Terpenoid Compositions of Resins from Callitris Species (Cupressaceae)

The environmental fate of conifer resins and their natural product compounds as mixtures is of importance for source, alteration, and transport studies. The compound compositions of resins of the common Callitris species (Cupressaceae) based on gas chromatography-mass spectrometry have not been reported. Results show that diterpenoids were the most abundant components and callitrisic acid was present in the resin extracts of all Callitris species analyzed. Significant amounts of 4-epi-pimaric and sandaracopimaric acids, with lesser communic, ozic, and lambertianic acids, were also in the mixtures. Phenolic diterpenoids, for example, ferruginol, hinokiol, were found in trace quantities in some samples. Thus, callitrisic acid and 4-epi-pimaric acid are the characteristic diterpenoids of Callitris species that are amenable to molecular biomarker analyses in geological or environmental applications.


Samples
The samples were collected as hardened, freshly bled resins from the stems of various Callitris species (Cupressaceae, subfamily Callitroideae). In the case of species with no obvious resin, a branchlet was sampled and air dried prior to extraction. The samples and their source locations are given in Table 1. The resins of other conifers were sampled and analyzed in the same manner.

Extraction and Gas Chromatography-Mass Spectrometry
The resin samples and dried branchlets were crushed and sonicated three times with dichloromethane:methanol (DCM:MeOH, 3:1, v/v) for 15 min. The total extracts were combined, filtered, and concentrated with a rotary evaporator and then with nitrogen blow down (to typically 1-3 mL). Aliquots (50 µL) of the total extracts were converted to trimethylsilyl (TMS) derivatives by reaction with N,O-bis(trimethylsilyl)trifluoroactamide (BSTFA) and pyridine for 3 h at 70 • C. Prior to GC-MS analysis, the excess silylating reagent was evaporated under a dry nitrogen stream and the sample mixture was dissolved in an equivalent volume of n-hexane. Other aliquots (50 µL in DCM:MeOH, 1:1 v/v) were treated with trimethylsilyldiazomethane (20 µL, 2 M in n-hexane, Sigma-Aldrich, St. Louis, MO, USA) to methylate carboxylic acids prior to analysis. This reaction proceeded at room temperature within 30 min, after which the excess reagent was removed with acetic acid (glacial grade), followed by blow down with nitrogen and dissolution in n-hexane.
GC-MS analyses of the underivatized and derivatized extracts were carried out using an Agilent model 6890 GC coupled to an Agilent model 5973 quadrupole MSD. GC-MS data were acquired with the associated Chemstation software. Identifications of compounds were based on comparisons with standards, literature mass spectra, Wiley 275 library data, and interpretation of mass spectrometric fragmentation patterns for unknown compounds. The mass spectra of novel compounds and their derivatives (methyl esters or TMS esters/ethers), with the basic fragmentation patterns, are also presented. The relative abundance of each significant compound was calculated using its peak area in the respective total ion current (TIC) trace and assuming the same response factor.

Results and Discussion
The sesqui-and diterpenoids identified in the resins of Callitris sp. and their relative abundances are listed in Table 2. The Kovats GC retention indices of the natural products or their derivatives relative to n-alkanes are given on the respective mass spectra [32].

Resin Compositions
The diterpenoid compositions of the samples are quite diverse, especially with regards to callitrisic acid (X, the chemical structures are given in Appendix A and follow the sequence in Table 2). Some examples of total resin compositions are shown in Figure 1. Callitrisic acid (X) is the dominant compound in resins from C. intratropica, C. macleayana, C. rhomboides, and C. verrucosa, a trace component in resin of C. oblonga, and minor in the other samples. Dehydroabietic acid (XI) is a trace component only in resin of C. muelleri and 16,17-bisnorcallitrisic acid is not detectable. Sandaracopimaric acid (XVI), 4-epi-pimaric acid (XII), communic acids (XIII-XV), and 12E-ozic acid (XVII) are the secondary major components (Table 2). Various hydroxycallitrisic acids (XXIII-XXVI), lambertianic acid (XXII), and 7-oxocallitrisic acid (XXI) are also significant in some of the resins. Callitrisol (V), ferruginol (VI), and sandaracopimara-8 (14),15-dien-3β-ol (VII) are minor hydroxylated components in some samples. In addition, C. preissii resin contains dominant lignans, as already reported [31]. Three sesquiterpenoids, i.e., callitrisin (I), columellarin (II), and dihydrocolumellarin (III), are present here only in resin of C. preissii (Figure 1c). These were reported before in heartwood of C. columellaris [25,26], but not detected in our resin sample.

Resin Compositions
The diterpenoid compositions of the samples are quite diverse, especially with regards to callitrisic acid (X, the chemical structures are given in Appendix I and follow the sequence in Table  2). Some examples of total resin compositions are shown in Figure 1. Callitrisic acid (X) is the dominant compound in resins from C. intratropica, C. macleayana, C. rhomboides, and C. verrucosa, a trace component in resin of C. oblonga, and minor in the other samples. Dehydroabietic acid (XI) is a trace component only in resin of C. muelleri and 16,17-bisnorcallitrisic acid is not detectable. Sandaracopimaric acid (XVI), 4-epi-pimaric acid (XII), communic acids (XIII-XV), and 12E-ozic acid (XVII) are the secondary major components ( Table 2). Various hydroxycallitrisic acids (XXIII-XXVI), lambertianic acid (XXII), and 7-oxocallitrisic acid (XXI) are also significant in some of the resins. Callitrisol (V), ferruginol (VI), and sandaracopimara-8(14),15-dien-3β-ol (VII) are minor hydroxylated components in some samples. In addition, C. preissii resin contains dominant lignans, as already reported [31]. Three sesquiterpenoids, i.e., callitrisin (I), columellarin (II), and dihydrocolumellarin (III), are present here only in resin of C. preissii (Figure 1c). These were reported before in heartwood of C. columellaris [25,26], but not detected in our resin sample.  Table 2. U = unknown.

Mass Spectrometry
The mass spectra of the compounds in Table 2, analyzed as the free and derivatized products, are shown in Figure 2. Additional mass spectra of related and derivatized natural products are collected and discussed in the Supplemental Materials.  Table 2. U = unknown.
The presence of 4-epi-pimaric acid (XII) is of interest. The identification was based on its early GC elution and the same mass spectrum as that of pimaric acid standard (Figures 2d and S1dd), coupled with a literature report [34]. The mass spectra of the communic acids (XIII-XV) and sandaracopimaric acid (XVI) match those of the respective standards (Figures 2e-h and S1ee-hh). The communic acids have been characterized for resin from C. columellaris [21]. Ozic acid (XVII, 4-epi-communic acid, assumed 12E-isomer) was a dominant component in two samples, and its mass spectra (Figures 2i and S1ii) were interpreted by comparison with literature data [37,38] and the GC retention indices versus those of the communic acids. Lambertianic acid (XXII) is a major component in most samples and its mass spectra (Figures 2k and S1nn) were interpreted by comparison with a surrogate standard from resin of Pinus lambertiana [39]. 7-Oxocallitrisic acid (XXI) is a significant oxidation product in many samples and its mass spectra (Figures 2j and S1mm) were interpreted by comparison with standard 7-oxodehydroabietic acid and GC retention index.

Environmental and Geological Implications
The environmental fate of conifer resins and their natural product compounds as mixtures is of importance for source, alteration and transport studies [10,[41][42][43]. The precursor-product relationship for diterpenoids based on the abietane and pimarane skeletons has been presented by numerous authors [1,42,44,45]. Thus, callitrisic acid, 4-epi-pimaric acid, ferruginol and lambertianic acid of the Callitris resins were proposed as the main environmental tracers. Over geological timespans, the fate of the communic and ozic acids is oxidation and incorporation into macromolecular polymers. The diagenetic fate of callitrisic acid is decarboxylation with subsequent aromatization, analogous as dehydroabietic acid, to the same hydrocarbons, i.e., dehydroabietin (18-or 19-norabieta-8,11,13-triene) and retene ( Figure 3). Also, 4-epi-pimaric acid may aromatize to 15,16-bisnorcallitrisic acid by loss of C 2 H 6 , or become incorporated into polymeric matter across the C-15 to C-16 double bond with subsequent release as the same diagenetic product (Figure 3). Bisnordehydroabietic acid may be derived by the same route from sandaracopimaric acid (Figure 3). These products are readily observed in pyrolysates of some ambers [18].

Environmental and Geological Implications
The environmental fate of conifer resins and their natural product compounds as mixtures is of importance for source, alteration and transport studies [10,[41][42][43]. The precursor-product relationship for diterpenoids based on the abietane and pimarane skeletons has been presented by numerous authors [1,42,44,45]. Thus, callitrisic acid, 4-epi-pimaric acid, ferruginol and lambertianic acid of the Callitris resins were proposed as the main environmental tracers. Over geological timespans, the fate of the communic and ozic acids is oxidation and incorporation into macromolecular polymers. The diagenetic fate of callitrisic acid is decarboxylation with subsequent aromatization, analogous as dehydroabietic acid, to the same hydrocarbons, i.e., dehydroabietin (18or 19-norabieta-8,11,13-triene) and retene ( Figure 3). Also, 4-epi-pimaric acid may aromatize to 15,16-bisnorcallitrisic acid by loss of C2H6, or become incorporated into polymeric matter across the C-15 to C-16 double bond with subsequent release as the same diagenetic product (Figure 3). Bisnordehydroabietic acid may be derived by the same route from sandaracopimaric acid (Figure 3). These products are readily observed in pyrolysates of some ambers [18].   The unknown factor is whether callitrisic acid can also isomerize to dehydroabietic acid in fossil resins. Dehydroabietic acid is generally the dominant compound in total extracts of certain ambers and fossil resins, with minor or trace amounts of callitrisic acid [12,18,19,46]. We also found the seco-derivatives of both callitrisic and dehydroabietic acids in some amber extracts and commonly in aged pine resins (see the mass spectra in the Supplemental Materials). The pine resins contained dehydroabietic acid, 10α(H)-and 10β(H)-9,10-seco-dehydroabietic acids, and 4,5,9,10-bis-seco-dehydroabietic acid [2,6-dimethyl-9-(3 -(2-methylethyl)phenyl)non-2-enoic acid]; whereas the ambers contained both sets of seco-derivatives, but the bis-seco-compound was not found. We propose that the 10α(H)-and 10β(H)-9,10-seco-callitrisic acids may also proceed to the 4,5,9,10-bis-seco-derivative ( Figure 4). Furthermore, we speculate if these reactions are reversible in amber, then ring reclosures may lead to epimerization at C-4.
The unknown factor is whether callitrisic acid can also isomerize to dehydroabietic acid in fossil resins. Dehydroabietic acid is generally the dominant compound in total extracts of certain ambers and fossil resins, with minor or trace amounts of callitrisic acid [12,18,19,46]. We also found the seco-derivatives of both callitrisic and dehydroabietic acids in some amber extracts and commonly in aged pine resins (see the mass spectra in the Supplemental Materials). The pine resins contained dehydroabietic acid, 10α(H)-and 10β(H)-9,10-seco-dehydroabietic acids, and 4,5,9,10-bis-seco-dehydroabietic acid [2,6-dimethyl-9-(3'-(2-methylethyl)phenyl)non-2-enoic acid]; whereas the ambers contained both sets of seco-derivatives, but the bis-seco-compound was not found. We propose that the 10α(H)-and 10β(H)-9,10-seco-callitrisic acids may also proceed to the 4,5,9,10-bis-seco-derivative ( Figure 4). Furthermore, we speculate if these reactions are reversible in amber, then ring reclosures may lead to epimerization at C-4. We found no callitrisic acid in the closely related species (e.g., Diselma archeri, Fitzroya cupressoides, Tetraclinis articulata, and Austrocedrus chilensis [47,48]). We were not able to detect any callitrisic acid in resins of Juniperus chinensis and J. phoenicea, as reported before [23,49,50]. However, we did find 4-epi-abietic and 4-epi-pimaric acids in the juniper resins we analyzed. They could dehydrogenate to the aromatic derivatives upon weathering, as for example the rapid oxidation of abietic acid to dehydroabietic acid. Macrofossils of Callitris species are rare [51], so further work on the preservation of the major resin tracer components by direct or extract analyses remains for the future.

Conclusions
Callitrisic acid was found in resin extracts of all Callitris species analyzed here. Significant amounts of 4-epi-pimaric and sandaracopimaric acids, with lesser communic, ozic, and lambertianic acids, were also in the mixtures. Phenolic diterpenoids, e.g., ferruginol, hinokiol, were found in trace amounts in some samples. Therefore, callitrisic acid and 4-epi-pimaric acid are the characteristic diterpenoids of Callitris species for molecular biomarker analyses in geological or environmental applications. Furthermore, callitrisic acid has not been found in closely related Cupressaceae species, although it is present in some Angiosperms.
Supplemental Material: Additional mass spectra of related and derivatized natural products are collected and discussed in the Supplemental Material section available with this paper.  We found no callitrisic acid in the closely related species (e.g., Diselma archeri, Fitzroya cupressoides, Tetraclinis articulata, and Austrocedrus chilensis [47,48]). We were not able to detect any callitrisic acid in resins of Juniperus chinensis and J. phoenicea, as reported before [23,49,50]. However, we did find 4-epi-abietic and 4-epi-pimaric acids in the juniper resins we analyzed. They could dehydrogenate to the aromatic derivatives upon weathering, as for example the rapid oxidation of abietic acid to dehydroabietic acid. Macrofossils of Callitris species are rare [51], so further work on the preservation of the major resin tracer components by direct or extract analyses remains for the future.

Conclusions
Callitrisic acid was found in resin extracts of all Callitris species analyzed here. Significant amounts of 4-epi-pimaric and sandaracopimaric acids, with lesser communic, ozic, and lambertianic acids, were also in the mixtures. Phenolic diterpenoids, e.g., ferruginol, hinokiol, were found in trace amounts in some samples. Therefore, callitrisic acid and 4-epi-pimaric acid are the characteristic diterpenoids of Callitris species for molecular biomarker analyses in geological or environmental applications. Furthermore, callitrisic acid has not been found in closely related Cupressaceae species, although it is present in some Angiosperms.

Conflicts of Interest:
The authors declare no conflicts of interest.

Appendix I.
Chemical structures cited.