High molecular weight chlorins in a lacustrine shale

Org. Geochem.Vol. 17, No. 6. pp. 877-886, 1991 Printed in Great Britain. All rights reserved

0146-6380/91 $3.00+ 0.00 Copyright © 1991 PergamonPress pie

High molecular weight chlorins in a lacustrine shale W. G. PROWSE and J. R. MAXWELL Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 ITS, England (Received 27 September 1990; returned for revision 14 December 1990; accepted in revised form 4 February 1991)

Abstract--The major esterified tetrapyrrole in a Miocene oil shale (Marafi, Brazil) is a flee base chlorin, most likely pyrophaeophorbide a, esterified to 24-ethyl-4~-methyl-5~t(H)-cholestan-3fl-ol, the structure being assigned by FAB-MS and t H NMR, and by GC-MS analysis of the sterol liberated by hydrolysis. FAB-MS and electronic absorption spectrophotometric data for the second most abundant component are consistent with the mesopyrophaeophorbide counterpart. Only one other minor esterified chlofin was detected, implying a highly specific and, hence, biologically-mediated esterification. It is not clear whether the reaction took place during biosynthesis of an unknown precursor chlorophyll or resulted from esterification of a hydrolysis product(s) from a known chlorophyll (most likely chlorophyll a) after cellular disruption, although the second possibility is considered more likely, Key words---4-methyl steroidal chlorins, fast atom bombardment-mass spectrometry of chlorins, IH NMR spectra of chlorins

INTRODUCTION Since Treibs (1936) reported the occurrence of porphyrins (as metal complexes) in a range of sediments and oils, and proposed that those with an exocyclic alkano ring arose from chlorophyll a (1), over 50 structures have been rigorously identified (reviewed by Chicarelli et al., 1987). An origin from chlorophylls for such components is now widely held, although in a number of cases, the structures are insufficiently specific to assign them to a particular precursor chlorophyll. For example, the metal complexes (mainly Ni and/or vanadyl) of the most abundant sedimentary porphyrin, desoxophylloerythroaetioporphyrin (DPEP, 2; Quirke et al., 1980; Fookes, 1983; Ekstrom et al., 1983), could have arisen from chlorophyll a (1) and/or a number of other chlorophylls. In a few cases, it appears that transformation of chlorophylls Cl and c2 (3) has given rise to the metal complexes of 4 via a rearrangement involving the C-17 acrylic side-chain and the exocyclic ring (Ocampo et al., 1984, 1985a). Likewise, rearrangement of chlorophyll c3 (3; Fookes and Jeffrey, 1989) with removal of the C-7 carbomethoxy group, could yield the metal complex of 5 (VerneMismer et al., 1990). The chlorophylls c occur in a variety of the brown algal classes (e.g. dinoflagellates, diatoms and chrysophytes; Jeffrey, 1980), so an algal source for the sedimentary components can be ascribed, although the specific type of source organism is difficult to deduce. There is, however, one example where the type of source organism has been assigned on structural comparison grounds. The metal corn-

plexes of the porphyrin acids (6), having extended alkylation at C-8 and C-12, appear to have arisen from the analogous bacteriochlorophylls d (7; Ocampo et al., 1985b), which occur only in green photosynthetic bacteria (Chlorobacteriaceae). More recently, comparison of the 613C values of components whose structures can be assigned to a specific type of chlorophyll, with the 613C values of cooccurring components with less specific structures, has provided information about the origins (algal or bacterial) of the latter (e.g. Hayes et al., 1987). A chlorophyll origin for most of the petroporphyrins identified to date suggests that functionalized "intermediate" compounds should occur in sediments. Such compounds might contain more "source" information, by way of characteristic functional groups or other structural features, which may be lost on full defunctionalization to alkyl porphyrins. Recent studies in this laboratory have involved the characterization of functionalized tetrapyrroles in immature sediments (e.g. Keely et al., 1990). The lacustrine organic-rich Marafi shale (Bahia state, Brazil) is one of the samples Treibs (1936) used to demonstrate the occurrence of chlorophyll derivatives in sediments. Details of the geology of the surrounding area and of the biological marker content have been discussed by Cardoso and Chicarelli (1983). We report here the isolation from this Miocene shale of two related chlorins esterfied with a sterol, and suggest an algal origin for these components.

877

W. G. I~OWSEand J. R. MAXWELL

878

R2

..,.o

0==~

OMe OPhytyl cI

1

c2 c3

RI

R2

R 1 = Me R 2 = Et R 1 = Me R 2 = vinyl R 1 = CO2Me R 2 =' vinyl

HO

R

4

5

R 3 = Me R 1 = Et R 2 = M e R 3 = C H 2 C O O H R I = Et, H R 2 = H R 3 -- Me R 1 = Et, H R 2 = M e

6

7

R = Et, Pr, iBu

OFarnesyl R = Et, Pr, iBu

R1

R

HO~

13

O

8

R = vinyl

9

R = Et

f

"",

10 11 12

R 1 = vinyl R 2 = H R 1 = Et R 2 = H R 1 = vinyl R 2 = phytyl

Scheme 1 EXPERIMENTAL

Isolation o f chlorin esters

The sediment (500 g) was ground by mortar and pestle and aliquots (c. 50 g) extracted by sonication (Mettler Electronics, ME4.6 Ultrasonic tank; 5min) in acetone (5 x 200ml each) followed by

centrifugation (10,000 rpm; 10 min). The combined dark green extracts were concentrated (to c. 2 ml) and subjected to gel permeation chromatography to give only one chlorin-containing fraction, which was methylated (CH2N2). Chiorins 8 and 9 were isolated by normal phase HPLC on a semipreparative scale, and were separated from each

879

High molecular weight chlorins in a lacustrine shale other using reversed phase HPLC on an analytical scale.

Hydrolysis of 8

of Repeta (1982). A Polymer Laboratories PL-Gel 50A column (600 x 7.5 mm i.d.) was used, eluting with CHz C12 (2 ml min- 1).

HCI (6 N) was added dropwise to a solution (4 ml) of 8 in diethyl ether until the pigment was transferred to the aqueous layer (c. 4 ml HCI). The ethereal layer was removed and the aqueous layer extracted with diethyl ether (3 x 4ml). The combined ether solutions were washed with water (6 x 10ml) and the solvent removed under reduced pressure. Prior to G C - M S analysis the product was derivatized by treatment with BSTFA (20°C, overnight).

Fast

High performance liquid chromatography (HPLC)

1H N M R spectra were recorded at 400 MHz on a Jeol GX400 instrument in d6-acetone and were referenced to the chemical shift of the solvent (2.05 ppm).

Normal phase HPLC was carried out on an analytical scale according to the method of Barwise et al. (1986), with monitoring at 400 nm. Normal phase HPLC on a semi-preparative scale was carried out using isocratic elution: Spherisorb S5W column (250 × 10mm i.d.); 25% A (CH2Cl2:acetone 4:1), 25% B (1% pyridine in hexane), 50% C (1% acetic acid in hexane); flow rate 3 ml min- 1. Reversed phase HPLC employed a Phase Separations $ 5 0 D S 2 analytical column (150 x 4.6 mm i.d.), and elution with 15% methanol in acetone (flow rate 1.5ml min-X).

Gel permeation chromatography (GPC) GPC was carried out with the HPLC instrumentation using a modification (Keely, 1989) of the method

atom

bombardment-mass

spectrometry

(FAB-MS) Spectra were recorded on a Finnigan TSQ 70 spectrometer, using a nitrobenzyl alcohol (NBA) matrix, and bombardment with a Xenon atom beam provided by a Ion Tech FAB gun operating at 8 keV with a current of 1 mA. The spectrometer was repeatedly scanned from m/z 100 to 1000 in 2 s.

IH Nuclear magnetic resonance (NMR) spectrocopy

Gas chromatography (GC) GC was carried out using a Carlo Erba Mega Series 5160 chromatograph equipped with an on-column injector and a DB-1701 column (60 m x 0.32 mm i.d.). Hydrogen was employed as carrier gas and the following temperature programme employed: 50-150°C at 10°C min i and 150-300°C at 4°C min ~.

Gas chromatography-mass spectrometry (GC-MS) G C - M S analyses were performed using a Varian 3400 gas chromatograph directly coupled to a Finigan TSQ 70 mass spectrometer and an OV-1

R R

......

OMa

/

I-Z Ill

F-

z

.

.

.

.

.

.2 0 .

.

. . . ' TIME (min)

.

. 4'0.

.

.

'

. . . .

Fig. 1. Normal phase analytical HPLC trace of the methylated extract from Marafi oil shale.

I

60

880

W.G. PROWSEand J. R. MAXWELL

column (50 m x 0.32 mm i.d.) with helium as carrier gas. The spectrometer was operated in the electron ionization (El) mode (electron energy 70 eV) with a scan (m/z 50-600) time of 1.0 s.

Electronic absorption speetrophotometry Electronic absorption spectra were recorded in acetone with a Perkin-Elmer 552 spectrophotometer using a scan speed of 120nm min -~, slit width of 2 mm, and cells of 1 cm path length. Spectral data for 8 and 9 were as follows: 8, 2max(rel. int.): 661 (40), 602 (8), 529 (8), 498 (12), 403 (100). 9, )'max(rel. int.): 651 (38), 595 (7), 526 (7), 494 (10), 401 (100). RESULTS

Figure 1 shows the normal phase HPLC distribution of tetrapyrroles in the methylated extract.

Apart from the polynuclear aromatic hydrocarbon perylene (identified after isolation by its electronic absorption spectrum and EI mass spectrum), two major peaks are apparent, the later eluting one being a mixture of two chlorins (as methyl esters), mesopyrophaeophorbide a (10) and pyrophaeophorbide a (11; Keely et al., 1990). The earlier eluting peak (c. 34 min) contains the esterified chlorins. These were isolated and separated by HPLC to give 8 and 9, and a third unidentified minor component. The electronic absorption spectrum of 8 (see Experimental) is typical (e.g. Keely, 1989) of a phaeophytin or pyrophaeophytin type of chlorin, whilst that of 9 shows a hypsochromic shift of l0 nm in the major visible band, as would be expected for the meso (C-3 ethyl) counterpart of 8. The FAB mass spectrum of 8 [Fig. 2(A)] shows M .+ and [M + H] + recorded at m/z 946.7 (40%) and 947.7 (40%) respectively; the

f 535

A)

43~

1

537

B) 463

o 449

948.7 949-7

35

lJ,,, ,

400

,

,

. . . .

I

. . . .

i

. . . .

I

. . . .

,

. . . .

I

. . . .

6OO

i

. . . .

I

. . . .

i

. . . .

I

8O0 m/z

Fig. 2. FAB mass spectra (NBA matrix) of: (A) 8, (B) 9.

. . . .

....

High molecular weight chlorins in a lacustrine shale molecular weight (946.7) also corresponds to a non integer value, mainly because of the mass defect arising from the large number of hydrogens. The base peak occurs at m/z 535, with other significant fragment ions at m/z 461,447 and 433. These ions have the same m/z values as those reported (Keely et al., 1988) for pyrophaeophytin a (12), for which MS/MS experiments subsequently showed (Keely and Maxwell, 1990) that m/z 535 (i.e. [M + H - 2 7 8 ] +) arises via loss of the esterifying phytyl chain as phytadiene (C20H38) in a rearrangement process. Hence, in the present case, m/z 535 may be assigned as loss of C30H52 from [M + H] +, suggesting that the esterifying group is a saturated sterol. The spectrum of 9 [Fig. 2(B)] reflects the replacement of the C-3 vinyl in 8 by a C-3 ethyl substituent, with a shift of + 2 Da in M .+ and [M + H] +, and all the significant fragment ions. The FAB spectrum of the third component (not shown) is very similar to that of pyrophaeophytin a (12; Keely et al., 1988), with [M + H] + at m/z 813 (base peak) and the same major fragment ions as 8. It did not, however, co-elute with a standard of pyrophaeophytin a on normal phase HPLC. It appears, therefore, that this esterified chlofin is closely related to 12, but with the esterifying alcohol being an isomer of phytol. The ~H N M R spectrum of 8 (Fig. 3 and Table l) reveals the presence of 3 meso protons (one of which resonates ¢. 1 ppm to lower frequency, indicating that

881

Table 1. 400 MHz I H NMR data for 8 recorded in dr-acetone Proton

6 ppm

Multiplicity (J Hz)

H-10 H-5 H-20 H-31 H-32 H-32" H-132 H-I 32' H-18 H- 17 CH:-81 CH3-121 CH3-21 CH3-71 CH3-181 CH3-82 NH

9.82 9.58 8.94 8.24 6.40 6.22 5.31 5.14 4.68 4.48 3.78 3.67 3.49 3.30 1.86 1.71 -1.76

s s s dd(12, 18) dd(18, 1) dd(12, l) d(20) d(20) dq(7, 2) m(2) q(8) s s s d(7) t(8) s

Coupled (6 ppm)

nOe" (6 ppm)

6.22, 6.40 8.24, 6.22 8.24, 6.40 5.14 5.3 l 1.86, 4.48 4.68 1.71

9.58

9.82, 1.71 9.82 8.94 9.58

4.68 3.78

a, Enhancement observed when 6 signal irradiated; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.

it is adjacent to the pyrroline ring), 3r-methyls, lfl-ethyl, lfl-vinyl, and CH2-132 protons, a doublet (1.86 ppm) assigned as the C-18 methyl substituent through decoupling, and one of the NH protons (the other being obscured by the resonances between 0 and 1 ppm). The trans relationship of the C-17 and C-18 substituents was deduced from the coupling constant (2 Hz) between H-17 and H-18 (Closs et aL, 1963). Below 2.8 ppm the spectrum contains an abundance of unassigned resonances, which presumably

CH382

~

8

-~

H20

S

tf+ , J,

i:H

H-10

H-5

H-~20

CH2-132

~

"'l NH

a (ppm)

Fig. 3. 400 MHz mH NMR spectrum of 8 recorded in d0-acetone. Arrows indicate nOe enhancements observed. S, residual solvent resonance.

882

W.G. PROWSEand J. R. MAXWELL

arise from impurities, and protons on the C-17 side-chain and sterol group. Coupling connectivities in the simple spin systems were analysed by selected decoupling experiments, whilst the relative substitution pattern around the macrocycle was investigated by nuclear Overhauser effect (nOe) experiments (summarized in Fig. 3 and Table 1). Although the low amount of material restricted the number of possible nOe experiments, the experiments carried out assign unambiguously the resonance of the CH3 group adjacent to the low frequency meso proton at C-20. They also show that the other two meso protons are flanked by a vinyl and a methyl substituent, and an ethyl and a methyl substituent, respectively. Furthermore, the chemical shift of CH3-21 (3.49ppm) strongly indicates that it is adjacent to the vinyl group, rather than to an alkyl substituent from comparison with the spectra of a range of pyro- and mesopyrochlorins (Keely, 1989). The only ambiguity lies, therefore, in the order of the substituents at C-8 and C-12, the structure shown in Fig. 3 being favoured from structural comparisons with known chlorophylls, none of which possess a CH3 substituent at C-8. Resonances appropriate to the sterol could not be assigned; however, the IH double triplet

IRIC

(3.92 ppm) may be tentatively assigned as the proton on the steroidal C-O carbon of the ester linkage. The nature of the ester side-chain was determined by examination of the alcohol (as the TMSi ether) produced on acid hydrolysis. The GC-MS traces (Fig. 4) show one major peak displaying the expected molecular ion in the mass chromatogram of 502 (corresponding to C30H53OSiMe3). The EI mass spectrum (Fig. 4) suggests a 4-methyl sterol on comparison with spectra (Fig. 5) of authentic standards (TMSi ethers) of 4~,23R,24R-trimethyl-5~(H)cholestan-3fl-ol (dinostanol, 13) and 24S-ethyl-4~methyl-Sct(H)-cholestan-3fl-ol (14). The spectra of the standards are similar to that of the hydrolysis product; however, the spectrum of 13 contains a greatly enhanced m / z 98 rearrangement ion compared to 14, consistent with the presence of 23,24dimethyl rather than 24-ethyl substitution (cf. Zielinski et al., 1983; Volkman et al., 1984; Goodwin et al., 1988), and a more intense ion at m / z 261 whose origin presumably involves fragmentation in the side chain. Hence, the spectrum of the hydrolysis product indicates the 24-ethyl component. This assignment was confirmed by GC co-injection studies on the semi-polar DB-1701 phase; the two standards were

1.,~

,

. . . . . .

tm/z 502

20 .......

,

3'0 ' 95

¢/

I

'

TIME (min) 4O

'

I

5O

~ss

I

l .si

- It.L1 130

.,u=..

100

373

229

l,. L,L., ,. j ....

300 m/z

412

[

,h

500

Fig. 4. Partial GC-MS traces (reconstructed ion chromatogram and m/z 502 chromatogram) and EI mass spectrum of hydrolysis product of 8 (TMSi ether). For conditions see text.

883

High molecular weight chlorins in a lacustrine shale

95

a) j

....

// ;ll.I I

/

TMSO

°

!

261

229 I lO0

373 ,~2 300

5OO

95

b,

I/

/

/ / / TMSO

. . . . . .

, ....

,

...

100

300

500

m/z

Fig. 5. El mass spectra of synthetic standards: (a) 4~,23R,24R-trimethyl-5~(H)-cholestan-38-ol (13), (b) 24S-ethyl-4~-methyl-5~(H)-cholestan-3/~-ol (14). Both as TMSi ethers. For conditions see text. separated almost to baseline and 14, which had the shorter retention time, co-eluted with the hydrolysis product. The C-24 stereochemistry cannot be defined, as the 24S and 24R isomers are reported to co-elute under such conditions (Robinson et aL, 1989). Insufficient amounts of the two minor components were available for such a detailed analysis but the structure of one of these (9) was tentatively assigned on the basis of the F A B - M S results above. DISCUSSION

The occurrence of the high molecular weight chlofins 8 and 9, with a sterol esterifying moiety, as the two most abundant chlorin esters in the shale is remarkable and unexpected since no chlorophylls with such an esterifying alcohol have been reported (see below). One possibility which was considered is

that they are artifacts, and that esterification of the two most abundant chlorin acids (10 and I1) occurred during extraction or isolation. We believe that this is unlikely since (i) the extract was obtained by ultrasonic extraction in acetone at room temperature, (ii) the components were present in a small aliquot of the extract which was methylated immediately and analysed by HPLC and (iii) the esters occur in a ratio of c. 4:1 whilst the co-occurring acids are present in approximately equal amounts. With one exception, 4-methylsterols have been identified, usually in high relative abundance, in all species of dinoflagellate algae in which sterols have been sought (Withers, 1983; Robinson et al., 1987 and references therein), and their presence in immature marine sediments has been used as an indicator of dinoflagellate inputs (Boon et al., 1979; Brassell and Eglinton, 1983; de Leeuw et al., 1983).

884

W.G. PROWSEand J. R. MAXWELL

In particular, among the C30 components, those with the 4,23,24-trimethyl (dinosteryl) skeleton (e.g. 13) are considered as dinoflagellate markers and the presence of 4-methyl dinosteranes in sedimentary rocks is also taken as evidence of a dinoflagellate input (Summons et al., 1987; Goodwin et al., 1988; Thomas, 1990). There appears to be, however, a difference between the C30 4-methyl sterane distributions in marine and lacustrine freshwater sediments, in that the former contain both dinosteranes and their 24-ethyl counterparts, whereas the latter are almost totally dominated by the 24-ethyl components. Although it appears that dinoflagellates are the major source of sedimentary C30 4,23,24-trimethyl steroids, the evidence for the origin of the 24-ethyl compounds is less certain. Recent results (N. Robinson, personal communication) have, however, shown that 14 (and not 13 as reported previously; Robinson et al., 1987) is a major sterol of the freshwater dinoflagellate Peridinium lomnickii and the underlying sediments of a lake from which the alga was obtained. It is also the major sterol of the lacustrine freshwater Messel shale, where it was ascribed to a dinoflagellate source (Robinson et al., 1989). Component 14 has been identified in two marine dinoflagellates (Withers, 1983; Bohlin et al., 1981). It has, however, along with its A22 unsaturated counterpart, also been reported in marine prymnesiophytes of the genus Pavlova (Ballantine et al., 1979; Volkman et al., 1990), and unidentified C30 24-ethyl4-methyl sterols have been detected in a marine diatom community (Nichols et al., 1990). Hence, based on the available evidence the esterifying sterol (14) in the pigments 9 and 10 may have a dinoflagellate source, since the Mara6 shale is a freshwater sediment. Other algal sources cannot, however, be discounted. The major chlorophyll reported in dinoflagellates is chlorophyll a (1) with lesser amounts of chlorophyll c2, and both occur in prymnesiophytes and diatoms along with chlorophyll c I (Jeffrey, 1980). However, the chlorophylls c are porphyrins. Furthermore, comparison of the major chlorin acids 10 and 11 (and of the same chlorin moieties in 8 and 9) with chlorophyll c and with the chlorin nucleus of chlorophyll a indicates that less structural modification would be required in the latter case to obtain the chlorins. Loss of the Mg ligand and of the C-132 carbomethoxy substituent can occur in the water column or during the very earliest stage of diagenesis (Keely et al., 1988), but chlorophylls c would also require reduction of the double bond in the C-17 acrylic chain and of the C-8 vinyl substituent for chlorophyll c2. Three possibilities can be considered for the esterification reaction, which gave rise to the high molecular weight chlorins: (i) It was a chemical reaction which occurred during sediment diagenesis. However, the fact that there is only one esterifying alcohol in the major

components argues against this and implies a high degree of specificity in the esterification. (ii) The reaction occurred during biosynthesis of an unknown chlorophyll. Esterification of the C-17 propionic acid group is the final step in chlorophyll biosynthesis, and is thought to be catalysed by the enzyme chlorophyll synthetase (Leeper, 1985 and references therein). To date the only esterifying groups identified in chlorophylls (reviewed by Svec, 1978) are the three terpene alcohols, phytol (C20), geranylgeraniol (C20) and farnesol (C15), and a straight chain aliphatic C~5 alcohol in a bacteriochlorophyll c (Gloe and Risch, 1978). In the eucaryote algae chlorophylls are located within the thylakoid system, complexed to proteins and organized in photosynthetic units (Jeffrey, 1980). If 8 and 9 do represent derivatives of a sterol-containing chlorophyll, the presence of such a bulky hydrophobic group would be expected to influence markedly the chlorophyll-protein interactions or in vivo aggregation in the light harvesting complexes. Such changes would alter the photosynthetic light absorption capabilities of the organism(s), perhaps as a physiological response to light availability (cf. Smith and Bobe, 1987). (iii) It was biologically mediated and occurred between a chlorophyll derivative (such as 10) and the sterol. In this case, it can be envisaged that the esterification occurred after cellular disruption caused by senescence, decay or herbivory, which are known to degrade chlorophylls by loss of magnesium and decarbomethoxylation, and to form free acids by ester hydrolysis (Owens and Falkowski, 1982; Scoch et al., 1981; Shuman and Lorenzen, 1975 and references therein). However, in the case of dinoflagellates, active chlorophyllase enzyme systems have not been observed to date (Jeffrey and Hallegraeff, 1987). CONCLUSIONS The two major esterified chlorins isolated from the lacustrine Marafi shale of Miocene age are pyrophaeophorbide a and mesopryrophaeophorbide a esterified with 24-ethyl-4~-methylcholestan-3fl-ol. The structure and specifcity of the sterol suggests a specific source for it and perhaps, therefore, for the associated chiorins. The esterification appears to have been a biological reaction. Whether it occurred during biosynthesis of an unknown chlorophyll or after cellular disruption is not clear at present, although in the absence of other evidence we consider the latter possibility more likely, with the chlorin macrocycle originating from chlorophyll a. Acknowledgements--We thank the SERC for a studentship

(W.G.P.), the NERC for MS facilities (GR3/2951 & GR3/3758), Dr M. I. Chicarelli for a sample of the Marafi shale and Professor C. Djerassi for sterol standards. We thank Dr B. J. Keely for valuable discussions and for a sample of pyrophaeophytin a, and Mr J. F. Carter for skilled mass spectrometric technical assistance.

High molecular weight chlorins in a lacustrine shale REFERENCES

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