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1978 845 Pyrroles and Related Compounds. Part 392 Structural and Biosyn-thetic Studies of the Chlorobiurn Chlorophylls-660 (Bacteriochloro-phylls c). Incorporations of Methionine and Porphobilinogen By George W. Kenner,' John Rimmer, Kevin M.Smith, and John F. Unsworth, The Robert Robinson Laboratories, University of Liverpool, P.O. Box 147, Liverpool L69 3BX Treatment of the phaeofarnesins (4) isolated from the Chlorobium chlorophylls-660 (ex. Chloropseudomonas ethylicum) with sulphuric acid in methanol gives the methyl phaeophorbides (1) which can be dehydrated by re- fluxing in benzene in the presence of acid to afford the 2-vinyl derivatives (2) ; prolonged refluxing gives the novel cyclised compounds (6). By-products from handling the pigments are the 8'-acetylbilitrienes (5).obtained by photo-oxygenation. [l4CH3]-, [l3CCH,J -, and [13C2H,] -L-methionine are efficiently incorporated into the Chlorobium chlorophylls-660, showing that the extra methylation present in the &methyl, 4-isobutyl, 4-n-propyl, and 5-ethyl groups of these pigments is methionine derived. Using 6-14C labelled material, porphobilinogen is also confirmed as an important building block in these bacterial chlorophylls. THEChlorobium chlorophylls-660 t (Figure 1) (recently renamed 3 as bacteriochlorophylls c) are a series of poly-OH H-/-Me Band R' R2 R3 yoof total 1 2 Bu Bu* Et Et Et(?)Me 0.5 0.5 3 4 Prn Prn Et Et Et(?)Me 2 16 5 Et Et Me 71 6 Et Me Me 10 1FIGURE Proposed 16 structures for Chlorobium chlorophylls-660 methylated chlorophylls derived from certain sulphur bacteria.A dispute has arisen over the precise struc- t The numbers 660 and 650 refer to the wavelength (in nm) of the maximum in the red region of the electronic absorption spectra of the pigments in ether solution. Part 38, G. W. Kenner, J. Rimmer, K. M. Smith, and J. F. Unsworth, J.C.S. Perkin I, 1977, 332. Part of this work was reported in the Royal Society Discus- sion Meeting on the Biosynthesis of Porphyrins, Chlorophyll, and Vitamin B,,, London, 1975; G. W. Kenner, J. Rimmer, K. M. Smith, and J. I?. Unsworth, Phil. Trans. Roy. SOC.,1976, 273B, 255. A. Jensen, 0. Aasmundrud, and K. E. Eimhjellen, Biochim. Biophys. Acta, 1964, 88, 466.M. B. AlIen, in ' The Chlorophylls,' eds. L. P. Vernon and G. R. Seely, Academic Press, New York, 1966, p. 516. A. S. Holt, in ' The Chemistry and Biochemistry of Plant Pigments,' ed. T. W. Goodwin, Academic Press, New York, 1965, p. 3, and references therein. V. N. Shaposhnikov, E. N. Kondrateva, and V. D. Fedorov, Nature, 1960, 187,167. tures of these chlorophylls, particularly with regard to the positioning and nature of the meso-alkyl substituent. The 650 t chlorophylls (bacteriochlorophylls d3), pro-duced 5 by Chiorobiurn thiosul$hatophilum strain L, lack the meso-alkyl substituent, but other strains of C. thiosulphatophiZum, as well as C. limicola and Chloro-Pseudomonas ethylicum (said to consist of C. limicola together with an unknown colourless non-photo-synthetic bacterium), all produce the Chlorobium chlorophylls-660. Degradative and synthetic *, work had clearly shown the meso-alkyl groups in the 660 series to be present in the &position, but an alternative positioning (either cc or p) was suggested by Mathewson et aZ.,1° who showed by n.m.r.spectroscopy that one of the two meso-protons was readily exchangeable with deuterium in acid; thus on the basis of previous work in the literature l1 it seemed reasonable to propose lothat the %proton (normally exchanged readily 11) was still present, and therefore that the alkyl substituent must be in the a-or p-position. On the basis of n.m.r. spectra, Holt et al.7 had already suggested 8-substitution because the highest field meso-proton l2 was missing in the Chlorobium phaeophorbides ; more recently, &sub-stitution has been conclusively confirmed by n.m.r.2713914 methods, and a rationalisation of the reasons for ex-change of the a-proton in 8-methylchlorins has been presented.2 In Part 19 9 we questioned the allocation of a 8-ethyl A. S. Holt and D. W. Hughes, J. Amer. Chem. Soc., 1961,83, 499; A. S. Holt and H. V. Morley, ibid., 1960, 82, 500; A. S. Holt, D. W. Hughes, H. J. Kende, and J. W. Purdie, ibid., 1962, 84, 2835; Plant Cell Physiol., 1963,4, 49; H. V. Morley and A. S. Holt, Canad. J. Chem., 1961, 39, 755. J. L. Archibald, D. M. Walker, K. B. Shaw, A. Markovac, and S. F. MacDonald, Canad. J. Chem., 1966,44, 345. M. T. Cox.A. H. Tackson. and G. W. Kenner. l. Chem. SOC. (C), 1971, 1974. lo T. W. Mathewson. W. R. Richards, and H. Rapoport, T. Arne; Chem. Soc., 1963; 85, 364; Biochemz. Biophys. R;s.-ComG., 1963, 18,1. R. B. Woodward and V. SkariC, J. Amcr. Ckem. SOC.,1961, 83, 4676. 12 For a review of porphyrin and chlorin n.m.r. spectroscopy, see H. Scheer and J. J. Katz, in ' Porphyrins and Metallo- porphyrins,' ed. K. M. Smith, Elsevier, Amsterdam, 1975, p. 399. 13 K. M. Smith and J. F. Unsworth, Tetrahedron, 1975,81,367. l4 H. Brockmann, jun., Phil. Trans. Roy. Sac., l976,273B, 277. group to bands 1 and 3 of the 660 chlorophylls, and there is no reason to expand on the stated arguments, or to alter our conclusions that structural assignments l5 featuring meso-ethyl groups are incorrect. In this paper we describe our work on the isolation and chemistry of the ChZorobium chlorophylls-660, and report on bio-synthetic experiments which, using radiochemical labels, establish methionine and porphobilinogen as primary building blocks.Structural Studies.-Initially, the source of Chloro-bium chlorophylls-660 used in the present work was Chlorobiumthiosulphatophilum (NCIB cat. no. 8346) , but after a short time this was replaced by Chloropseudo-moms ethylicum, a hardier anaerobe which produces the pigments much more prolifically. Attempts to isolate the chlorophylls themselves were thwarted by ready demetallation, so in order to facilitate the examination of products from feeding experiments, as well as to prepare the pigments for separation of the individual homo-logues,* it was decided to develop efficient degradative procedures to give the methyl phaeophorbides (1), methyl 2-vinylphaeophorbides (2) and methyl meso-phaeophorbides (3).R Me (2) R =CH=CH;! (3lR=Et '0 CO2 Farnesyl (41 Extraction of the growing cultures with acetone after filtration on Celite was found to be preferable to the methanol used by previous workers because smaller volumes were required. Partition between water and methylene chloride, followed by evaporation of the methylene chloride gave the phaeofarnesins (4). Mass spectrometry indicated the presence of a series of homo-logues with mass ions corresponding to the proposed l5 structures (Figure 1) with the exception of band 1. Characteristic losses in the spectra were 18 and 204 mass units due to dehydration and removal of the farnesyl ester respectively.The phaeofarnesins were trans-* Holt and his co-workers ' have reported band separations using distribution between hydrochloric acid and ether on Celite columns ; however, this separation is not completely efficient.16 Ail work described in the present paper was carried out on the intact mixture, the composition of which is shown in Figure 1. 15 A. S. Holt, J. W. Purdie, and J. W. F. Wasley, Canad. J. Chem., 1966, 44, 88. J.C.S. Perkin I esterified in 5% concentrated sulphuric acid in methanol to give the methyl phaeophorbides (1). In this pro- cedure ca.5% of the l-hydroxyethyl material was 0.D. -1 I II 11 300 400 500 600 Alnm FIGURE Electronic absorption spectrum (in CHC1,) of the2 total mixture of 8'-acetylbilitrienes (5) from C. ethylicum transformed into vinyl, but the compounds (1) and (2) were readily separable by column chromatography on alumina; lH and 13C n.m.r. spectroscopy confirmed the proposed structure for band 5 (Figure 1, major compo- nent). A characteristic feature of the t.1.c. of either the methyl (1) or farnesyl phaeophorbides (4)when elution was carried out with 30%acetone in light petroleum (b.p. 60-80') was resolution of a purple band which was more polar than the green-black phaeophorbide band. Pre-parative t.1.c. (elution with 5% tetrahydrofuran in methylene chloride) gave a clear separation and the purple material possessed an electronic absorption spec- trum (Figure 2) characteristic of an open-chain bile pigment or a macrocycle with interrupted conjugation.lH N.m.r. showed two high field methine protons, and the methyl group resonances were at higher field than those of the methyl phaeophorbides (1) attesting to the absence of a ring current.12 Mass spectrometry con- firmed that the purple pigment was a homologous mix- ture of 8'-acetylbilitrienes (5), derived presumably by photo-oxygenation at the 8-methyl-bearing methine OHH-Me Et ,Prn,Bu' Me,Et 602Me (5) position. Similar photo-oxygenations of magnesium porphyrins l7 and zinc chlorins have been reported] R. A. Chapman, M.W. Roomi, T. C. Morton, D. T. Kraj-carski, and S. F. MacDonald, Canad. J. Chem., 1971, 49, 3544. l7 J.-H. Fuhrhop and D. Mauzerall, Photochem. Photobiol., 1971, 13, 453. J.-F. Fuhrhop, P. K. W. Wasser, J. Subramanian, and U. Schrader, Annalen, 1974, 1450. 1978 and in the latter case both possible formylbilitrienes have been isolated. Moreover, we have synthesised l9 6-meth yl-trans-oc t ae thylchlorin and have obtained both possible acetylbilitrienes.20 However, in the case of the ChZorobium-660 methyl phaeophorbides only one isomer is obtained, namely that with the acetyl group attached to the reduced ring, and similar conclusions have been reached by Br0~kmann.l~ Oxygenation of methyl phaeophorbides in daylight during one week accom-plishes transformation into the acetylbilitrienes (5) in 71% yield.Hydroxyethylporphyrins can be transformed into vinylporphyrins by heating with toluene-9-sulphonic acid in benzene 21 or o-dichlorobenzene.22 Treatment of the methyl phaeophorbides (1) for 1 h in refluxing ben- zene containing toluene-p-sulphonic acid gave a 77% yield of the 2-vinyl product (2). However, more pro- longed heating in benzene and acid, followed by treat- ment with diazomethane to rectify some partial hydro- lysis of the methyl ester, gave a different product in 79% yield. The electronic absorption spectrum showed a hypsochromic (1 1 nm) shift of the long wavelength peak from that of the 2-vinyl compounds (2), and the mass spectrum featured the same mass ions as in (2).lH N.m.r. spectroscopy, however, showed only one meso- proton and no vinylic hydrogens, but some extra badly defined signals in the T 6-7 region were present. From this data it was apparent that cyclisation of the 2-vinyl group in (2)to the a-meso-position had occurred, and that the new products were the a-ethylenephaeophorbides (6). 13C N.m.r. spectra also confirmed this structural assignment. It is notable that when methyl pyro-phaeophorbide n (7) (obtained 23 by degradation of HCH2 n CH ,Prn, Bui C02Me COzMe (6) (7) phaeophytins) was subjected to the same reaction con- ditions no similar vinyl cyclisation was apparent ; this therefore establishes that the &methyl substituent is M. J.Bushell, B. Evans, G. W. Kenner, and K. M. Smith, Heterocycles, 1977, 7, 67. 2o M. J. Bushell, unpublished results. 21 P. S. Clezy and J. Barrett, Biochem. J., 1061, 78, 798. 22 G. W. Kenner, S. W. McCombie, and K. M. Smith, Annalen, 1973, 1329. 23 G. W. Kenner, S. W. McCombie, and K. M. Smith, J.C.S. Pevkin I, 1973, 2517. 24 E.g., J. Barrett, Natuve, 1959, 183, 1185; H. H. Inhoffen, H. Brockmann, jun., and K.-M. Bliesener, Annalen, 1969, 730, 173. essential for the reaction. Several explanations for this novel cyclisation can be advanced, and these range from purely electronic to mainly steric effects. For example, one could argue that the well established electron-donating capacity of the methyl group was raising the overall electron density in the macrocycle and that cyclisation with a primary carbonium ion obtained by protonation of the 2-vinyl (followed by loss of the proton originally on the a-carbon) would give (6); cyclisation of the more likely secondary carbonium ion would give a sterically impossible four-membered ring.In support of this type of electronic argument it has been noted l3 that change from CDC1, solvent to trifluoro- acetic acid in the carbon n.m.r. spectra of chlorins causes a downfield shift of the vinyl CH, of ca. 8 p.p.m. com- pared with no shift for the vinyl CH peak. On the other hand, steric effects of the &methyl group would cer-tainly be expected to distort the macrocycle so as to render the a-position susceptible to electrophilic attack.A final possibility is that protonation could occur at the &carbon, and this might be expected to relieve steric compression in that area of the molecule; a simple electrocyclic reaction, followed by deprotonation, would give the required product. Both the photo-oxidative ring cleavage to give the acetylbilitrienes (5),and the acid catalysed vinyl cyclis- ation to give (6), are important reactions from the structural standpoint. The presence of a 8’-acetyl group in (5) further confirms that the meso-methyl group is in the &position in the chlorophylls. The fact that the vinyl group in the 2-position can be cyclised to give (6) adds yet more evidence that the meso-methyl group is not present at the a-position. Possibly more important, compounds (6) no longer possess the photochemically labile 24 vinyl substituent, and their ready accessibility makes them useful as substrates upon which to attempt band separations without fear of decomposition during handling.The mesophaeophorbides (3), which are equally useful in this context, are also readily available by hydrogenation of the methyl 2-vinylphaeophorbides (2) (96% yield). Biosynthetic Studies .-Methionine. Though met hio- nine has been shown 25 to enhance the growth of Chloro-bium bacteria, and ethionine has been shown26 to be an inhibitor, no conclusive proof of the involvement of methionine (as S-adenosylmethionine) in the biosyn- thesis of the Chlorobium chlorophylls using modern labelling techniques has been gathered.[14CH3]- and [13CH3]-~-methionine were prepared 27 from L-homo-cystine using sodium in liquid ammonia, and the appro- priat ely labelled methyl iodide. [14CH3]-L-Methionine was administered to C. ethylicum using a variety of 25 V. E. Uspenskaya and E. N. Kondrateva, Doklady Akad. Nauk S.S.S.R.,1964,157, 678; T. N. Godnev, E. N. Kondrateva, and V. E. Uspenskaya, Izv. Akad. Nauk S.S.S.R. (Ser.Biol), 1966, 31, 525. See also ref. 26. 28 W. R. Richards and H. Rapoport, Biochemistry, 1966, 5, 1079; 1967, 6, 3830. 21 V. du Vigneaud, H. M. Dyer, and J. Harmon, J. Biol. Chem., 1933, 101, 719. methods. Feeding to growing cultures, whether at the time of subculturing or batchwise during growth (and particularly during the exponential region of a regularly measured time-growth curve), gave reproducible incor- A 150 100 50 0 6 FIGURE 13C N.1n.r.spectrum (Varian XL-100, proton dc-3 coupled) of the total mixture of methyl phaeophorbides (1) from C. ethylicum: A, obtained from feeding [13CCH,]-~-methionine; B, at natural abundance porations into the farnesyl phaeopliorbides (4)of ca. 9”,. Incubations with concentrated cell suspensions to which lysozyme had been added (to disrupt the cell wall) also gave 9% incorporations, but disruption of the cell wall by freeze-thawing before feeding gave a decreased incorporation of only 3%. A blank experiment was performed involving administration of [14CH,]-~-niethio- nine to a solution of phaeofarnesins, quinones, and caro- tenoids from C.ethyliczm; an ‘ incorporation ’ of 0.00576 gave an indication of the level of cross contamination and helped to confirm the incorporations laid out above. Encouraged by the high radiochemical incorporations, we decided to feed [13CH,]-~-methionine to growing cultures, this being the most straightforward method as well as the one giving the best incorporations. [l3CCH,j-I--hfethionine (91% mol) was added to a growing culture (13 l), and the pigments were extracted. The 13C n.1n.r. spectrum of the derived methyl phaeopliorbides (1) is shown in Figure 3A, and Figure 3B shows tlie spectrum of the phaeophorbides at natural abundance. lwo greatly enhanced peaks are immediately apparent in Figure 3A; the resonance at 20.1 p.p.m.is attributed to the %methyl group, and that at 16.6 1i.p.m. to the ’1methyl of the 5-ethyl function in 660 bands 1-5 (1”gure 1). The peaks are 17-19 times more intense than the natural abundance resonances (measured with respect to the line at 51.5 p.p.m.). A closer examination of the * Material kindly supplied by Drs. A. S. Holt and J. W. I;. Wasley. ** J. Rimmer, work in progress. J.C.S. Perkin 1 spectrum shows two other signals to be significantly enhanced, namely those at 14.4 and 23.1 p.p.m. These peaks could not be assigned to band 5 (Figure l), but comparison (Figure 4) of a greatly intensified spectrum (Figure 4B) with that of fairly pure band 4 methyl phaeophorbide * (Figure 4A) enables the peak at 14.4 p.p.m.to be assigned to the terminal methyl of thc 4-11-propyl group in 660 band 4 (Figure 1) ; good coniparison with the terminal methyl in the 13C n.m.r. spectrum (14.0 p.p.m.) of n-propylbenzene is also obvious. The signals at 23.1 p.p.m. could also be assigned to the terminal methyl groups of an isobutyl function (cf.isobutylbenzene, 22.4 p.p.m., though a better model Is required 28). Therefore, the enhancements at 14.4 and 23.1 p.p.m. are due to incorporation of methyl units from methionine into the terminal positions of n-propyl and isobutyl groups present in bands 1-4 (Figure 1) of the total phaeophorbide mixture. No evidence for the existence of meso-ethyl groups (which, if produced from two labelled methyl groups would show large 13C-13C couplings) in the complete mixture was provided from these results.Thus, this experiment clearly establishes the origin of the &methyl group and the terminal methyl groups in the 4-n-propyl, 4-isobutyl, and 5-ethyl func- tions of the Chlorobizm chlorophylls-660 (ex. C. ethylicum) as L-methionine. We have proposed2 a route (Scheme 1) by which methyls can be incorporated into the 660 chlorophylls to give n-propyl or isobutyl functions. It is possible, by analogy with the known29 formation of the 2-and 4-vinyls in protoporphyrin-IX from the 2-and 4-propion- ates in coproporphyrinogen-111, that unsaturated groups I 0 ,, ,% 150 100 50 0 ITIGURE 4 13C N.m.r. spectrum (Varian XL-100, proton dc-coupled) of the Chlorobium chlorophyll methyl phaeophor- bides: A, 660 series, band 4 (see Figure 1) from Chlorobizm tlzios.Lilphatophzlunz ; B, the total mixture from C.ethylicuntafter feeding of [13CH,]-~-methionine (note that this spectrum is the same as shown in Figure 3.4, but plotted at much higher intensity) (Scheme 1) are precursors of the propyl and but171 functions. The same is true for the 5-ethyl group, and a pathway (assuming 30 hydroxylation followed by decarb- 29 E.g. J. A. S. Cavaleiro, G. W. Kenner, and K. M. Smith, J.C.S. Perkin I, 1974, 1188 and references therein. 30 S. Sano, J. Biol. Chem., 1966, 241, 5276. oxylation and dehydration) is outlined in Scheme 2. In order to investigate the possibility presented in Scheme 2, we prepared [13C2H&~-methionine from Me Me SCHEME1 13C2H31 as described earlier, and fed this to a growing medium.The pigments produced were transformed into the methyl phaeophorbides and their 13C n.m.r. spectrum was measured, using deuterium decoupling. * Figure 5 I I I I I I 1 1 I40 30 20 10 0 b FIGURE5 13C N.m.r. spectrum (Varian XL-100, deuterium decoupled) of the total mixture of methyl phaeophaeophor- bides (1) from C. ethylicum after feeding with [13C2H3]-~-methionine. Only the 0-40 p.p.m. range is shown, and the spectrum was measured with an external lH lock; the spectrum was referenced to ' imaginary ' tetramethylsilane by assigning the CDCl, line a value of 6 76.9 shows the spectrum (040 p.p.m. region only) obtained; the peak at 19-29 P.P.m.is assigned to the 6-methyl group, incorporated as expected, intact. The triplet at 15.63 P.P.m. is also immediately assigned to a 13C2H3 * We thank Professor E. A. V. Ebsworth and Dr. A. S. Boydfor this spectrum. function attached to a CH,; the coupling (J 4.8 Hz) is as expected3l for a carbon-13 nucleus coupling with protons attached to an adjacent carbon atom (i.e. 13C2H3-12CH,). Thus, both methyl groups are incor-porated intact and the proposition advanced in Scheme 2 is excluded in favour of the general type of mechanism COZH SCHEME2 involved in the transformation of uroporphyrinogen-111 into coproporphyrinogen-III.32 The additional peaks in Figure 5 at 22.07and 13.54 p.p.m. can also be assigned to the terminal methyl groups in the isobutyl and n-propyl side-chains in bands 14 (Figure 1j. Porphobilinogen (PBG) (7).It was recognised that feeding to growing cultures would be unsatisfactory for substrates such as porphyrinogens. They would require a more active cell preparation. [6-14CjPBG was syn-thesised and used to develop such a versatile system. 31 G. C. Levy and G. L. Nelson, ' 13C Nuclear Magnetic Resonance for Organic Chemists,' Wiley, New York, 1972; J. B. Stothers. ' l3C Nuclear Magnetic Resonance SPectrOscOpy,'Academic Press, New York, 1972; H. Scheer and J. j.Katz, in ' Porphyrins and Metalloporphyrins, ed. K. It%. Smith, Elsevier, Amsterdam, 1975, p. 490. 32 A. R. Battersby and E. McDonald, in ' Porphyrins amd Metalloporphyrins,' ed.K. M. Smith, Elsevier, Amsterdam. 1975, p. 61. Administration of [6-14C]PBG to growing cultures and cell suspensions in much the same way as described above for methionine gave only very small incorporations into the Chlorobiunz chlorophylls-660. Feeding to sonicated cell suspensions, however, proved more successful, the derived methyl phaeophorbides (1) showing incorpor- ations of between 3 and 7%. Experiments using [6-13C]PBG, designed to confirm these results, are in hand. EXPERIMENTAL Electronic absorption spectra (solutions in methylene Chloride or ether) were measured using a Unicam SP 800 spectrophotometer. Since the materials used were a mixture of at least six homologous fractions, combustion analyses on the products have not been obtained.For the same reason, accurate extinction coefficients are not quoted ; however, relative intensities are given. lH N.m.r. spectra were determined (usually in CDCl, solution with tetra-methylsilane as internal standard) with a Varian HA- 100 instrument, and 13C n.m.r. spectra (conditions as previously described 13) with a Varian XL-100 spectrometer. Mass spectra were measured using an A.E.I. MS 12 instrument (direct insertion probe; 70 eV, 50 PA, and source temper- atures ca. 200"). The activities of radioactive samples were determined with a Packard (model 3003) Tri-Carb liquid scintillation counter. To maximise counting efficiency, coloured samples were bleached before counting. This was done by dissolving in tetrahydrofuran (ca. 3 nil), adding benzoyl peroxide (10.1 nigj, and allowing the solution to stand in sunlight ur=til colourless. Then, an organic scintillator solution (cu;. 7 rnl of O.S0/h butyl-PI3D in toluene) was added and the sample counted. The efficiency of counting was determined using :14Cc]-n-hesadecane as internal standard. Background counts were obtained over long periods prior to wanting radioactive samples. Incor-porations are calculated as (counts recovered x loo)/ (counts fed). Growth of Chloropseudomonas ethy1icum.-The strain was obtained from the Botany Pepartment, King's College, London. The medium used consisted of four solutions A-D in distilled water which were autoclaved at 15 lb in-, for 15 min before use.Solution A contained potassium dihydrogen phosphate ( 1.5 g), ammonium chloride ( 1.5 g), magnesium chloride hexahydrate (7.5 g), sodium chloride (30 g), calcium chloride (68 ma), iron(m) chloride hexa- hydrate (3.6 mg), boric acid (0.84 mg), zinc sulphate hexa- hydrate (0.66 nig), cobalt(I1) nitrate hexahydrate (0.38 mg), copper(r1) sulphate pentahydrate (0.03 mg), and manganese(r1) sulyiiate (0.03 mg) in water (I 500 ml). Solution B contained iron(11) sulphate heptahydrate (3.8 mg) in water (7.5 ml). Solution C contained sodium sulphide nonahydrate (0.15 g) in water (7.5 ml). Solution D contained sodium hydrogencarbonate (2 g) in water (75 ml). Solutions B-D were added under sterile condi- tions to solution A.The pH was adjusted to 7.3 if neces- sary using sterile dilute phosphoric acid. The resulting medium was inoculated with well grown C. ethyZicum (10-20 ml) and grown under 30 W fluorescent lamps at 30". The cells were harvested after 7 days or else subcultured into fresh medium after 10 days to maintain the culture. Isolation of Chlorobium ChlorophylZs-660.-Seven day old culture (4.5 1) was filtered through a Celite bed (4 cm deep, 40 cm diameter; 3 parts Celite 545 to 1 part 505). After drying the Celite bed under vacuum for 1 h it was stirred J.C.S. Perkin I with acetone (2 1). The resulting suspension was filtered and the Celite re-extracted with acetone (3 x 2 1). The green filtrates were combined, methylene chloride was added, and the mixture was washed with saturated aqueous sodium chloride.The organic layer was dried (Na,SO,) and evaporated to dryness to give chlorophylls contaminated with carotenoids, quinones, etc., Amx. (ether I, 660; 11, 625; 111, 431; IV, 411 nm (I11 > I > IV > 11),together with absorptions at 462 and 493 nm due to carotenoids, etc. A very concentrated solution of crude chlorophyll showed an additional Am%,-(CH,Cl,) at 765 nm. Mass spectra measured on this material were uninterpretable. Farnesyl 5-Ethyl-2- ( l-hydroxyethyl) -5-demethyZ-6-methyl-2- devinylpyrophaeophorbide a and Homologues (Farnesyl Phaeophorbides OY Phaeofarnesins from C. ethylicum) (4) .-A solution of crude Chlorobiurn chlorophyll-660 from C. ethylicurn (20 1 of medium) in methylene chloride (200 ml) was shaken vigorously with 1% v/v concentrated HC1 in water (200 ml).An immediate colour change from green to brown was observed. The organic layer was washed with water (2 x 100 ml), dried (Na,SO,), and evaporated to dryness. The residue was chromatographed on neutral alumina (grade V) ; elution was first with petroleum (b.p. 60-8O0) to remove carotenoids, quinones, etc., and then with methylene chloride-petroleum (1 : 1) to remove the black phaeofarnesin band. The eluates were then eva-porated to dryness, T (0.1~in CDCl,) 0.20 and 0.58 (2 meso-H), 3.70 (CHOH), 4.86 (2 H, s, CH,CO), and 6.25, 6.64, and 6.82 (3 x Me); other resonances were unresolved; A,,,. (CH,Cl,) I, 669; 11, 614; 111, 557; IV, 525; V, 492; and VI, 424 nm, (VI > I > I11 > IV > I1 > V); m/e, 812 (&I+,6%), 798 (M+,28), 796 (7), 794 (5), 784 (iW+,loo), 782 (26), 780 (23), 768 (74), 766 (74), 754 (6), 752 (5),and a corresponding set of peaks from m/e 608 to 548.iVIethy1 5-EthyZ-2-(l-hydroxyethy2)-5-demethyZ-6-methyl-2-deuiny lpyrophaeophorbide a and Homologues (Methyl Phaeo- phorbides from C. ethylicum) (1).-The foregoing phaeo- farnesins were left overnight at 25" in 5% v/v concentrated sulphuric acid in methanol (100 ml). Methylene chloride (100 nil) and water (100 nil) were added and the organic layer was washed with saturated aqueous sodium hydrogen- carbonate, then with water, dried (Na,SO,), and finally evaporated to dryness to give a black oil.Chromatography on alumina (Fluka, neutral, Brockmann Grade 11) (elution with methylene chloride) gave two fractions. The first was a minor product, the dehydrated phaeophorbides (2). The second band was collected and rechromatographed on six 20 cm x 20 cm x 1.5 mm preparative t.1.c. plates (silica G), developing with 5% tetrahydrofuran in methylene chloride. Two bands were obtained; the black methyl phaeopliorbides (1) (RF 0.4) were removed from the silica gel by stirring in 5% methanol in methylene chloride, and after evaporation of the solvent, they were crystallised by scratching under hexane to give a black powder (180 mg), T (0.1~)0.19 (p-meso-H), 0.56 (a-meso-H), 3.69 (q, CHOH), 4.88 (s, CH,CO), 5.54 and 6.03 (m, 7-and 8-H), 6.03 (q, 5-CH2), 6.25 (&Me), 6.46 (OMe), 6.54 and 6.81 (1-and 3-Me), 6.37 (9, 4-CH2), 7.5-8.2 (m, CH,CH,), 8.00 and 8.33 (t, 4- and 5-Me), and 8.64 (d, 8-Me); A,,,, (CH,Cl,) I, 669; 11, 614; 111, 557; IV, 525; V, 492; and VI, 424 nm (VI > I > I11 > IV > I1 > V); m/e 622 (M', l%), 608 (M+,6), 606 (7), 604 (B), 594 (M', 15), 592 (31), 590 (30), 580 (38), 578 (loo), 576 (88), 564 (19), 562, (21), and a corresponding set of peaks from nz/e 535 to 475. The second product (Rp 0.1) was removed from the silica 1978 gel as above.Filtration and evaporation gave a purple solid (20 mg) identified as 1,3,8-trimethyl-4,5-diethyl-2-( 1 -hydroxyethyl) -6-c-( 1-oxoethylene) -7,8-dihydro- 7- (2- methoxycarbonylethyl)-8'-acetylbilitrieneand homologues (5), 7 (0.05~)3.16 and 3.58 (methine Hs), 4.9-5.0 (m, CHOH), 6.34 (OMe), 6.46 (10-CH,), 7.2-8.1 (8 H, m, CH,CH, and 4-and 6-CH,), 7.82, 7.93, and 8.14 (1-and 3-Me and CHs- CO), 8.41 (d, 8-Me), and 8.75 (6 H, t, 4- and 5-Me); Am= (ether) I, 590; 11, 561; 111, 364; IV, 314 nm (IV > I11 > I1 > I); m/e 654 (M+,4%), 640 (M+, 19), 626 (M+,loo),612 (M', 16), 611 (€9, 597 (23), 583 (69), 569 (ll),539 (9), 497 (4), 419 (4), 368 (7), 356 (a),325 (9), 313 (8),and 260 (17), m* 556.9, 543.0, and 529.0.The 8'-acetylbilitrienes were prepared more efficiently as follows. A solution of methyl phaeophorbides (1) (80 mg) in methylene chloride (200 ml) was purged with oxygen for 1 week. The solution was evaporated to dryness and the residue was chromatographed on four 20 x 20 cm x 1.6 mm preparative t.1.c.plates of Kieselgel G (elution with 5% tetrahydrofuran in methylene chloride). A small amount of starting material soon separated from the required product, and after removal from the silica and standing in hexane, the purple band afforded a purple solid (60 mg, 71y0), identical in all respects with the material (5) described above. Methyl 5-Ethyl-5-demethyl-6-methylpyrophaeo~horbidea and Homologues (Methyl 2- Vinylphaeophorbides from C. ethyli- cum) (2) .-Methyl phaeophorbides (1) (180 mg) in benzene (150 ml) containing toluene-p-sulphonic acid hydrate (1 g) were refluxed under nitrogen for 1h. Chloroform (300 ml) was added and the solution was washed with saturated aqueous sodium hydrogencarbonate and water.The organic layer was dried (Na,SO,) and evaporated to dryness. Analytical t.1.c. showed that partial hydrolysis of the methyl esters had occurred, so the product was treated with ethereal diazomethane. The resulting solution was evaporated and the residue chromatograplied on alumina (Grade 11) (elution with methylene chloride). Evaporation of the eluates and scratching of the residue under hexane gave a black powder (140 mg, 77%), 't (0.1~)0.55 (p-meso-H), 0.63 (a-meso-H), 2.1-2.4 and 2.8-3.1 (m, CHXH,), 4.80 (10-CH,), 5.50 and 5.95 (m, 7-and 8-H), 5.95 (9, S-CH,), 6.21 (%Me), 6.41 (4, 4-CH2), 6.46 (OMe), 6.46 and 6.86 (1- and 3-Me), 7.4-8.2 (m, CH,CH,), 8.06 and 8.35 (t, 4-and &Me), and 8.55 (d, %Me), A,,,. I, 671; 11, 615; 111, 553; IV, 522; V, 491; and VI, 420 nm.(VI > I > 111 > IV > I1 > V); m/e 604 (M+,6%), 590 (M', 22), 576 (M+,loo),and 562 (M+,10). Methyl 2-a-Ethylene-5-ethyl-5-demethyl-6-methyl-2-devinylpyrojjhaeophorbide a and Homologues (Methyl 2-a- Ethylenephaeophorbides from C. ethylicum) (6) .-A solution of methyl phaeophorbides (1) (100 mg) in benzene (100 ml) containing toluene-9-sulphonic acid ( 1 g) was refluxed overnight under nitrogen. The product (80 mg, 79%) was isolated as for the methyl 2-vinylphaeophorbides (2), 7 (0.1~)0.76 (p-meso-H), 4.82 (10-CH,), 5.5-5.7 and 5.8-6.0 (m, 7-and 8-H), 6.0-7.3 (8 H, m, 2-, 4-, 5-, and a-CH,), 7.5-8.2 (CH,CH,), 6.36 (&Me), 6.46 (OMe), 7.07 and 7.57 (1- and 3-Me), 8.08 and 8.49 (t, 4- and 5-Me) and 8.49 (d, 8-Me); Lx.(CH,Cl,) I, 660; 11, 608; 111, 563; IV, 529; V, 420 nm (V > I > I11 > IV > 11); m/e 604 (Mf, 3%), 590 (M+,20), 576 (M+,loo), and 562 (M+,18).Methyl 5-Ethyl-5-demethyl-8-methylmesopyrophaeophorbide a and Homologues (Methyl Mesophaeophorbides from C. ethylicum) (3).-Methyl 2-vinylphaeophorbides (2) (140 mg) in acetone (25 ml) were hydrogenated during 1 h at 25" and 1 atm over 10% palladised charcoal (50 mg). Filtration through Celite and evaporation gave a black residue which soon solidified in hexane to afford a black powder (135 mg, 96%), 7 (0.1~)0.55 (P-meso-H), 0.75 (a-meso-H), 4.82 (10- CH,), 5.5-5.7 and 5.9-6.1 (m, 7- and 8-H), 5.95 (9,5-CH,), 6.39 (4 H, q, 2-and 4-CH2), 6.24 (&Me), 6.49 (OMe), 6.71 arid 6.81 (9 H, t, 2-, 4-, and &Me), and 8.58 (d, 8-Me); A,,,, (CH,C12) I, 667; 11, 613; 111, 557; IV, 525; V, 492; and VI, 419 nm (VI > I > I11 > IV > I1 > V); mle, 6015 (M+,a%), 592 (M+,20), 578 (M+,loo), and 564 (M+, 6).[WH3]-~-Methionine.-Liquid ammonia (30 ml) was distilled into L-homocystine (750 mg, 1equiv.) while stirring and cooling in a dry-ice-acetone bath.Nitrogen was passed through the solution and pieces of sodium were added until there was a permanent blue colouration. [13C1H3]Methyl iodide (91% g atom; 1.0 g, 1.2 equiv.) was added. After 20 min the ammonia was allowed to evapo- rate and water (20 ml) was added. The solution was filterted and brought to pH 6.0 with hydriodic acid. Boiling ethanol was added and the solution was cooled to 0".Platelets (550 mg, 64%) were collected and the material was shown to be identical with authentic unlabelled L-methionine by t.1.c. An amino-acid analysis of the labelled material showed L-methionine to be the only amino-acid present, 't (D,O; unlabelled methionine) 5.69 (t, CH), 6.8-7.4 (m, CH,CH,), and 7.40 (s, Me); 't (D,O; 1sC labelled methionine) 5.7(m, CH), 6.9-7.5 (m, CH,CH,), and 7.50 ((1, 13CH3,J 140 Hz) (shifts appeared to be concen- tration dependent). A sample of [14CH3]-~-methioninewas prepared in a similar fashion using L-homocystine and I4CH,I. Feedzng Experi~ents.-[~~CH,]-~-Methionine.(i) Incub-ation in the presence of lysozyme.C. ethylicum (1.5 1; grown i'or 70 h after subculturing) was centrifuged at 10 000 r.p.m. i'or 40 min at 0". The centrifugate was resuspended in O.1M-potassium phosphate buffer (20 ml; pH 7.4) and to this was added magnesium chloride hexahydrate (24 mg), adenosine triphosphoric acid disodium salt (ATP) (64 mg) , lysozyme (70 mg), and a known quantity (ca.200 mg) of aqueous [14CH3]-~-methionine solution (specific activity 4 mCi m0t-l). The resulting suspension was incubated during 12 h under normal growth conditions, then stirred for 15 min with methanol (200 ml). Methylene chloride (250 ml) was added and the organic layer was washed with saturated aqueous sodium chloride and 1% v/v concentrated HCl in water (500 ml), then dried (Na,SO,), and evaporated to dryness.The residue was chromatographed on alumina (Grade V) (elution with petroleum first and then with 1 : 1 petroleurr-methylene chloride). The dark eluates were evaporated and rechromatographed on two 20 x 20 cm x 1.5 mm preparative silica t.1.c. plates [elution with 307; acetone in petroleum (b.p. 60-8O0)]. Two green-black bands were observed, the methyl and farnesyl phaeophor- bides. A radiochemical scan of the plates showed activity in both bands. The actual amounts were estimated from spectrophckometry of the Soret bands and red absorption maxima. This indicated that the two compounds had approximately the same specific activity, and therefore that methionine had been incorporated into the chlorin nucleus, and not just into the farnesyl group.The methyl and farnesyl phaeophorbides were removed from the silica gel by stirring in 576 methanol in methylene chloride. Both samples were then dissolved in tetrahydrofuran (2 ml), bleached in the presence of benzoyl peroxide, and counted : total activity fed in the form of [14CH,]-~-methionine 3.!5 pCi, total activity recovered in the form of methyl and farnesyl phaeophorbides 0.32 pCi, incorporation 9.2%. (ii) Incubation with growing cultures. Two batches [(a) and (b)] of C. ethylicurn (1.51) were subcultured. To (a) was added, just prior to subculturing, a solution (ca. 200 mg) of [14CH,]-~-methioninein water (specific activity 4 mCi mol- 1) diluted with inactive L-methionine (5 mg).To (b) was added a similar solution in four equal batches, at the time of subculturing, and 24, 48, and 72 h later. (The total activity administered in each feeding was approximately equal.) The cultures were harvested after 7 days growth by filtration through Celite and extraction with methanol. The methyl and farnesyl phaeophorbides were bleached and counted as before; (a) total activity fed 4.9 pCi, total activity recovered in pigments 0.46 pCi, incor- poration 9.5%; (b) total activity fed 4.6 pCi, total activity recovered 0.43 pCi, incorporation 9.2%. (iii) Incubation with a freeze-thawed cell extract. The procedure was as described for the lysozyme incubation except that prior to addition of cofactors, the buffered cell suspension was frozen at -30" and then allowed to thaw at 25O, and the lysozyme was omitted: total activity fed 1.1 $3, total activity recovered in pigments 0.032 pCi, incorporation 3.0%.(iv) Blank experiment. To a solution of farnesyl phaeophorbides, quinones, carotenoids, etc., obtained from C. ethylicum culture (1.5 1) was added [14CH,]-~-methionine in aqueous solution (ca. 200 mg; specific activity 4 mCi mol-1). Methanol (200 ml) was added and the piE;ments were isolated as for the lysozyme incubation : total axtivity ' fed 3 pCi, total activity recovered in pigments 1.6 x pCi, ' incorporation ' 0.005~0. [13CH,]-~-Methionine. Four batches of C. etiiylicurn (3.25 1 each) were subcultured. Just prior to subcinl.turing, [l3CH3]-~-methionine(50 mg) was dissolved in distilled water and added to each batch.The cultures were harvested after 9 days growth and the Clalorobium chlorophyll-660 was extracted and degraded to the methyl pliaeophorbides ( 120 mg) and 8'-acetylbilitrienes ( 10 mg), as described earlier. The 13Cn.m.r. spectrum of the methyl phaeophorbides is shown in Figures 2 and 3. [6-l4C]PorphobzZinogen. (i) Incubation with growing cul- ture. To C. ethylicurn culture (400 ml) (48 h after sub- culturing) was added [6-14C]PBG (10 mg, specific activity 9.8 mCi mol-l). The culture was harvested after 5 days growth and the pigment was isolated as described earlier to obtain the methyl and farnesyl phaeophorbides which were bleached and counted: total activity fed 0.46 pCi, total activity recovered in pigments 1.1 x pCi, incorporation 0.02y0.J.C.S. Perkin I (ii) Incubation in the presence of lysozyme. The pro- cedure was the same as for the incubation of [14CH3]-~-methionine except that (a) 400 ml of culture was centrifuged, and (b) to the buffered cell suspension (5 ml) was added magnesium chloride hexahydrate (6 mg), ATP (16.5 mg), lysozyme (30 mg), [6-14C] PBG (500 mg, specific activity 0.16 mCi mol-l) in ammonium acetate solution, and the reduced form of glutathione (5 mg): total activity fed 0.32 pCi, total activity recovered in pigments 2.24 x lo-* pCi, incorporation 0.07yo. (iii) Incubation with a sonicated cell extract. C. ethylicum culture (250 ml) grown for 48 h after subculturing was centrifuged at 10 000 r.p.m.for 40 min at 0". The centrifugate was resuspended in 1M-Tris buffer (8 ml; pH 7.3) in a sonication tube. ATP (16 mg), magnesium chlo- ride hexahydrate (6 mg), and L-methionine (25 mg) were added and the suspension was ssnicated (8 microns peak to peak) for 30 s while cooling in an ice-salt bath (in 5 s bursts of sonication with 15 s intervals). A known amount of [6-14C]PBG (ca. 2 mg; specific activity 7.5 mCi mol-l) dissolved in ammonium acetate solution (0.5 ml) was added immediately after sonication and the cell suspension was incubated for 18 h under normal growth conditions. This was then shaken with acetone (100 ml), diluted with methylene chloride (100 ml), and washed with water (3 x 100 ml) and saturated aqueous sodium chloride (2 x 250 ml). The organic layer was dried (Na,SO,) and eva- porated to dryness to give a residue which was trans-esterified to the methyl phaeophorbides (1) as previously described. Chromatography (one 20 x 20 cm x 1.5 mm silica plate ; elution with 5% tetrahydrofuran in methylene chloride) gave a clean phaeophorbide band which was extracted from the silica gel, evaporated to dryness, and the residue was dissolved in tetrahydrofuran (3 ml), bleached, and then counted. The results from four separate experi- ments are as follows: 1 2 3 4 Total activity fed (pCi)Totalactivityin recovered 0.07 0.059 0.076 2.66 x lo-* 2.4 x loe3 2.4 x 0.07 0.49x methyl phaeo- phorbides ($3)Incorporation(70) 0.4 4.0 3.1 7.1 We thank Drs. A. S. Holt and J. W.F. Wasley for provid- ing a sample of Cldovobiurn chlorophyll-660, band 4, Pro- fessor E. A. V. Ebsworth and Dr. A. s.Boyd for the carbon- 13 spectrum with deuterium decoupling, and the S.R.C. for studentships (to J. R. and J. F. U.). [7/1425 Received, 4th August, 19771

ISSN:1472-7781    

DOI:10.1039/P19780000845

出版商:RSC    

年代:1978

数据来源: RSC