Energy 13Fuels 1990,4,628-634
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Articles The Treibs Hypothesis: An Evaluation Based on Structural Studies B. J. Keely, W. G. Prowse, and J. R. Maxwell:.; Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's ('lose, Bristol BS8 ITS, United Kingdom Received May 2, 1990. Revised Manuscript Received September 18,1990
The Treibs hypothesis linking sedimentary 13,15-ethanoporphyrinsto an origin from chlorophylls requires the natural occurrence of a suite of intermediates on the proponed degradative pathway. The hypothesis was suggested over 50 years ago, yet despite the now extensive literature on the structural characterization of sedimentary porphyrins, few of the putative intermediates have been isolated and identified. Through examination of the tetrapyrrole distributions of three selected sediments, we have now assigned the minimum number of "intermediates" necessary for the operation of a pathway of defunctionalization, linking a precursor such as chlorophyll a to a sedimentary porphyrin with the same carbon skeleton (deoxophylloerythroetioporphyh). This provides further structural evidence for the defunctionalization of chlorophylls to give sedimentary 13,15-ethanoporphyrins.
Introduction Background. The Treibs hypothesis was proposed over 50 years ago.' Since then numerous identifications of sedimentary alkyl porphyrins and their carboxylic acid counterparts have been reported, many based on rigorous structural proof (recently reviewed).* Hence, the hypothesis, often with extension or modification depending on the carbon skeletons of the porphyrins and of the chlorophylls invoked as precursors, has been used to explain the occurrences of a variety of porphyrins in sediments and petroleums. Despite this interest in sedimentary porphyrins and their presumed origins from chlorophylls, in particular chlorophyll a (1 in Figure l),an analogous level of structural characterization has not been accorded to the putative chlorin intermediates on the degradative pathway. In this paper we attempt to reevaluate the Treibs hypothesis, mainly through consideration of the structures of a number of chlorins and porphyrins we have isolated from three specifically selected sediments. We consider here only components that possess a fivecarbon exocyclic ring, a feature that is characteristic of chlorophylls. The Treibs Hypothesis. Treibs' proposed that chlorophylls (e.g., chlorophyll a; 1) were the source of the sedimentary porphyrin deoxophylloerythroetioporphyrin (DPEP; 2 in Figure 1)and implied that they were degraded via a series of intermediates (shown for chlorophyll a in Figure 2), through both biological and chemical reactions. He separated the transformations into those that would be expected to occur readily (demetalation, ester hydrolysis, reduction of the vinyl group, decarboxylation of the (1) Treibs, A. Angew. Chem. 1936,49,682-686. (2) Chicarelli, M. I.; Kaur, S.;Maxwell, J. R. In Metal Complexes in Fossil Fuels; ACS Symposium Series 344; American Chemical Society: Washington, DC. 1987; pp 4C-67.
C-13*substituent, and aromatization) and those that he thought would result from greater thermal stress (Le., reduction of the C-13l ketone, decarboxylation, and metal insertion (sedimentary porphyrins occurring mainly as metallo species)). Circumstantial evidence for the derivation of cycloalkanoporphyrins from chlorophylls comes from the occurrences of sedimentary components with the five-membered exocyclic ring3 and similarities in the carbon skeletons of the macrocycle substituents. Convincing evidence that aromatization of chlorins can occur comes from the identification of nickel porphyrins from the Messel shale which bear extended alkylations a t C-8 and/or C-l!L4 These components are most likely derived from green sulfur bacteria of the order Chlorobiaceae which are known to produce bacteriochlorophylls d with the same extended alkylations a t these positions on the macr~cycle.~Evidence for an origin of petroporphyrins from chlorophylls c (3, the only known porphyrinic chlorophylls; chlorophyll c3 recently determineds) has been provided by the recognition of the nickel complexes of cycloalkanoporphyrins such as 4 in the Messel shale, in which the unusual exocyclic ring is considered to arise from a rearrangement involving the C-17 side chain of a chlorophyll c (3).' Further evidence comes from the use of stable carbon isotope ratios, which have indicated that the (3) (a) Quirke, J. M. E.; Ivfaxwell, J. R.; Eglinton, C.;Sanders, J. K. M. Tetrahedron Lett. 1980,21, 3887-2990. (b) Krane, J.; Skjetne, T.; Tenaes, N.; Bjoray, M.; Solli, H. Tetrahedron 1983,24, 4109-4119. (c) Fookes, C. J. R. J . Chem. Soc., Chem. Commun. 1983, 1472-1473. (4) Ocampo, R.; Callot, H. J.; Albrecht, P. J. Chem. Soc., Chem. Commun. 1985, 200-201. (5) Smith, K. M.; Bobe, F. W. J . Chem. Soc., Chem. Commun. 1987, 276-277 and references therein. J. Chem. Soc., Chem. Commun. (6) Fookes, C. J. R.; Jeffrey, S. I,?. 1989, 1827-1828. (7) Ocampo, R.; Callot, H. J.; Albrecht, P.; Kintzinger, J. P. Tetrahedron Lett. 1984,25, 2589-2592.
0 1990 American Chemical Society
The Treibs Hypothesis
Energy & Fuels, Vol. 4, No. 6, 1990 629
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bacteriochlorophyll d derived porphyrins from Messel are depleted in 13C with respect to the algal (chlorophyll c derived) component.8 Baker and Louda have reviewedgJOtheir extensive investigations of the tetrapyrrole assemblage of samples from the deep sea drilling project (DSDP).These studies included examination of chlorins. On the basis of absorption spectrophotometry, a modified Molisch phase test, chromatographic behavior, and in some cases through recognition of molecular ions in the mass spectra, these workers have tentatively assigned many of the intermediates proposed in the Treibs scheme. However, given that several chlorophylls can be precursors, a variety of possible intermediates could be implicated and could show similar behavior under these analytical conditions. Hence, more extensive structural studies of sedimentary chlorins are necessary, especially of intermediates implied by the scheme, in order to allow a more detailed assessment of the possible transformations involved. Such identifications could also provide information about the order in which the reactions leading to the intermediates occur. Accordingly, we consider here, in terms of the operation of a Treibs-type scheme, the structures of tetrapyrrole components isolated from a lake bottom sediment (Priest Pot) and two highly immature older sediments (Marad shale and Willershausen lake sediment). Priest Pot is a small eutrophic freshwater lake in Cumbria (U.K.)whose bottom sediments contain a mixture of chlorins."J* The lacustrine Marad shale (Miocene) from Bahia State (Brazil) has also been shown to contain chlorins.'J3 Willershausen sediment was laid down in a small meromictic lake (Pliocene) with a saline hyp~limnion'~ and is known to contain chlorins and porphyrins.15J6
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Figure 1. Structures cited in the text with compound names.
Methods. Low-resolution electron ionization (EI) mass spectra were recorded with the use of a direct insertion probe, on a Finnigan 4500 quadrupole mass spectrometer (70 eV, 200 PA). The probe temperature was raised to ca. 150 "C and then ballistically t o 300 O C and spectra were obtained by repeatedly scanning from m / z 50 to 700 (total cycle 3 s). Electronic absorption (UV/vis) spectra were obtained using a Perkin-Elmer 555 spectrophotometer(scan speed 120 nm min-', slit width 2 nm, path length 1 cm). The 400-MHz 'H NMR spectra were recorded at ambient temperature in (CD3)&0 on a Jeol GX400 instrument and were referenced to the chemical shift of the solvent. Analytical scale, normal-phase high-performance liquid chromatography (HPLC) wa3 carried out according to a published method," with monitoring at 400 nm. Semipreparative scale HPLC was carried out employing isocratic elution: Spherisorb S5W column (250 X 10 mm i.d.);25% A (dich1oromethane:acetone 4:1), 25% B (1% pyridine in hexane), 50% C (1% acetic acid in hexane);flow rate 3 mL m i d . Preparative reversed-phaseHPLC (8) Hayes, J. M.; Takigiku, R.; Ocampo, R.; Callot, H. J.; Albrecht, P. Nature 1987,329,48-51. (9) Baker, E. W.; Louda, J. W. In Aduances in Organic Geochemistry 1982; Bjoray, et al., Eds.; Wiley: Chichester, 1983; pp 401-421. (10) Baker, E. W.; Louda, J. W. In Biological Markers in Sediments; Johns, R. B., Ed.; Elsevier: Amsterdam, 1986; Methods Geochem. Geop h p . 1986, 24, 125-225. (11) Kee1y;B. J.; Brereton, R. G . In Advances in Organic Geochemistry 1985;Leythaeuser, D., RullkBtter, J., Fds.; Pergamon Press: Oxford 1986; Org. Geochem. 1986, 10, 975-980. (12) Keely, B. J.; Brereton, R. G.; Maxwell, J. R. In Aduances in Organic Geochemistry 1987; Mattavelli, L., Novelli, L., Eds.; Pergamon Press: Oxford, 1988; Org. Geochem. 1988, 13, 801-805. (13) Chicarelli, M. I. Personal communication, 1988. (14) Meischner, D.; Paul, J. CFS,Cow. Forschungsimt. Senckenberg 1982,56, 147-152. (15) Tibbetta, P. J. Ph.D. Thesis, University of Bristol, 1980. (16) Liebezeit, G.; Eglinton, G. Personal communication, 1985. (17) Barwise, A. J. G.; Evershed, R. P.; Wolff, G.; Eglinton, G.; Maxwell, J. R. J. Chromatogr. 1986, 368, 1-9.
Keely et al.
630 Energy & Fuels, Vol. 4 , No. 6, 1990
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Figure 2. Schematic representation of the Treibs hypothesis for the conversion of chlorophyll a (1) to sedimentary DPEP (2). As noted by Treibs,' the order of the reactions could be different from that shown.
employed two columns (Phase Separations S5 ODS2; each 150 X 4.6 mm) connected in series and elution with methanol (1.5 mL min-'). Isolation of Marati Shale Chlorins. An aliquot of the frozen shale (600 g) was allowed to thaw, ground by mortar and pestle, and divided into six equal aliquots. Each was extracted by sonication (5 min; Mettler Electronics ME 4.6 Ultrasonic tank) in acetone (5 X 200 mL). Centrifugation (10 min, 10000 rpm) and decantation of the supernatant afforded a dark green extract. The combined extracts were concentrated (to ca. 2 mL) and subjected to gel permeation chromatography using a modification'* of a previously described yielding only one chlorin-containing
fraction, which was methylated with CH2N,. Chlorins 5 and 6 were isolated as the methyl esters by preparative-scale normalphase HPLC and were separated from each other by use of reversed-phase HPLC (Figure 2). UV/vis ,A, (5,Me ester) 664 (re1 intensity loo), 586 ( l l ) , 534 (14), 504 (17),and 409 nm (230); A,, (6,Me ester) 654 (loo), 578 (121,530 (14), 501 (151, and 405 nm (230).
(18) Keely, B. J. Ph.D. Thesis, University of Bristol, 1989. (19) Repeta, D. J. Ph.D. Thesis, Massachusetts Institute of Technology/Woods Hole Oceanographic Institute WHOI-82-46, 1982.
The Treibs Hypothesis
Energy & Fuels, Vol. 4, No. 6, 1990 631
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Table I. 400-MHz 'H NMR Data for 5 and 6 (Methyl Esters) in (CD,),CO component 5 (Me component 6 (Me ester) ester) proton 6, ppm multiplicitv 6, DDm multidicitv" H-10 9.82 S 9.76 S H-5 9.57 9.41 H-20 8.92 S 8.79 H-3' 8.23 dd (12, 18) H-3' 6.40 dd (18, 1) H-3" 6.22 dd (12, 1) H-13' 5.27 5.24 d (20) H-132' 5.13 5.10 d (20) H-18 4.67 4.63 dq (7, 2) H-17 4.43 4.40 m (2) CHz-3l 3.94 CHr8l 3.78 3.76 q (8) CH3-121 3.66 S 3.64 S CH303.55 3.55 CH3-z1 3.49 S 3.36 CH3-7l 3.29 S 3.28 CH2-171,172 2.8-2.3 m 2.8-2.2 CH3-181 1.84 1.83 t (7) CH3-3* 1.75 CH3-8' 1.69 1.69 t (8) NH 0.43 S 0.58 NH -1.77 S -1.69 a ( J , Hz); s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.
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Results Only the assignments of the major Marafi chlorins a r e described since details of the Priest Pot and Willenhausen components are given elsewhere (see below). Figure 3 shows the HPLC distribution of tetrapyrroles in an aliquot of the methylated total extract. The UV/vis spectra of the two isolated constituents (Figure 3, inset) were identical with those of standards of 5 and 6 (as methyl esters) preparedla from chlorophyll a, and the E1 spectra (Figure 4) showed base peak molecular ions ( m / z 548, 550, respectively) corresponding to the masses of these compo-
Keely et al.
632 Energy & Fuels, Vol. 4, No. 6,1990
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nents. The major fragment ions (m/z 461 and 463) result from &cleavage of the C-17 substituent, with m/z 433 and 435 resulting from a subsequent loss of CO. The 'H NMR spectrum of 5 (methyl ester; Figure 5 , Table I) is virtually identical with that of the standard,18 showing the presence of three &methyls, one ðyl, one 0-vinyl, the CH2-132 protons, and a doublet assigned through decoupling as the C-18 methyl substituent. The protons of the (2-17 substituent were assigned by comparison with the spectrum of the standardla and consideration of literature data.20 The trans relationship of the
C-17 and C-18 substituents was deduced from the coupling constant (2 Hz) between H-17 and €3-18 (Table 1).l8 The relative substitution pattern around the macrocycle was unambiguously determined by nuclear Overhauser enhancement (NOE) experiments (summarized in Figure 5 and Table I) using the CH3-181doublet (1.84 ppm) as a reference point as described previously.18 The spectrum of the meso counterpart (6, methyl ester; Figure 6, Table (20) Smith, K.M.;Goff, D.A.; Abraham, R. J. J. Org. Chem. 1987,52, 176-180.
The Treibs Hypothesis
Energy & Fuels, Vol. 4, No. 6,1990 633
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Figure 7. Possible transformation pathway for chlorophyll a (1) to DPEP (2) based on components isolated from Priest Pot bottom sediment. MaraO shale, and Willershausen sediment. Solid arrows indicate reactions that must occur.
I) was virtually identical with that of the standardls and closely resembled that of 5 (methyl ester), with signals corresponding to a @ethyl substituent (q,3.94 ppm; t, 1.75 ppm) replacing those of the vinyl group (dd, 8.23 ppm; dd, 6.40 ppm; dd, 6.22 ppm). The substitution pattern was again unambiguously assigned by selected NOE experiments (summarized in Figure 6 and Table I) using the CH3-181 doublet (1.83 ppm) as a reference point (see above).
Discussion The components identified in the three sediments represent the minimum number necessary for the operation of a Treibs-type scheme, linking chlorophyll a (1) to DPEP (2). Hence, for convenience, the results are discussed in order of increasing extent of defunctionalization of chlorophyll a (l),which has been recognized previously in the bottom sediments of Priest Pot lake.” The components
634 Energy & Fuels, Vol. 4, No. 6, 1990
occur in these sediments as the free bases, so tetrapyrrole metalation is not considered here. Demetalation, Decarbomethoxylation, and Deesterification. These three reactions are grouped together since the components resulting from them have been identified in the surface sediment of Priest Pot lake.",'* Phaophytin a (7)and pyropheophytin a (8) have been fully assigned.18*21Pyrochlorophyll a (9) and pheophorbide a (10)were assigned on the basis of HPLC coinjection," and pyropheophorbide a ( 5 ) was assigned from HPLC coinjection," its E1 mass spectrum, and comparison of the 'H NMR spectrum with that of a standard preparedla from chlorophyll a. However, it has now also been isolated from the Marad shale in sufficient quantity for full unambiguous assignment (see above). Pheophytins are recognized products of cellular disruption,22which may arise from prolonged darkness, senescence, or h e r b i v ~ r y . ~In~ the - ~ ~latter case pheophytins are often dominated by pheophorbides which may be the main products of g r a ~ i n g Pyro . ~ ~components ~ ~ ~ ~ ~ may also be formed from chlorophylls by enzymatic degradation, as demonstrated by the accumulation of pyropheophytin a (8) in the green alga Euglena when subjected to periods of prolonged darkness.30 Due to the greater abundance of pheophytins than pheophorbides in Priest Pot lake, it seems probable that, in the main, the pheophytin components were formed through the enzymatic degradation of chlorophyll a (I). The pheophorbides, which represent about 30% of the abundance of their respective pheophytins, could also be formed in this manner from organisms containing the enzyme chlorophyllase. The possible derivation of components 5 and 7-10,which all seem to have formed in the water column, or at the very earliest stages of diagenesis, is outlined in Figure 7. Thus removal of the C-132carbomethoxy group can occur as an early transformation reaction of chlorophylls that contain this substituent. Notably, this reaction results in complete removal of the carbomethoxy substituent and not in the formation of a hydrolyzed component bearing an acid functionality at C-132as suggested by Treibs.' Reduction. Mesopyropheophorbide a (6) and deoxomesopyropheophorbide a (11)occur in the Marad Shale (see above) and Willershausen sediment,3l respectively. In both of these sediments a secondary origin for the chlorins via porphyrin reduction can be disregarded due to the absence32of porphyrins containing a C-13' ketone group (Marad) and absence3' of the chlorin counterpart of DPEP, which is the major tetrapyrrole in Willershausen sediment. Neither sediment has experienced any appreciable thermal effects and both are highly i m m a t ~ r e ~ '(for " ~ example, Willershausen sediment still contains18intact sterols). The occurrence of these two components in such immature (21) Keely, B. J.; Maxwell, J. R. Org. Ceochem. Submitted for publi-
cation.
(22) Owens, T. G.; Falkowski, P. G. Phytochemistry 1982,21,979-984. (23) Daley, R. J.; Brown, S. R. Arch. Hydrobiol. 1973, 72, 277-304. (24) Daley, R. J. Arch. Hydrobiol. 1973, 72, 409-439. (25) Burkhill, P. H.; Mantoura, R. E'. C.; Llewellyn, C. A.; Owens, N. J. P. Mar. Biol. 1987, 93, 581-590. (26) Currie, R. I. Nature 1962, 193, 956-957. (27) Lorenzen, C. J. Deep-sea Res. 1967, 14, 735-745. (28) Moreth, C. M.; Yentsch, C. S. J . Exp. Mar. Biol. Ecol. 1970, 4 , 238-249. (29) Shuman, F. R.; Lorenzen, C. J. Limnol. Oceanogr. 1975, 20, 580-586. (30) Scoch,S.; Scheer, H.; Schiff, J. A.; RBdiger, W.; Siegelman, H. W. Z. Naturforsch. 1981,36c, 827-833. (31) Keely, B. J.; Popp, B. N.; Hayes, J. M.; Meischner, D.; Maxwell,
J. R. Unpublished results. (32) Prowse, W. G.; Maxwell, J. R. Unpublished results.
Keely et al. sediments indicates that reduction of the vinyl group and of the C-13l ketone may occur early on in chlorophyll diagenesis. It is perhaps noteworthy that reduction of the C-3 vinyl group of tetrapyrroles has been suggested to occur as a result of ingestion by terrestrial herbivore^.^^ This perhaps suggests a precedent in the sedimentary situation for the occurrence of a biological reduction giving rise to the meso components. Decarboxylation. The cooccurrence of DPEP (2) and its corresponding carboxylic acid ( in Willershausen sediment indicates that decarboxylation has occurred, and accordingly both components are linked in Figure 7. The cooccurrence of these components in such an immature sediment indicates that the reaction can occur before or during the earliest stages of diagenesis. Indeed, Treibs' suggested that decarboxylation might occur via a biological transformation. Aromatization. The chlorin (11) and its porphyrin counterpart (12)cooccur in Willershausen ~ediment.~'On this basis it would appear that chlorin to porphyrin aromatization has occurred, especially since convincing evidence for such a reaction comes from the occurrence in the Messel shale of porphyrins considered4to be derived from bacteriochlorophylls d. If the Willershausen porphyrins have resulted from aromatization, consideration of the tetrapyrrole assemblage purely on the basis of structural comparisons would suggest that aromatization preceded decarboxylation (Figure 7). However, it is equally possible that the porphyrins and chlorins could have arisen independently from defunctionalization of precursors represented by porphyrins (i.e., chlorophylls c) and dihydroporphyrins (e.g., chlorophyll a ) , respectively.
la3'
Conclusions The minimum number of intermediates necessary for the operation of a Treibs-type transformation scheme linking chlorophyll a (1) and sedimentary DPEP (2) have been identified from examination of the tetrapyrrole distributions in a recent and two older sediments. In fact an origin from chlorophyll a (1)can be envisaged for all of the components (Figure 7). Their occurrence in sediments does not, however, prove such an origin, and other chlorophylls could equally well be sources, by way of additional defunctionalization reactions. Nevertheless, the sedimentary occurrences of the components in Figure 7 also extend the circumstantial structural evidence for the defunctionalization of chlorophylls to form sedimentary alkyl porphyrins. In accordance with expectations the more highly functionalized components occur in the youngest sediment, and the least functionalized components were identified in the two older, though still highly immature sediments. This leads to the conclusion that the defunctionalization reactions can occur during the very early stages of the degradation of the precursor chlorophylls.
Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. We also thank British Petroleum plc for HPLC facilities, the Science and Engineering Research Council for a studentship (W.G.P.), the Natural Environment Research Council for MS facilities (GR3/2951 and GR3/3758), and Dr. M. I. Chicarelli for a sample of Marad Shale and for helpful discussions. (33) Hendry, G . A. F.; Houghton, J. D.; Brown, S. B. New Phytol. 1987, 107, 255-302. (34) Fookes, C. J. R. J. Chem. SOC.,Chem. Commun. 1983,1474-1476.
