connectivity. There are a number of different types of 2D NMR experiments which provide complimentary evidence of C-H and H-C n -H connectivity. HMQC analyses are used to observe correlations between 13C and 1H through a single covalent bond, usually abbreviated as 1JC-H. HMBC analyses also provide information concerning C-H connectivity, but in this case NMR parameters are set such that two and three bond (2JC-H and 3JC-H) correlations are detected. That is, correlations are observed between 1H nuclei and 13C adjacent to (and twice removed from), the carbon to which the proton is directly covalently bound. COSY data detect 2JH-H and 3JH-H correlations and hence show correlations between geminal protons (protons attached to the same carbon) and protons attached to adjacent carbons (note: in order to be observable, geminal protons must be magnetically inequivalent). When taken together, these data sets provide compelling and complementary evidence for C-C and C-H 1, 2 and 3 bond connectivity.
Although the process can be somewhat complex, by examining correlations and cross-correlations in this manner, contiguous chain information may be built up, and structural elements can often be identified with confidence, even in complex systems. Unfortunately, in the polymer systems characterized in this study it is not possible to completely establish all connectivities due to coincidental overlap of resonances arising from chemically similar but discontinuous structures. Nevertheless it is possible to unambiguously establish the nature of a number of key structural elements, most notably olefinic structures and several of the oxygenated structures. The remainder of this report will focus on analysis of olefinic structural elements.
1H and 13C spectra (Fig. 2 and 3 respectively) clearly indicate the presence of a number of olefinic structures in both the modern and fossil polymers (1H = 6.0–3.1 ppm, 13C = 155–105 ppm). The 13C spectra for the modern Dammar polymer shows 2 predominant olefin resonances at 127.2 and 135.3 ppm (with some indication of multiple overlapping resonances), and with secondary olefinic resonances at 124.7 and 131.6 ppm. Additional minor olefinic resonances are also observed. Chemical shift considerations and DEPT data (Fig. 4) indicate that the resonances at 135.3 and 131.6 ppm are due to nonprotonated olefinic carbons and the 127.2 and 124.7 ppm resonances arise from protonated olefinic carbons.
The absence of correlations between the 1H and 13C of the protonated olefinic carbon of this structure and α 1H indicates that there are no protons on the carbon α to the protonated olefinic carbon. That is, the aliphatic carbon alpha to the protonated olefinic carbon is quaternary. This assignment is further supported by the observation of 2JC-H and 3JC-H couplings between the olefinic proton (1H = 5.47 and 5.50 ppm) and four aliphatic 13C resonances (Fig. 9c, 33.2, 38.5, 46.9 and 49.7 ppm). The existence of four such couplings confirms the quaternary nature of the carbon α to 13C = 127.2 ppm. Hence the structural nature of the most abundant (primary) olefin must be as indicated in Fig. 8.
As noted above, analyses such as those used in the present study depend on the transfer of magnetization from one nuclei to adjacent or more remote nuclei. The magnitude of any given coupling is determined by the coupling constants between the nuclei, and by loss of magnetization due to relaxation. Therefore, to ensure that the absence of detectable couplings to the 13C 127.2 ppm resonance does not result from rapid relaxation of the 1H magnetization, 1H T1 measurements were taken for the olefinic protons of both the primary and secondary olefinic structures. The results of these measurements are as follows: 1Hprimary Olefinic = 670 ms, 1Hsecondary olefinic = 1100 ms. Both of these measurements are well within the range detectable in these experiments. (The HMBC sequence used for these experiments requires only 107 ms before acquisition.) To even further ensure that correlations would be observable if they were present, 1H T1 experiments were also performed on a soluble polymer isolated from an immature Class I (polylabdanoid) resinite. 1H T1 values measured for the olefinic protons in this (polymeric) sample were found to be of the same order (800–1100 ms) as those of the olefinic 1H of the present samples. COSY and HMBC correlations were readily observable in all cases, including 2JC-H to the protonated 13C of the side-chain olefin from the adjacent methylene protons. It therefore appears that the absence of observable 2+3JC-H correlations to the protonated 13C of the primary olefin in these samples is not an experimental artifact.
The structure proposed by van Aarssen and coworkers [3, 12–15] (Fig. 1, III) includes only one quaternary aliphatic carbon (carbon 1). In order to place the double bond illustrated in Fig. 8 into this skeleton it would be necessary to place it between carbon 6 and carbon 13'. This bond is integral to the backbone of the macromolecular structure. If the double bond were in this position then cleavage of the olefin by ozonolysis, or treatment with other oxidants such as RuO4 would lead to complete depolymerization of the structure. This is not observed.
HMQC data (Fig. 10c) indicate that these protons (1.63 and 1.69 ppm) are directly coupled to 13C resonating at 17.5 and 25.7 ppm, respectively. It can be seen from the HMBC data for this sample (Fig. 10d) that these protons cross-couple. That is, (in addition to the olefinic carbons resonating at 124.5 and 131.6 ppm), the 1H resonance at 1.63 ppm correlates 3JC-H only with the 13C resonance at 25.7 ppm, and likewise, the protons resonating at 1.69 ppm correlate only with the 13C resonance at 17.5 ppm. This cross-correlation confirms that the carbons giving rise to the resonances at 13C = 17.5 25.7 ppm are connected through a single quaternary carbon (viz.13C = 131.6 ppm).
The secondary olefin observed in the modern sample, however, is much less abundant in the fossil polymer. It is not possible from the present data to determine if this difference is sample or maturity related. The 1H spectrum of this sample shows an apparent strong resonance centered at ~5.33 ppm which is not significant in the corresponding data for the modern sample. HMQC data for the fossil polymer suggest that this proton is coupled to the 13C resonating at 124.3 ppm. However, one dimensional 13C data for this sample indicate no strong single resonance at this chemical shift. Rather, these data indicate at least 4 partially resolved 13C resonances between 125 and 124 ppm. Furthermore, higher resolution HMQC measurements of this correlation show that the 1H resonance observed in the 1D 1H data for this sample is in fact composed of at least 4 unresolved resonances.
As a result of the coincidence of these resonances it is not possible to develop detailed connectivity arguments for these signals. Clearly these signals derive from a family of closely related structural elements (at least in terms of 1H and 13C chemical shifts). However, it is possible to derive limited information about the structural characteristics of the olefins contributing to these overlapping resonances from COSY data. In this case, 3JH-H correlations with the multiple resonance at ~5.33 ppm are observed at 5.47 ppm (suggesting the presence of a 1,2-diprotonated olefin), and 2.11 and 2.15 ppm (doublet) suggesting that at least one of these olefinic protons is located α to a protonated (probably methine) carbon. Further analysis of these olefins is limited by the overlap of their respective 13C and 1H resonances, but their diversity itself suggests that a variety of possible pathways are being followed during maturation of these polymers.
One and two dimensional spectra from both the modern and fossil polymers indicate the presence of a number of other minor olefins. In addition to evidence for 1,2-disubstituted (diprotonated) olefins, (discussed above) resonances attributable to minor amounts of terminal and/or exo-olefins are also observed. However, due to the weakness of the resonances attributable to these structures, no further analysis of these structures has been attempted at this time.
The nature of the olefinic structures present in the high MW fraction of both modern and fossil Dammar resins is inconsistent with the current widely held "polycadinene" model for these materials. Nor can the data be accommodated within any simple double bond isomer of the existing "polycadinene" model. Additional investigations are necessary to define the precise structural characteristics of these polymers, but it is clear that the existing model is inadequate and requires revision. Data generated in these analyses also suggest that although individually minor, in sum, functionalized structures (especially O-containing ones) may contribute significantly to the overall characteristics and reactivity of these polymers and should not be wholly neglected when describing these materials.
† For Part IX see ref. 1
The authors wish to express their appreciation to Mr William Lambert for technical assistance in preparation of the polymers, and to Dr David J. Clifford for helpful comments during preparation of this manuscript. Mr Nabil Mailloux of Advanced Chemistry Development Inc. http://www.acdlabs.com is thanked for his assistance in the preparation of JCAMP-DX format NMR data files. The support of the U.S. Dept. of Energy, Office of Basic Energy Sciences, under contract No W-31-109-ENG-38 is also gratefully acknowledged.
- Clifford DJ, Hatcher PG, Muntean JV, Botto RE, Anderson KB: The Nature and Fate of Natural Resins In the Geosphere. IX. Structure and maturation similarities of soluble and insoluble polyabdanoids isolated from Tertiary Class I resinites. Org Geochem. 1999, 30 (7): 635-10.1016/S0146-6380(99)00018-2.View ArticleGoogle Scholar
- van Aarssen BGK, Zhang Q, de Leeuw JW: An unusual distribution of bicadinanes, tricadinanes, and oligocadinanes in sediments from the Yacheng gasfield, China. Org Geochem. 1992, 18: 805-10.1016/0146-6380(92)90049-4.View ArticleGoogle Scholar
- van Aarssen BGK, Hessels JKC, de Leeuw JW: The occurrence of polycyclic sesqui-, tri-, and oligoterpenoids derived from a resinous polymeric cadinene in crude oils from southeast Asia. Geochim Cosmochim Acta. 1992, 56 (3): 1231-10.1016/0016-7037(92)90059-R.View ArticleGoogle Scholar
- Curiale JA, Kyi P, Collina ID, Din A, Nyein K, Nyunt M, Stuart CJ: The Central Myanmar (Burma) oil family-composition and implications for source. Org Geochem. 1994, 22 (2): 237-10.1016/0146-6380(94)90172-4.View ArticleGoogle Scholar
- Murray AP, Summons RE, Boreham CJ, Dowling LM: Biomarker and n-alkane isotope profiles for tertiary oils: relationship to source rock depositional setting. Org Geochem. 1994, 22 (3–5): 521-10.1016/0146-6380(94)90124-4.View ArticleGoogle Scholar
- Armanios C, Alexander R, Sosrowidjojo IB, Kagi RI: dentification of bicadinanes in Jurassic organic matter from the Eromanga Basin, Australia. Org Geochem. 1995, 23 (9): 837-10.1016/0146-6380(95)80004-B.View ArticleGoogle Scholar
- Killops SD, Raine JI, Woolhouse AD, Weston RJ: Chemostratigraphic evidence of higher-plant evolution in the Taranaki Basin, New Zealand. Org Geochem. 1995, 23 (5): 249-10.1016/0146-6380(95)00019-B.View ArticleGoogle Scholar
- Stout SA: Resin-derived hydrocarbons in fresh and fossil Dammar resins and Miocene rocks and oils in the Mahakam Delta, Indonesia. ACS Symp Ser. 1995, 617 (Amber, Resinite, and Fossil Resins): 43-View ArticleGoogle Scholar
- Summons RE, Boreham CJ, Foster CB, Murray AP, Gorter JD: Chemostratigraphy and the composition of oils in the Perth Basin, western Australia. APEA J. 1995, 35 (pt. 1): 613-Google Scholar
- Sosrowidjojo IB, Murray AP, Alexander R, Kagi RI, Summons RE: Bicadinanes and related compounds as maturity indicators for oils and sediments. Org Geochem. 1996, 24 (1): 43-10.1016/0146-6380(96)00007-1.View ArticleGoogle Scholar
- Brackman W, Spaargaren K, Van Dongen JPCM, Couperus PA, Bakker F: Origin and structure of the fossil resin from an Indonesian Miocene coal. Geochim Cosmochim Acta. 1984, 48 (12): 2483-10.1016/0016-7037(84)90299-0.View ArticleGoogle Scholar
- van Aarssen BGK, Cox HC, Hoogendoorn P, de Leeuw JW: A cadinene biopolymer in fossil and extant Dammar resins as a source for cadinanes and bicadinanes in crude oils from South-East Asia. Geochim Cosmochim Acta. 1990, 54 (11): 3021-10.1016/0016-7037(90)90119-6.View ArticleGoogle Scholar
- van Aarssen BGK, de Leeuw JW, Horsfield B: A comparative study of three different pyrolysis methods used to characterize a biopolymer isolated from fossil and extant Dammar resins. J Anal Appl Pyrolysis. 1991, 20: 125-10.1016/0165-2370(91)80067-I.View ArticleGoogle Scholar
- van Aarssen BGK, de Leeuw JW: High-molecular mass substances in resinites as possible precursors of specific hydrocarbons in fossil fuels. Org Geochem. 1992, 19 (4–6): 315-10.1016/0146-6380(92)90002-F.View ArticleGoogle Scholar
- van Aarssen BGK, de Leeuw JW, Collinson M, Boon JJ, Goth K: Occurrence of polycadinene in fossil and recent resins. Geochim Cosmochim Acta. 1994, 58 (1): 223-10.1016/0016-7037(94)90459-6.View ArticleGoogle Scholar
- Mills JS, Werner AEA: The chemistry of Dammar resin. J Chem Soc. 1955, 3132-10.1039/jr9550003132.Google Scholar
- Mills JS: The constitution of the neutral, tetracyclic triterpenes of Dammar resin. J Chem Soc. 1956, 2196-10.1039/jr9560002196.Google Scholar
- Rene de la Rie E: Stable varnishes for old master paintings. PhD Thesis. 1988, University of AmsterdamGoogle Scholar
- Anderson KB, Winans RE: The nature and fate of natural resins in the geosphere. I. Evaluation of pyrolysisgas chromatography-mass spectrometry for the analysis of natural resins and resinites. Anal Chem. 1991, 63: 2901-10.1021/ac00024a019.View ArticleGoogle Scholar
- Doddrell DM, Pegg DT, Bendall MR: Distortionless enhancement of NMR signals by polarization transfer. J Magn Reson. 1982, 48: 323-Google Scholar
- Shaka AJ, Keeler J, Freeman R: Evaluation of a new broadband decoupling sequence: WALTZ-16. J Magn Reson. 1983, 53 (2): 313-Google Scholar
- Shaka AJ, Keeler J, Frenkiel T, Freeman R: An improved sequence for broadband decoupling: WALTZ-16,. J Magn Reson. 1983, 52 (2): 335-Google Scholar
- Starcuk Z, Sklenar V: Composite pulse sequences with variable performance. J Magn Reson. 1985, 62 (1): 113-Google Scholar
- Aue WP, Bartholdi E, Ernst RR: Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J Chem Phys. 1976, 64 (5): 2229-10.1063/1.432450.View ArticleGoogle Scholar
- Nagayama K, Kumar A, Wuethrich K, Ernst RR: Experimental techniques of two-dimensional correlated spectroscopy. J Magn Reson. 1980, 40 (2): 321-Google Scholar
- Bax A, Griffey RH, Hawkins BL: Correlation of proton and nitrogen-15 chemical shifts by multiple quantum NMR. J Magn Reson. 1983, 55 (2): 301-Google Scholar
- Bax A, Subramanian S: Sensitivity-enhanced two- dimensional heteronuclear shift correlation NMR spectroscopy. J Magn Reson. 1986, 67 (3): 565-Google Scholar
- Bax A, Summers MF: Proton and carbon-13 assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J Am Chem Soc. 1986, 108 (8): 2093-10.1021/ja00268a061.View ArticleGoogle Scholar
- Shaka AJ, Barker PB, Freeman R: Computer-optimised decoupling scheme for wideband applications and low-level operation. J Magn Reson. 1985, 64 (3): 547-Google Scholar
- Atta-ur-Rahman , Ahmad VU: 13C-NMR of natural products. Monoterpenes and Sesquiterpenes. 1991, Plenum Press, New York, 1:Google Scholar