716
Energy & Fuels 1990, 4 , 716-719
in TLC methodology, and comparisons with synthetic haem reference compounds, become available. This is now in hand. It also seems likely that the use of paramagnetically shifted spectra of the dicyanoiron(II1) complexes may be of value in the assignment of the structures of porphyrins isolated from crude oil and its relatives.
Conclusions The results demonstrate the presence in Colorado coal of etioheme 111 and suggest strongly that it is the head substance for a series of cracking products formed by cleavages involving peripheral substituents during the catagenesis of coal. The mixtures formed are complex but can be separated with some degree of success by careful TLC using multiple and sequential development. The fractions so formed have been examined by paramagnetically shifted 'H NMR spectroscopy of the dicyanoiron(II1)
complexes. Some of the fractions are still complex mixtures. Nevertheless for the less degraded homologues the spectra provide clear structural information and the first experimental evidence in the coal porphyrin series for aromatic dealkylation (cleavages a and c) and benzylic demethylation (cleavage b). The results exemplify for the first time the application of paramagnetically shifted NMR spectra in porphyrin geochemistry and provide important confirmation for current views on porphyrin catagenesis during coal maturation.
Acknowledgment. The award by the Royal Society of a Guest Research Fellowship (to F.C.) and of a visitors' grant (to L.L.-G.) is gratefully acknowledged. We are indebted to Mr. P. Cook and Mr. G. Coumbarides for their expertise in the measurement of the mass spectra and the NMR spectra, respectively.
NMR Studies of Sedimentary Tetrapyrroles B. J. Keely and J. R. Maxwell* Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom Received May 23, 1990. Revised Manuscript Received September 18, 1990
This paper briefly reviews recent developments in 'H NMR studies of chlorins and porphyrins of sedimentary origin, with particular reference to correlated spectroscopy (COSY), which allows elucidation of spin-coupled systems.
Introduction Structural assignments of sedimentary biological markers rely mostly on the use of GC and GC-MS techniques, involving both chromatographic and mass spectral comparisons with synthetic standards in addition to de facto mass spectral interpretation. Difficulties experienced in the analysis of sedimentary porphyrins by GC-MS' (which until very recently2 required derivatization), and the desire to understand the origins and geochemical transformations of these components and their precursors, have led to more extensive structural investigations than usually accorded to other classes of biological marker. These studies typically involve HPLC isolation and purification of individual components, which are then examined by use of absorption spectrophotometry (UV/vis), mass spectrometry (MS), and 'H nuclear magnetic resonance (NMR) spectroscopy. In the past decade this has led to numerous reports of the structures of sedimentary porphyrins. Background. The earliest report of the application of 'H NMR spectroscopy to the structural determination of sedimentary porphyrins was the characterization of etioporphyrin I11 ( 1 in Figure 1) by direct comparison of the spectrum (after demetalation of the naturally occurring nickel complex) of its bis[ (porphyrinato)mercury(II)ace(1) Marriott, P. J.; Gill, J. P.; Eglinton, G. J . Chromatogr. 1982, 236, 395-401. (2) Blum, W.; Eglinton, G. J . High. Resol. Chromatogr. Chromatogr. Commun. 1989, 12, 621-623.
0887-0624/90/ 2504-07 16$02.50/0
tato]mercury(II) complex with those of standards of the four possible isomer^.^ Later studies have for the most part relied on the application of nuclear Overhauser enhancement (NOE) difference spectroscopy in conjunction with spin decoupling, to distinguish among the theoretically possible isomers for individual sedimentary porphyrin structures. Initially this approach allowed partial confirmation of the structure of demetalated DPEP (2 in Figure 1)as its Zn complex, indicatine the presence of the five-carbon exocyclic ring. From additional consideration of the products of chromic dcid oxidation, the number of possible isomers was limited to three.4 Subsequently, unambiguous assignment of the structure was a ~ h i e v e d . ~ , ~ Since these contributions, a wide variety of sedimentary porphyrin structures, both as metallo (Ni) and demetalated species, have been determined (see, for example, refs 7 and 8). In contrast to the porphyrins, in spite of an early interest involving examination by UV/vis, EI-MS, and chromatographic comparisons with standards, sedimentary chlorins (putative precursors of the porphyrins) have not (3) Quirke, J. M. E.; Maxwell, J. R. Tetrahedron 1980,36,3453-3456. (4) Quirke, J. M. E.; Maxwell, J. R.; Eglinton, G.; Sanders, J. K. M. Tetrahedron Lett. 1980, 21, 2987-2990.
(5) Krane, J.; Skjetne, T.;Telnaes, N.; B j o r ~ yM.; , Solli, H. Tetrahedron 1983, 24, 4109-4119. (6) Fookes, C. J. R. J. Chem. SOC.,Chem. Commun. 1983,1472-1473. (7) 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. (8) (a) Fookes, C. J. R. J . Chem. Soc., Chem. Commun. 1983, 1474-1476. (b) Verne-Mismer, J.; Ocampo, R.;Callot, H. J.: Albrecht, P. Tetrahedron Lett. 1990, 31, 1751-1754.
0 1990 American Chemical Society
N M R Studies of Sedimentary Tetrapyrroles
p
....
0
TOM. phytyl
C33 BicycloeUranochlorin (a) c33 BicycloPlhnoporphyrin 0 Figure 1. Structures cited in the text with compound names.
been investigated in such a rigorous manner until recently (see below).
Correlated Spectroscopy (COSY) COSY is a two-dimensional (2D) NMR technique that allows examination of the connectivity of spin systems. Since its inceptiong several modifications of the technique have been demonstrated. The basic pulse sequence involves the creation of transverse magnetisation, in allowed spin transitions, by application of a 90" pulse. Following an evolution time, a 90" mixing pulse is applied to redistribute the coherence corresponding to this magnetization (9) Aue, W. P.; Bartholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64, 2229-2246 and references cited therein.
Energy & Fuels, Vol. 4, No. 6,1990 717
among all possible coherences within the spin system^.^ Due to the application of a 90" mixing pulse, this technique is often referred to as COSY-90. A variation on this pulse sequencelo involves the application of a 45" mixing pulse (hence COSY-45). This results in an increase in the relative intensities of the components of cross-peaks related to directly connected transitions within a spin system. From this, it follows that the relative signs of coupling constants can be determined by analysis of the tilt of the cross-peak relative to the diagonal.1° A further, and perhaps the most useful, modification of the basic COSY-90 experiment is phase-sensitive COSY.g Recently" it has been demonstrated that the two proposed techniques12for obtaining the required phase modulation of the signals are equivalent. The technique results in cross-peaks that contain both negative and positive components. Analysis of the phase information allows recognition of the coupling that gives rise to the cross-peak (the active coupling) since it will have components that are antiphase in nature. Hence, from analysis of the phase-sensitive COSY spectrum, coupling networks may be confidently analyzed, and coupling constants can be directly abstracted. Despite the early demonstration of the application of COSY spectroscopy5 to assist in elucidation of the structure of a sedimentary alkyl porphyrin, the main technique to date for the investigation of the spin systems in such compounds has been decoupling, which has proved adequate for components with relatively simple spin systems.' Example. Figure 2 demonstrates the applicability of the 2D COSY-45 technique to the analysis of the spin systems of a chlorin standard, mesopyropheophytin a (3), prepared from chlorophyll a according to published methods.13J4 This 400-MHz 'HJH COSY-45 spectrum (recorded in deuteriated acetone) was acquired over the portion of the full spectrum (Table I) which demonstrated spin couplings, i.e., a frequency range of 2000 Hz. A data matrix of 512 data points in the F2 dimension and 256 data points in the F1 dimension was acquired. The spectrum was zero filled once in F2 and twice in F1, and was Fourier transformed using a negatively shifted sine-bell window. Following symmetrization, the spectrum was plotted using an exponential contour mode and chemical shifts were referenced to that of the solvent. The spectrum (Figure 2) shows the presence of many coupled spins. Couplings are established through the recognition of off-diagonal elements and include three isolated spin systems, Le., an AB system (132;13"),and two A2X3 systems (31;32and 8l;g2) (Table I). A connective network (Figure 2) was established from the methyl at position 18 to the 172diastereotopic methylene protons; coupling between protons 18 and 17 was only apparent when a lower threshold than shown in Figure 2 was used. Further couplings from 17-H to 17l-H and 17l'-H were observed, and analysis of the region of the COSY spectrum between 2 and 3 ppm revealed couplings of the diastereotopic 17l and 172protons that are entirely consistent with the assignments. Further connective networks were observed for resonances associated with the phytyl chain. Thus, a coupling was observed from the mutually coupled diastereotopic P1 protons to the P2 (10) (a) Bax, A.; Freeman, R. J. Magn. Reson. 1981,44,542-561. (b) Bax, A. Two Dimensional Nuclear Magnetic Resonance in Liquids; Delft University Press: Delft, 1982. (11) Keeler, J.; Neuhaus, D.J. Magn. Reson. 1985, 63, 454-472. (12) (a) States, D.J.; Haberkorn, R. A.; Ruben, D.J. J. Magn. Reson. 1982,48,286-292. (b) Marion, D.;Wuthrich, K. Biochem. Biophys. Res. Commun. 1983,113,967-974. (13)Furhop, J.-H.; Smith, K. M. In Porphyrins and Metalloporphyrins; Smith, K. M., Ed.; Elsevier Scientific: New York, 1975. (14) Pennington, F. C.; Strain, H. H.; Svec, W. A,; Katz, J. J. J. Am. Chem. SOC. 1964,865, 1418-1426.
Keely and Maxwell
718 Energy & Fuels, Vol. 4, No. 6, 1990 82
Pl5'
1-(P2; P3')
F1
P F2
Figure 2. 'H-lH COSY-45 spectrum of mesopyropheophorbide a (3). Cross-peaks are joined by solid lines to indicate couplings between isolated spin systems and broken lines to indicate couplings between connected spin networks. Assignments in parentheses are those of allylic and homoallylic long-range couplings.
proton. Furthermore, although the experiment was not set up to examine them, allylic and homoallylic long-range couplings of these three protons to those of the methyl P3l are evident (Figure 2). Additional couplings within the 0.8-2 ppm region allowed discrimination of the methyls P7l and P11' from the gem-methyls at P15 (P15l and P16) by the observation of the coupling of the latter two to a methine proton septet (P15) at ca. 1.42 ppm. Examination of the spectrum by using a lower threshold reveals that the P15 proton is further coupled to a poorly resolved signal at ca. 0.97 ppm, thus locating the chemical shift of one or both of the P14 methylene protons. On the basis of chemical shift argument the P4 protons are assigned at ca. 1.8 ppm, and are observed to couple to the P5 protons at ca. 1.2-1.35 ppm. Hence, analysis of the COSY-45 spectrum of mesopyropheophorbide a allowed full assignment of the diastereotopic methylene protons of the C-17 propionate substituent in deuteriated acetone. Previously in the 'H NMR spectra of chlorophylls, pheophytins, and pheophorbides these resonances have either not been individually assigned,15 or assignments have been based16 on comparison of the subspectra with those generated through (15) Katz, J. J.; Brown, C. E. Bull. Mugn. Reson. 1983,5, 3-49. (16) Smith, K. M.; Goff, D. A.; Abraham, R. J. J. Org. Chem. 1987,52, 176-180.
spectral simulations. Furthermore, the COSY-45 spectrum shows that the previous tentative assignment of the methyls P15l and P16, based on consideration of spin lattice relaxation times (T,'s)," is correct. Furthermore, additional assignments of the chemical shifts of protons in the phytyl chain have been made. This demonstrates the utility of the 2D COSY technique to the study of compounds of this class. Sedimentary Components. The chlorins pheophytin a (4) and pyropheophytin a (5) from the bottom sediment of a small eutrophic lake were fully assigned'* using a combination of FAB mass spectrometry and 'H NMR techniques, including NOE difference and COSY-45 spectroscopy. In both cases assignment of the esterifying alcohol was based on recognition of a fragment ion in the FAB mass spectrum corresponding to loss of the phytyl chain as phytadiene. Further evidence was obtained from the presence in the NMR spectra (1D and 2D) of resonances and spin couplings that correspond to phytyl resonances as seen in the spectra of standards (e.g., mesopyropheophytin a , 3). (17) Sanders, J. K. M.; Waterton, J. C.; Dennis, I. S. J. Chem. Soc., Perkin Trans. I 1978, 1150-1157. (18) Keely, B.J.; Maxwell, J. R. Org. Geochem. Submitted for publication.
NMR Studies
of Sedimentary Tetrapyrroles
Table I. The 400-MHz *€I NMR Data for Mesopyropheophytin a (3) in Acetone-d6 chemical shift b, proton(s) PPm 10-H 9.68 5-H 9.35 20-H 8.76 13'-H 5.20 P2-H 5.15 13"-H 5.06 18-H 4.60 P1-H 4.50 P1I-H 4.42 17-H 4.36 3l-CHz 3.91 8l-CH2 3.72 12'-CH3 3.60 2l-CH3 3.34 7l-CH3 3.25 17l-H 2.13 17*-H 2.65 17"-H 2.40 17l'-H 2.23 P4-CHz 1.88 18l-CH3 1.82 3'-H 1.74 8'-CH3 1.67 P 3 - CH 3 1.60 P15-H 1.42 P5-CHZ 1.36 P7, P11 2 X H 1.28, 1.22 P14-CHz 0.97 PCHz'sd 6 X CHz 0.93-0.85 P15', P16 2 X CH3 0.79 P7l, P11' 2 X CHS 0.70, 0.66 NH 0.51 NH -1.73
'
multiplici t p (J, Hz)
coupled spin b, PPmb
S S
S
d (20) tq (7, 1) d (20) dq (2, 7) dd (7, 12) dd (7, 12) d t (2, 9) q (8) q (8)
5.06 4.50, 4.42 (1.60) 5.20 4.36,' 1.82 5.15, 4.42 (1.60) 5.15, 4.50 (1.60) 4.60,c 2.73, 2.23 1.74 1.67
S
S
Energy & Fuels, Vol. 4 , No. 6, 1990 719
both containing extensively coupled spin systems. In the former case, complete analysis of the phase information and abstraction of coupling constants directly from the 2D spectrum allowed determination of the relative stereochemistry and the conformation of the seven-membered exocyclic ring system. In the case of the porphyrin component the phase-sensitive COSY did not allow determination of the conformation, because of the presence of only one chiral center and the isochronous nature of key methylene protons of the seven-membered exocyclic ring. However, analysis of the COSY spectrum allowed a more complete assignment of the resonances of this component than had previously been possible using decoupling experiments on a structurally identical component that had been isolated from another sediment.20 In these examples the estimated amounts of chlorin and porphyrin isolated were between 50 and 100 pg.
S
m m m
4.36, 2.65, 2.40, 2.23 2.73, 2.40, 2.23 2.73, 2.65, 2.23 m 4.36, 2.73, 2.65, 2.40 m 1.35-1.25 4.60 (7) 3.91 (7) 3.72 S (7) (5.15, 4.50, 4.42) septet (7) 0.97: 0.79 m (br) 1.88 m (br) 0.70, 0.66 m (br) 1.42c m (br) d (7) 1.42 2 X d (7) 1.28-1.22 s (br) s (br)
= singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad. *Long-range couplings given in parentheses. Crosspeak detected using a lower threshold than shown in Figure 2. dP6, P8-Pl0, P12, P13. O s
Similarly, in conjunction with NOE difference spectroscopy, the application of phase-sensitive COSY to the analysis of a sedimentary chlorin (6) and its porphyrin counterpart (7) from a highly immature Pliocene clay allowed full assignmentlg of the spectra of the compounds,
Summary Since the earliest applications of NMR studies to the characterization of sedimentary tetrapyrroles, several developments have been important for the advancement of the ability to characterize these components, a prerequisite for understanding their origins. We regard the application of 2D COSY techniques in the determination of components with extensively coupled spin systems as a further advance, since they allow for a comprehensive analysis of spin-coupled networks in a single experiment, and avoid perturbation of closely resonating signals. In the case of sedimentary chlorins full structural assignments could greatly enhance our understanding of the processes involved in the degradative transformations of chlorophylls. Acknowledgment. We are grateful to the NERC for a studentship (B.J.K.), to the SERC for NMR instrumentation, and to British Petroleum plc for HPLC equipment. Dr. M. Murray is thanked for assistance with the NMR studies and for helpful discussions. (19) Keely, B. J.; Maxwell, J. R. J . Chem. Soc., Perkin Trans. 1. Submitted for publication. (20) Prowse, W. G.; Chicarelli, M. I.; Keely, B. J.; Kaur, S.; Maxwell, J. R. Geochim. Cosmochim. Acta 1987,51, 2875-2871.
