Note
A novel sedimentary chlorin: structural evidence for a
chlorophyll origin for aetioporphyrins
Ruth L. Airs, Chi Jie, Brendan J. Keely *
Department of Chemistry, University of York, Heslington, York, YO10 5DD, UKReceived 31 July 2000; accepted 12 September 2000 (returned to author for revision 25 August 2000)
Abstract
A novel sedimentary transformation product of chlorophyll, purpurin-7 phytyl ester, has been identi®ed by atmo-spheric pressure chemical ionisation liquid chromatography mass-spectrometry in sediments from Lake Baikal, Russia, Loch Ness and Priest Pot, UK. This product of oxidative transformation is an intermediate on the transformation pathway leading to sedimentary aetioporphyrins and its occurrence lends strong support to the view that aetiopor-phyrins derive mainly from precursor chlorophylls.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Tetrapyrroles; Chlorins; Purpurins; Chlorophyll transformation; Oxidation; Sedimentary pigments; Aetioporphyrins; Priest Pot; Lake Baikal
1. Introduction
The identi®cation of two tetrapyrroles based on the purpurin-18 macrocycle in sediments from Lake Baikal and Loch Ness demonstrated the generation of oxidation products of chlorophylls early in the diagenetic fate of chlorophylls (Naylor and Keely, 1998). Subsequent to our initial report, purpurin-18 phytyl ester (1) has also been reported in a variety of other sedimentary environ-ments (Ocampo and Repeta, 1999) and in organisms grown in culture (Louda and Baker, 1998). The wide-spread occurrence of chlorophyll derivatives possessing the six-membered anhydride ring-E structural feature indicates that chlorophyll oxidation, involving reaction centred on ring-E (type I reactions according to Brown et al., 1991), occurs readily in the natural environment and is probably involved in the formation of many aetiopor-phyrin structures (Naylor and Keely, 1998, 1999). We describe here the identi®cation of a chlorophyll oxidation product, modi®ed in ring-E, that has not been reported previously in sediments.
2. Experimental
Frozen sediments (c. 10±15 g) from Lake Baikal, Russia (central basin), and Priest Pot, UK (55 cm depth from a core analysed previously; Keely and Brereton, 1986) were extracted as described previously (Naylor and Keely, 1998). The extracts were analysed by high perfor-mance liquid chromatography (HPLC) and atmospheric pressure chemical ionisation liquid chromatography± mass spectrometry (APCI LC±MS) as described in Airs and Keely (2000). A standard of purpurin-7 dimethyl phytyl ester (2), as the magnesium complex, was isolated preparatively from the products of the allomerisation of chlorophylla(Woolley et al., 1998a) and characterised by electronic absorption spectroscopy (UV/vis), APCI LC±MS and 1H NMR spectroscopy before and after
demetallation.
2.1. HPLC
HPLC was carried out using a system described pre-viously (Airs and Keely, 2000) and employing a gradient solvent programme comprising acetonitrile, water, methanol and ethyl acetate at a ¯ow rate of 0.7 cm3minÿ1
(Atkinson, 1996).
0146-6380/00/$ - see front matter#2000 Elsevier Science Ltd. All rights reserved. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 1 3 7 - 6
Organic Geochemistry 31 (2000) 1253±1256
www.elsevier.nl/locate/orggeochem
2.2. APCI LC±MS
APCI LC±MS was performed in the positive ion mode using conditions described previously (Airs and Keely, 2000). The interface conditions were: vaporiser 450C; capillary 150C; discharge current 5 mA, sheath
gas ¯ow 60 arbitrary units; auxiliary gas o. HPLC conditions were as described above.
3. Results and discussion
The major components in the pigment distribution from Priest Pot sediment were assigned previously from on-line UV/vis spectra and negative-ion thermospray LC±MS analysis of fractions isolated by gel permeation chromatography (Eckardt et al., 1991). In re-examining the distribution using an improved HPLC method
(Atkinson, 1996), we noted the presence of a minor component eluting between hydroxyphaeophytin a (3) and phaeophytina(4) in the 400 nm HPLC chromato-gram and exhibiting a UV/vis spectrum characteristic of tetrapyrroles that lack the cyclopentanone ring system (ring-E) of chlorophyll. As a consequence of the reduc-tion in ring strain arising from disrupreduc-tion of ring-E, such components do not exhibit satellite bands at wavelengths to the blue of the Soret band (Woolley et al., 1998b). The same component was also observed in sediment extracts from Loch Ness and Lake Baikal (Fig. 1a). Mass chromatography of the extract from Lake Baikal con®rmed the component as that having MH+
atm/z917 and distinguished it from that with MH+at
m/z 956, eluting slightly later and exhibiting stronger response in the base peak chromatogram (Fig. 1b). The protonated molecule atm/z917 (Fig. 2), observed in all three sediment extracts, corresponds to purpurin-7 phy-tyl dimethyl ester (2). The resonance excitation-induced MS spectra fromm/z917 (MS2; Fig. 2) and its product
ion atm/z 551 (MS3; not shown) gave identical ions for
the compound in all sediment extracts. The fragment ions in MS2 (Fig. 2b) are assigned formally as arising
from loss of methanol (m/z885), loss of the 13 or C-15 CO2Me with hydrogen abstraction (m/z857), loss of
the C-15 COCO2Me (m/z830), loss of the phytyl ester
with back transfer of a proton (m/z 639), subsequent loss of methanol (m/z607), and loss of both the phytyl and C-15 substituents (m/z551). The ions observed in MS3and their abundances correlate closely with those
seen for the standard. These include losses of methanol (m/z519) followed by expulsion of water (m/z501) or CO2 (m/z 475), and loss of the remaining C-17
sub-stituent as propenoic acid with back transfer of a proton (m/z 479). Co-elution with a standard con®rmed the identi®cation of this ring-opened chlorophyll derivative as purpurin-7 dimethyl phytyl ester (2). The improved sensitivity aorded by MS detection (Airs and Keely, 2000) also revealed the presence of minor amounts of purpurin-18 phytyl ester (1) in Priest Pot sediment (cf. Naylor and Keely, 1998).
The identi®cation of purpurin-7 phytyl ester (as the dimethyl ester) represents the ®rst unambiguous char-acterisation of a ring-opened oxidative transformation product of chlorophyll in sediments. As discussed in our initial report on chlorophyll oxidation products in Loch Ness and Lake Baikal (Naylor and Keely, 1998), earlier reports of ring-opened chlorophyll derivatives relied on partial characterisation in crude fractions, and extrac-tion procedures that may have led to oxidaextrac-tion. Fur-thermore, purpurin-7 phytyl ester was not among the structures reported in the earlier studies.
By analogy with the formation of cycloalk-anoporphyrins from chlorophyll derivatives such as phaeophytina(4) and pyrophaeophytina(5; Keely et al., 1990), dehydration and reduction of OH-phaeophytina (3), present in the sediments examined, may also yield porphyrin products in which ring E survives. By con-trast, purpurin-18 and other compounds that contain anhydride-type ring-E systems would be expected to yield aetioporphyrinsviaa combination of (formally base-cat-alysed) hydrolytic reactions centred on the anhydride ring followed by defunctionalisation (Naylor and Keely, 1998; 1999). Notably, however, because of its ring-opened structure, purpurin-7 phytyl ester may be assumed to be a direct precursor of aetioporphyrins via the normal
Fig. 2. APCI MS spectra of purpurin-7 phytyl dimethyl ester (2) obtained during on-line LC±MS analysis of Lake Baikal sediment extract. (a) MS and (b) MS2fromm/z917.
defunctionalisation reactions involved in the Treibs scheme. Thus, the presence of purpurin-7 and purpurin-18 type structures attests to the oxidative ring-opening of chlorophylls in the natural environment and lends ®rm support to the view extolled by Corwin (1960) that aetio-porphyrins must, for the most part, derive from cleavage of ring-E of chlorophylls. Further details, including rationalisation of the mechanistic detail of chlorophyll oxidation reactions, will be presented elsewhere.
4. Conclusions
Chlorophyll oxidation products feature as minor components in a number of sedimentary environments. Purpurin-7 phytyl ester represents a signi®cant compo-nent of the chlorophyll oxidation products in three lake sediments where it is accompanied by allomer and anhydride-type oxidation products. The occurrence in sediments of purpurin-7 phytyl ester as a ring-opened pigment makes it a strong candidate as a precursor for sedimentary aetioporphyrins.
Acknowledgements
The EPSRC is thanked for provision of studentship support for R.L.A.
Associate EditorÐA.G. Douglas
References
Airs, R.L., Keely, B.J., 2000. A novel approach for sensitivity enhancement in atmospheric pressure chemical ionisation
liquid chromatography/mass spectrometry of chlorophylls. Rapid Communications in Mass Spectrometry 14, 125±128. Atkinson, J.E., 1996. Pigment Analysis and the Recognition of
Bacterial Inputs to Sediments. MSc thesis, University of York.
Brown, S.B., Houghton J.D., Hendry G.A.F., 1991. Chlor-ophyll breakdown. In: Scheer, H. (Ed.), ChlorChlor-ophylls. CRC Press, pp. 465±489.
Corwin, A.H., 1960. Petroporphyrins. In: Proceedings of the 5th World Petroleum Congress, pp. 119±129.
Eckardt, C.B., Keely, B.J., Waring, J.R., Chicarelli, M.I., Maxwell, J.R., 1991. Preservation of chlorophyll-derived pigments in sedimentary organic matter. Philosophical Transactions of the Royal Society, London B 333, 339±348. Keely, B.J., Brereton, R.G., 1986. Early chlorin diagenesis in a
recent aquatic sediment. Organic Geochemistry 10, 975±980. Keely, B.J., Prowse, W.G., Maxwell, J.R., 1990. The Treibs Hypothesis: an evaluation based on structural studies. Energy and Fuels 4, 628±634.
Louda, J.W., Baker, E.W., 1998. Chlorophyll-a degradation during cellular senescence and death. Organic Geochemistry 29, 1233±1251.
Naylor, C.C., Keely, B.J., 1998. Sedimentary purpurins: oxi-dative transformation products of chlorophylls. Organic Geochemistry 28, 417±422.
Naylor, C.C., Keely, B.J., 1999. Sedimentary purpurins: oxi-dative transformation products of chlorophylls. Organic Geochemistry 30, 471±472.
Ocampo, R., Repeta, D.J., 1999. Structural determination of purpurin-18 (as methyl ester) from sedimentary organic matter. Organic Geochemistry 30, 189±193.
Woolley, P.S., Moir, A.J., Hester, R.E., Keely, B.J., 1998a. A comparative study of the allomerization reaction of chlor-ophyllaand bacteriochlorophylla. Journal of the Chemical Society Perkin Transactions 2, 1833±1839.
Woolley, P.S., Keely, B.J., Hester, R.E., 1998b. Chlorophyll and phaeophytin derivatives in geochemical transformation pathways: a surface-enhanced resonance Raman spectro-scopic study. Biospectroscopy 4, 147±159.
