Diagenetic Products of Iron Porphyrins Isolated from Texas Fairfield Lignite.

Franciszek Czechowski1 R. Paul Philp2

1

Wroclaw University of Technology, Institute of Organic Chemistry, Biochemistry and Biotechnology, 27 Wybrzeze Wyspianskiego St., 50-370 Wroclaw, Poland; Czechowski@nafta1.nw.pwr.wroc.pl tel. 0048-71-3206502; fax 0048-71-3221580 2 School of Geology and Geophysics, University of Oklahoma, Norman, OK 73019, USA

INTRODUCTION Tetrapyrrole pigments occurring in geological sediments as nickel and vanadyl complexes are represented by several structural types. The two most common skeletal types are polyalkylporphyrins and readily correlated with chlorophyll compounds 13,15-ethanopolyalkylporphyrins [1-3]. In coal occur mainly polyalkyl porphyrins as metal free bases or complexes with iron, gallium, and in some coals, with manganese and copper [4-7]. Mesoheme and mesoporphyrin are the dominant structures of soft brown coals. Products of defunctionalisation of coal porphyrins biological precursors generated upon coalification typically are represented by homological series showing domination of even carbon numbered homologues. Extend of their defunctionalisation relates to coal maturation level, and at advanced catagenesis even carbon numbered homologues predomination disappears [4, 8]. Structural analysis of intermediate compounds of coal porphyrins formed upon coalification are scare [9-12]. The paper reports on the isolation of narrow TLC fractions of heme and chlorophyll a origin iron porphyrins separated from Texas Fairfield lignite and, using mass spectrometry in combination with 1D and 2D 1H NMR technique, identification of intermediate polyalkyl porphyrins generated from the precursors. MATERIALS AND MATHODS The sample investigated was subbituminous C coal, from deposit of Texas Fairfield lignite from Freestone County (Eocene, Upper Wilcox group, formation Upper Calvert Bluff). Bed moisture (31.94%), mean ulminite reflectance 0.45% and carbon, hydrogen and nitrogen content (Cdaf = 74.29%, Hdaf = 4.85%, Ndaf = 1.42%) of the coal indicate advanced diagenetic stage of coalification. The iron porphyrins were isolated from 15 kg of finely pulverised and dried coal sample by repetitive exhaustive batch extraction with 7% (v/v) sulfuric acid in methanol at room temperature. Obtained crude porphyrin extract was purified on Merck silica gel 60H TLC plates (20 x 20 cm) using various solvents as described earlier [9]. Isolated iron porphyrins mixture amounts 12.97 g/g of the raw coal. Homological composition of the mixture, as seen by mass spectrometry, is illustrated in Fig. 1. Final separation into 46 narrow TLC fractions was achieved using various solvent systems. Each fraction was characterised by mass spectrometry. Bis-cyanide iron(III) porphyrin complexes of the fractions were characterised by 1D and 2D 1H NMR spectra, which were aquired at temperature of 293 K on a Bruker AMX 300 spectrometer operating in a quadruple detection mode at 300 MHz [11]. RESULTS AND DISCUSSION Homological composition of the isolated heme mixture shows high predominance of even carbon numbered dicarboxylic C32d and C30d homologues (isolated as dimethyl esters), monocarboxylic C32m, C30m and C28m homologues (isolated as methyl esters) and polyalkyl C32 - C28 homologues (Fig. 1). This pattern is consistent with the most frequent precursor defunctionalisation steps in coal porphyrins generation, involving preferential decarboxylation as well as -cleavage of ethyl groups attached to porphyrin macrocycle. It rationalises assumption

protoheme IX as a source structure. In 22 narrow heme fractions mass spectrometry revealed domination of a single homologue, which might consist a single compound or isomeric mixture. The heme fractions were analysed by 1 H NMR spectroscopy as low-spin six coordinate bisC 30m cyanide iron(III) complexes. 100 Such complexes give convenient way to identify nature of peripheral 80 pyrrole substituents. This is because spectral window C 28m 60 C 32m ranges for -pyrrole substituents are shifted C 32d outside the range of signals C 29m 40 C 31m C 30d C 29 deriving from diamagnetic C 28 C 30 C C contaminants in isolated 32 31 20 C 29d heme fractions. Chemical C 31d shift ranges are: for methyls from 12 to 20 ppm, 0 for -methylenes from 6 to 9 450 490 530 570 610 650 ppm and for -hydrogens m/z from -12 to -18 ppm. Signals Figure 1. Mass specrum of the iron porphyrins isolated from Fairfield lignite. of -methyls and Number in C index corresponds to number of carbons in the homologue, hydrogens are singlets, while while index m describes monocarboxylic acids methyl esters and index d of -methylenes from ethyls dicarboxylic acids dimethyl esters. are quartets and from propionic acid substituents are triplets. Furthermore, the chemical shift values of -substituents show strong influence on their nature as well as overall arrangement around the porphyrin macrocycle [9, 10]. Therefore the 1-D 1 H NMR spectral pattern on set of all eight porphyrin macrocycle -pyrrole substituents is characteristic and unique for each heme structure. It allowed interpretation of all -pyrrole substituents of the hemes in analysed fractions. Based on MS and 1H NMR data three specific spectral groups of isolated structures were distinguished. To the first group are assigned hemes of which each structure gives characteristic -pyrrole resonances of four methyls and four methylenes (1H NMR spectra are not shown). They are represented by C32d, C32m and C32 homologues. The proposed reaction pathway of their generation from protoheme IX via vinyls hydrogenation and stepwise decarboxylation is shown in Fig. 2 - compounds (1), (2), (3) and (4). Loss of one carboxylic group is manifested by change of one -methylene multiplicity from triplet to quartet, as well as lowering of homologue molar mass by 58 mass units (because heme carboxylic acids were separated as methyl esters). The both homologues C32d and C32, each represented by one compound, were earlier recognised by comparison with spectral pattern of the standards to be mesoheme IX (1) and etioheme III (4) [9, 10]. Spectrum of C32m homologue shows eight -methyl resonances in comparable intensity as well as six -methylene quartets and two triplets. Number of the observed resonances is consistent with presence of the two C32m heme isomers in similar concentration, each possessing four methyls, three ethyl groups and one propionic acid (separated and characterised as methyl esters). These structures are two isomeric intermediates of mesoheme IX monodecarboxylation at propionic acid substituent on the route to etioheme III. The both compounds were unambiguously assigned by 2D 1H NMR (COSY and NOESY) experiments as 172-methoxycarbonyletioheme III (2) and 132-methoxycarbonyletioheme III (3). Second spectral group is represented by mono--H hemes (C30d, C30m, C30) lacking one ethyl substituent at -pyrrole. Particularly important information derives from the spectrum of C30d homologue, which consists of two isomeric forms in the equivalent concentration. -Substituents of each isomer are four methyls (eight singlets in the spectral window from 15 to 18 ppm), one ethyl and two propionic acids (two quartets and four triplets in the spectral
Relative abundance

window from 6 to 8 ppm) and one hydrogen (two singlets in the range from -16 to -14 ppm). It is indicative that the two structures occurring in the same concentration and possessing both propionic acid substituents, as well as lacking one ethyl, could only be obtained from protoheme IX (protoporphyrin IX) as a precursor. Therefore, relating to biogenetic grounds, the possible structures of two isomers characterised by observed substitution patterns 16 4 are 3-desethylmesoheme IX (5) and 8desethylmesoheme IX (6) (Fig. 2). They are the result of two competitive reactions of protoheme IX degradation taking place in humic (phenolic reach) environment, where one vinyl was hydrogenated while another cleaved 3 2 14 15 giving comparable concentration of both C30d isomers. Propionic acids substituents monodecarboxylation of (5) and (6) resulted in generation of four isomers of C30m homologue, i.e. structures (7), (8), (9) and (10). In fact, five structures of substitution pattern adequate to the C30m homologues are observed by 1H NMR 1 13 (four singlets from -methyls, two quartets from methylenes of -ethyls, one triplet from methylene of -propionic acid and one singlet from -hydrogen, each). We recognised that additional isomer of C30m homologue is due to contribution -phylloheme XV (characterised as protoheme methyl ester) deriving from chlorophyll a degradation under oxidative conditions. Complete decarboxylation of C30m isomers resulted in isomeric mixture of three isomers (four methyl singlets, three methylene quartets and one hydrogen singlet, each) assigned to 5 homologue C30. We suggest, that two of them, 6 (11) and (12), are the decarboxylation products of (7) and (8) as well as (9) and (10), respectively, and are represented by 11 and 12. The heme homologues with two ethyls displaced by hydrogens represent the third spectral group of compounds analysed (C28d, 10 8 7 9 C28m and C28). The observed one dideethylated compound possessing two preserved propionic acid substituents (four -methyl singlets, two -methylene triplets and two -hydrogen singlets) appeared to be deuteroheme IX (13), the product of two vinyls loss in protoheme IX 12 11 Figure 2. Diagenetic heme products in Fairfield lignite generated (Fig. 2). Pattern of all -pyrrole substituents is virtually identical with that determined earlier via defunctionalisation of protoheme IX. for deuteroheme obtained from protoporphyrin
N N N N N Fe Fe N N N - CO2 - CO2 N N N N N N N N Fe Fe Fe Fe N N N N N N N N CO2 H CO2 H CO2 H CO2 H - CO2 - CO2 N N N N N Fe Fe N N N CO2H CO2 H CO2 H CO2 H vinyls hydrogenation didevinylation N N N Fe N CO2H CO2 H vinyl hydrogenation and monodevinylation N N N N Fe Fe N N N N CO2H CO2H CO2H CO2 H - CO2 - CO2 N N N N N N N N Fe Fe Fe Fe N N N N N N N N CO2 H CO2 H CO2 H CO2 H - CO2 - CO2 N N N N N N Fe Fe N N

IX by didevinylation in resorcinol and subsequent iron insertion [10]. Unambiguous identification of this key structure with the reference to model compound gives strong evidence of its origin from protoheme IX. Loss of two ethyl groups at the positions C-3 and C-8 is possible only when in the parent compound these groups were vinyls. Devinylation reaction of protoheme IX in the humic matrix can be rationalised by the presence of various phenolic compounds in this material, which are known to induce this reaction. Instead of expected two isomers, (14) and (15), of C28m homologue generated by deuteroheme monodecarboxylation, similarly to their C30m counterparts, additional isomer in higher relative concentration has been found. The didevinylation and didecarboxylation product of protoheme IX, i.e., deuteroetioheme IX (16), was not observed in the coal investigated, although it was found in Colorado coal of higher coalification stage [11]. We presume that at Fairfield lignite maturation stage the highly degraded compound (16) generated from low quantity of parent intermediate structures (14) and (15), is present only in trace amount. In the heme fractions from Fairfield lignite are also evidenced diagenetically altered products of chlorophyll a formed on non-oxidatine diagenetic pathways i.e. compounds bearing five-membered isocyclic ring as Fe(III) deoxophylloerytroetioporphyrin and its Fe(III) 17-propionic methyl ester analogue [13]. CONCLUSIONS Geoporphyrins present in humic Fairfield lignite are represented by iron complexes containing intermediate diagenetic products generated from precursors: protoheme IX and chlorophyll a. Porphyrin skeleton of the isolated Fairfield lignite hemes is represented by ETIO compounds, and in much smaller concentration by DPEP and phyllotype porphyrins possessing methyl substituent at C-15 position. The major part of intermediate ETIO-type compounds constitute: mesoheme IX (1), its two isomeric forms of monodecaroboxylation (2) and (3), and the product of mesoheme IX didecarboxylation - etioheme III (4). Further intermediates of source molecules transformation are two isomeric forms of mesoheme IX monodeethylation at C3 (5) or C8 (6) positions and its dideethylation product at both these positions i.e. deuteroheme IX (13). DPEP and phyllo-type polyalkyl hemes are less abundant in Fairfield lignite. The above compounds are direct products of protoheme IX and chlorophyll a transformation. The reaction pathways of their generation under mild thermal conditions involve preferential loss of carboxylic groups, partial or complete vinyls hydrogenation and simultaneous concurrent monodevinylation or didevinylation. Compounds deriving from chlorophyll a are formed on oxidative and non-oxidative diagenetic pathways. The 1D and 2D 1H NMR technique applied here to coal iron(III) porphyrins proved to be effective in obtaining structural information on substituents arrangement of individual porphyrin molecules. REFERENCES
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