in the Biochemistry of Micro-organisms

Aldridge, W. N. (1945). Analyst, 70, 474. Black, W. A. P. (1949). J. Soc. chem. Ind., Lond., 68, 183. Bowler, R. G. (1944). Biochem. J. 38, 385. Brown-Grant, K. (1957). J. Physiol. 135, 644. Broyer, T. C. & Overstreet, R. (1940). Amer. J. Bot. 27,425. Cowie, D. B., Roberts, R. B. & Roberts, I. Z. (1949). J. cell. comp. Physiol. 34, 243. Epstein, E. (1953). Nature, Lond., 171, 83. Epstein, E. & Hagen, C. E. (1952). Plant Physiol. 27, 457. Fletcher, K., Honour, A. J. & Rowlands, E. N. (1956). Biochem. J. 63, 194. Freinkel, N. & Ingbar, S. H. (1955). J. clin. Endocrin. Metab. 15, 598. Freinkel, N. & Ingbar, S. H. (1956). Endocrinology, 58, 51. Halmi, N. S., Stuelke, R. G. & Schnell, M. D. (1956). Endocrinology, 58, 634. Jacques, A. G. & Osterhout, W. J. V. (1938). J. gen. Physiol. 21, 687. Kelly, S. (1953). Biol. Bull., Wood's Hole, 104, 138. Kelly, S. & Baily, N. A. (1951). Biol. Bull., Wood's Hole, 100, 188. Krebs, H. A., Gurin, S. & Eggleston, L. V. (1952). Biochem. J. 51, 614. Lineweaver, H. & Burk, D. (1934). J. Amer. chem. Soc. 56, 658. Logothetopoulos, J. H. & Myant, N. B. (1956). J. Physiol. 133, 213. Lyman, J. & Fleming, R. H. (1940). J. Mar. Re8. 3, 134. Mitchell, P. (1953). J. gen. Microbiol. 9, 273. Mitchell, P. (1954). J. gen. Microbiol. 11, 73. Osterhout, W. J. V. (1952). J. gen. Physiol. 35, 577. Pitt-Rivers, R. (1950). Physiol. Rev. 30, 194. Roche, J., van Thoai, N. & Lafon, M. (1949). C.R. Soc. Biol., Paris, 143, 1327. Roche, J. & Yagi, Y. (1952). C.R. Soc. Biol., Paris, 1468 642. Rogina, B. & Dubravoic, M. (1953). Analyst, 78, 594. Stanley, M. M. & Astwood, E. B. (1948). Endocrinology, 42, 107. Sutcliffe, J. F. (1954). J. exp. Bot. 5, 313. Taurog, A., Tong, W. & Chaikoff, I. L. (1950). J. biol. Chem. 184, 83. Tong, W. & Chaikoff, I. L. (1955). J. biol. Chem. 215, 473. Vanderlaan, J. E. & Vanderlaan, W. P. (1947). Endocrinology, 40, 403. Wood, J. L. & Kingsland, N. (1950). J. biol. Chem. 185,833. Wyngaarden, J. B., Stanbury, J. B. & Rapp, B. (1953). Endocrinology, 52, 568. Wyngaarden, J. B., Wright, B. M. & Ways, P. (1952). Endocrinology, 50, 537.

(Received 1 April 1957) Raistrick, Stickings & Thomas (1953) have described the isolation of two metabolic productsalternariol (I) and one of its monomethyl ethersfrom the mycelium of two strains of Alternaria tennui8 auct. grown on Czapek-Dox solution. These 0<0 HOOH CH3 OH (I) two compounds appear to constitute practically the whole of the ether-extractable, but petroleuminsoluble, fraction of the dried mycelium.
It was noted during this work that the metabolism solution of these strains of A. tenui8 gave a reddish brown colour reaction with ferric chloride. While small amounts of alternariol and its methyl ether could be isolated from the solution, it was soon clear that the material responsible for this ferric reaction was a complex mixture of these two compounds with a number of new substances.
It is the purpose of this paper to describe some of the main constituents of this mixture, and their separation and characterization. The study has been extended to two other strains of A. tenUi8. Detailed considerationofthe structures ofthe new compounds will be published later. It will be clear from the following account that the list of metabolic products is probably very incomplete, and indeed paper chromatography indicates the presence of * Part 102: Birkinshaw, Chaplen & Lahoz-Oliver (1957). several other constituents not yet isolated. None of the new compounds appears to resemble alternaric acid (Grove, 1952) or the products of Alternaria aolani described by Darpoux, Faivre-Amiot & Roux (1950).

The altenuic acid8
The first experiments were carried out with strain no. 94. The products were isolated by adsorption on charcoal, followed by elution with ethanol. In this way three crystalline compounds were isolated: two colourless acidic substances, m.p. 183-184°and 245-246°respectively, which we have called altenuic acid I and altenuic acid II; and a yellow compound, m.p. 189-190°, which is discussed in the next section. Strain no. 94 deteriorated rapidly on subculturing, and attention was switched to strain no. 430, in particular to a sandculture of this strain which produced high yields of altemariol and its methyl ether (see Raistrick et al. 1953).
Although varying amounts of altenuic acids I and II have been obtained from cultures of no. 430, many batches produced substantial quantities of another colourless acidic compound, whose reactions and molecular formula indicated a close relationship with altenuic acids I and II. It has been established that both the latter acids are readily converted into this new compound, simply by dissolving in aqueous sodium hydroxide and reacidifying. The new product is therefore called altenuic acid III. All three compounds have the molecular formula CL5H1408, containing one methoxyl and one carbon-methyl group.
Altenuic acid I gives a pale purple-brown ferric colour in ethanol, scarcely affected by adding water. The acid forms a colourless dimethyl derivative, m.p. 177-178°, which no longer shows any ferric reaction. Altenuic acid II is a very sparingly soluble substance. In dioxan-ethanol it gives a pale-brown colour with ferric chloride, again scarcely affected by addition of water. This acid also forms a colourless dimethyl compound, giving no ferric colour; the m.p. is 172-173°, depressed on admixture with the dimethyl derivative of altenuic acid I. Altenuic acid III has a variable melting point, of little value diagnostically. Its ethanolic solution gives only a pale wine colour with ethanolic ferric chloride, but on the addition of water this immediately deepens to an intense purple. The acid forms a colourless dimethyl derivative, m.p. 143-5-144.5o, soluble in cold aqueous sodium hydroxide but not in aqueous sodium carbonate, and giving a positive ferric reaction; this dimethyl compound can readily be monoacetylated. More complete methylation yields a colourless neutral trimethyl derivative, m.p. 125-126.5o, which gives no ferric colour.
Mixtures of altenuic acids I and III are difficult to separate by crystallization. Their behaviour on paper chromatography has been studied and they can be clearly separated on a buffered paper. The paper chromatograms also served as a model for separations on a preparative scale by means of a Craig countercurrent-distribution apparatus, and excellent separations were achieved.

Altenu8in and dehydroaltenuein
The yellow compound, m.p. 189-190°, isolated from strain no. 94, has the molecular formula C15HI2O^. It was not obtained from other strains when solvent-extraction methods were used. However, ether extraction of the concentrated culture filtrate from no. 108 at pH 5 yielded a fraction from which a related colourless product, m.p. 202-203°, could be isolated by crystallization from chloroform. Smaller amounts of the same material were obtained from no. 430. When this substance, whose molecular formula is C15H14016, is dissolved in aqueous ethanol and treated carefully dropwise with dilute aqueous ferric chloride, an initial violet colour is observed which disappears on shaking, umtil with increasing amounts of ferric chloride a permanent deep-brown colour develops.
This brown-colour reaction is identical with that given by the yellow compound isolated from no. 94, and, in fact, if the addition of ferric chloride to the colourless substance is stopped when the permanent brown colour just begins to appear, a yellow crystalline product can easily be isolated from the reaction solution. This compound is identical with the yellow substance from no. 94. It appears therefore that the colourless compound C15H1406 gives a violet colour with ferric chloride, but is oxidized at once to the yellow C15H1206, Fe3+ being reduced to Fe2+; this is confirmed by testing with potassium ferricyanide. The yellow product can be reduced by sodium dithionite to the colourless C15H1406 .
Although both compounds are soluble in aqueous sodium bicarbonate, the acetyl derivative of the yellow substance is insoluble even in sodium hydroxide solution, making the presence of carboxyl or other strongly acidic groups improbable. We therefore propose the name altenusin for the colourless C15H1406, and dehydroaltenusin for the yellow C15H1206 -While both may be formed by the mould, and indeed their ready interconversion may play a part in the metabolism of the organism, we have no certain evidence of the presence of dehydroaltenusin in the culture filtrates. The isolation of this compound from cultures of no. 94 may well be the result of oxidation of altenusin on the charcoal (cf. Raistrick & Stickings, 1951). Unfortunately, strain no. 94 no longer produces either altenusin or dehydroaltenusin, so the question must remain open.

Vol. 67
The molecular formula of altenusin includes one methoxyl group and one carbon-methyl group. Methylation yields a neutral, colourless tetramethyl compound, not reacting with ferric chloride. Dehydroaltenusin gives a red precipitate with Brady's reagent (2:4-dinitrophenylhydrazine in aqueous hydrochloric acid), shown to be a monoderivative. It forms a colourless triacetate with acetic anhydride-sodium acetate.

Altertenuol
The concentrated culture filtrate from strain no. 108, extracted at pH 7, yields a solid giving a green ferric chloride reaction; small quantities of the same material were also obtained from no. 430. A pure substance has been isolated, which shows this ferric colour; it is insoluble in aqueous sodium bicarbonate and yields a neutral triacetate. We propose for it the name altertenuol. It crystallizes from acetic acid in buff-coloured rods, m.p. 284-2850. The molecular formula, C14H1006, includes one methoxyl group, but no carbon-methyl group. In addition to the triacetate the compound gives a neutral colourless trimethyl compound.
Tenuazonic acid and isotenuazonic acid In early experiments with strain no. 430, it was noted that some acidic fractions gave a strong orange-red ferric colour, not attributable to any of the compounds described so far. This material is -very soluble in organic solvents, even in cold light petroleum. It has been purified in various ways, but has not been obtained as a solid. The material is ketonic and strongly acidic. It is also laevorotatory; of the metabolic products described in this paper, it is the only one to show optical activity. Analyses of the compound and of its derivatives indicate the -molecular formula CloH1503N. A green crystalline chloroform-soluble copper salt is readily obtained, and can be used for isolation and purification; on recrystallization it attains a constant rotation, [oc] 1961 -1240 in methanol. The acid recovered from the purified copper derivative has [0C]2°61-1320 in chloroform.
On long standing the rotation of the product slowly becomes less negative, and eventually crystallization starts. The purified crystalline substance also has the formula C10H15O3N, and has very similar chemical properties to the original material.
However, it is dextrorotatory, [OC] 5461 + 230 in chloroform, and its copper salt has [ac] 226 + 24.50 in methanol. There is no reason to suppose that this isomer is present in the freshly isolated metabolic material, and we regard it as an artifact.
We propose the name tenuazonic acid for the metabolic (liquid) product, and isotenuazonic acid for the crystalline isomer. Tenuazonic acid is produced also by strain no. 628, which does not appear to give any substantial amounts ofthe other culturefiltrate products.
Tenuazonic acid titrates sharply as a monobasic acid and contains no methoxyl group. It forms a crystalline mono-derivative with Brady's reagent, and a semicarbazone, m.p. 187-189°with effervescence; the latter gives a deep-blue colour with ferric chloride. Tenuazonic acid can be converted into the iso-acid by boiling with aqueous alkali. isoTenuazonic acid has properties very similar to those of tenuazonic acid, but crystallizes from a small volume of low-boiling light petroleum in colourless needles, m.p. 61-63.50. The crystals are not very stable, and frequently decompose on keeping, to a liquid which does not appear to be tenuazonic acid. It is conveniently kept as the copper salt. Brady's reagent gives an amorphous precipitate, which is a mixture containing some tenuazonic acid 2:4-dinitrophenylhydrazone. The semicarbazone, m.p. 206-206 5°with effervescence, gives the same intense blue ferric colour as the semicarbazone of tenuazonic acid. Alternariol and its methyl ether Alternariol and its methyl ether have been extracted from the mycelium of strains nos. 94, 430 and 108. Nos. 94 and 430 yield mixtures in which the methyl ether predominates (see Raistrick et al. 1953), but no. 108 gives large yields of a mixture which is mainly alternariol. No. 628 gave only a small amount of solid, from which a little impure alternariol methyl ether was isolated, with perhaps a trace of alternariol.
These substances have also been shown to be present in small amounts in the culture filtrate of no. 430. General The products isolated from the four strains of Alternaria tenuis are shown in Table 1.
It would be premature at this stage to speculate on the chemical or biochemical relationship between these compounds. It is, however, noteworthy that, with the exception of tenuazonic acid, all these products of A. tenuis contain either a carbonmethyl group or a methoxyl group or both, attached in each case to a C13 residue. Strain no. 628, from which we have isolated tenuazonic acid, but none of the C14 or C15 culture-filtrate products, likewise produces in its mycelium only a small amount of alternariol methyl ether and a negligible quantity of alternariol. Altenusin and altertenuol were most conveniently obtained from no. 108, from which the other culture-filtrate products have not been isolated. After the deterioration of no. 94, no. 430 was used as a source of the altenuic acids and tenuazonic acid, although the other compounds are also produced by this strain. Tenuazonic acid was also obtained from no. 628.

History of cultures
Details of strains nos. 94 and 430 have been given previously (Raistrick et al. 1953). We are indebted to Mr G. Smith of this department for the following mycological notes on the remaining cultures.
No. 108. This strain was isolated from a mouldy orange by Mrs S. Marcus in April 1950. It has remained morphologically very stable. Whereas many isolates of A. tenuis deteriorate and rapidly become sterile in laboratory cultures, no. 108 still produces abundant and typical spores.
No. 628. This was isolated by Mr G. Agosti, in December 1956, from roadside soil collected near Limerick, Eire. This also is a strongly sporulating strain.
Both isolates produce long, often branched, chains of extremely polymorphous spores, which have both cross and longitudinal septa. According to Neergaard's (1945) classification they are to be regarded as Alternaria tenuis auct. sensu stricto.

Cultural conditions
The mould was grown on Czapek-Dox medium as described previously (Raistrick et al. 1953), usually in batches of 100 flasks. These were harvested after 4-5 weeks, when the pH was about 7-5, and the glucose about 0 5 %. The mycelium was separated from the culture filtrate and dried and extracted as in the earlier work.
Isolation of alternariol methyl ether from the mycelium of strain no. 628 No solid separated from the ether extracts of the dried mycelium. On removal of the solvent and washing the residue with cold light petroleum, there remained a brown, sticky solid (yield 3 g./100 flasks). This was triturated with aq. Na2CO3 and filtered; the filtrate on acidification yielded a solid (27 mg.), m.p. >3100 (decomp.), which was possibly crude alternariol. The Na2COS-insoluble portion, m.p. 255-2660, was extracted in a Soxhlet apparatus with light petroleum (b.p. 40o60') and the insoluble residue (1-5 g.) was sublimed in high vacuum. The bulk of the material sublimed below 2100; after recrystallization from ethanol and acetone it melted at 268-271°, not depressed on admixture with alternariol methyl ether, and showing the same fluorescence and ferric colour.
Isolation of products from culture filtrates of strain no. 94 A sample of the metabolism solution gave an intense brown ferric reaction, although in the later batches, when the yield of extractable crystalline material fell almost to zero, the only colour obtained on addition of FeCl3 was a dull olive-green. The solution was filtered through cotton wool, then kieselguhr, and acidified to pH about 2. Adsorbent charcoal (5 g./l.) was added, and, after shaking, the solution was filtered and the charcoal washed with a little water; the filtrate no longer gave a ferric reaction. The charcoal was dried in vacuo over conc. H2SO4, then suspended in ethanol and loosely packed in a vertical glass tube in the manner of a chromatographic column. Elution with ethanol gave an intense ruby-red eluate which was concentrated at 35-40' under reduced pressure. A crop of yellow crystals first separated, and were removed; further concentration of the filtrate yielded colourless crystals. The maximum combined yield was 5-2 g./100 flasks, but this fell rapidly in succeeding batches. The yellow crystals were readily recrystallized from ethanol, yielding dehydroaltenusin in yellow plates.
The colourless solid was boiled with water and filtered: crude altenuic acid II remained on the filter, and the filtrate on cooling deposited colourless needles of altenuic acid I. Altenuic acid I was recrystallized from water, and altenuic acid II by dissolving in boiling dioxan and adding 3 vol. of water.
Isolation of products from culturefiltrates of strain no. 108 When a sample (10 ml.) of the culture filtrate was treated with FeCls, the first drops produced a greenish colour, with a precipitate, but the addition of more FeCl3 changed the green to brown. The filtered solution was acidified to pH 6-5 with conc. HCI, then concentrated to 1-51. in a climbing-film evaporator (internal temperature 35-40°). Four volumes of ethanol were added, and the sticky precipitate (which gave no ferric reaction) was removed by filtration through glass wool. The ethanol was removed in the climbing-film evaporator, and the pH adjusted to 7-0 with NaOH. This procedure helped to reduce emulsification during the subsequent extractions. The concentrate was then extracted continuously with ether until the extract no longer gave a green ferric reaction (about 24 hr.). The pH was adjusted to 5 with HCI, and the solution further extracted continuously with ether until the extracts no longer gave an evanescent violet colour with FeCl3 (about 40 hr.); the pH was readjusted to 5 when necessary.
Extract at pH 7. Evaporation yielded a brown solid, which was washed with ether; wt. 1-2 g./100-flask batch. It gave an intense green colour with FeCl3 and consisted mainly of altertenuol. The presence of other substances, including probably alternariol and its methyl ether, made the compound difficult to purify. In one experiment, the solid (1-26 g.) was triturated first with ether, which removed a little material giving a purple ferric colour, and then dissolved as far as possible in boiling ethyl acetate. The hot solution was filtered quickly. The insoluble part gave a purple ferric reaction and melted above 3000. The filtrate was concentrated under reduced pressure until it became turbid. After 16 hr. the crystalline altertenuol was collected; a second crop was obtained from the filtrate (total yield, 0-7 g., m.p. 284-287°). Extract at pH 5. Removal of the ether gave a slightly coloured crystalline solid. It was triturated with cold CHCl3 and filtered. The crude altenusin was dissolved in ether, and passed through a column ofacid-washed alumina. Washing was continued with ether until the eluate ceased to give the typical ferric reaction. The ethereal solution was reduced to a small volume, and CHC1, was added until a slight turbidity resulted. On allowing to stand, colourless crystals separated. These were filtered, and the filtrate concentrated to give further crops. The altenusin, fairly pure at this stage, weighed 2-5-6-5 g./100-flask batch.

I957
Isolation of products from culture filtrates of strain no. 430 The culture filtrate behaved towards FeCl3 in a manner similar to the filtrate from no. 108, but the final colour was a deeper brown. Earlier batches were concentrated as described for no. 108. Later, it was found convenient to acidify to pH 1-2 with HCI before evaporation; the dark amorphous material precipitated was removed by filtration (fraction A), and the filtrate was readjusted to pH 6-5 with NaOH. After evaporation, the concentrated culture filtrate was adjusted to pH 7, and extracted continuously with ether for 30-35 hr. A cream-coloured solid separated from the boiling ether; this was filtered (fraction B) and the filtrate evaporated (fraction C).
The solution was then acidified to pH 1-2 with conc. HCI, and extracted several times with half-volumes of benzene, until the extracts no longer gave an appreciable orange colour with FeCl3. Evaporation of the benzene yielded fraction D.
The aqueous concentrate was then further extracted three times with ether and the ether evaporated to give fraction E. Fraction A. This gave a brown ferric colour. It has not yet been investigated. Fraction B (0-1 g.) dissolved in ethanol (1 ml.) was diluted with water (9 ml.) and acidified to Congo red with 2N-HCl. The solution was extracted three times with light petroleum (b.p. 60-800). The petroleum was evaporated, leaving a colourless gum (0-05 g.), with the properties of tenuazonic acid as isolated from fraction D. The solid is therefore almost certainly a magnesium salt of tenuazonic acid. The yield was 0-6-0-9 g./bateh of 100 flasks.
Fraction C. The material removed during the first few hours of continuous extraction was largely solid, and was worked up separately. In the earlier batches, this gave a green ferric reaction, no doubt due to altertenuol (see below). In the case of the later batches, in which the preliminary acidification was carried out, the ferric reaction was purple, indicative of alternariol or its methyl ether. This part of fraction C has not been studied extensively, but the isolation of altertenuol, alternariol and alternariol methyl ether, by somewhat different separation procedures, is described below.
After the first few hours the continuous extracts yielded gums on evaporation, but some of these slowly crystallized. The crystals were very soluble in ether but much less so in CHC13, from which they could be recrystallized. The fractions were therefore allowed to stand with CHC13 (10 vol.) until crystallization was complete, and then filtered; further crystalline material was obtained on evaporation of the filtrate. In this way 2-3 g. of crude altenusin was obtained per batch of 100 flasks. After purification, it melted at 201.5-2030 (decomp.) and did not depress the m.p. of altenusin from strain no. 108. Fraction D. The benzene extract, after removal of the solvent under reduced pressure, left an orange-coloured gum. It was dissolved in a little ethanol, titrated to pH 7 with aq. N-NaOH, then treated with a chemically equivalent volume of 01 N-copper acetate. The mixture was extracted with CHCl3 until the extract no longer gave the orange ferric reaction. Evaporation of the CHCl3 yielded a green gum, which was dissolved in warm methanol (20 vol.) and treated with warm water (50 vol.). On cooling, the green copper salt crystallized; further quantities were obtained from the filtrate. The yield was 5-7 g./batch of 100 flasks. If prepared from freshly isolated tenuazonic acid, it was fairly pure. If necessary it was recrystallized from aq. methanol.
It is essential for this method of purification that altenusin should first be removed, since this substance is oxidized by copper acetate, resulting in pH changes and contamination of the copper tenuazonate. Exhaustive continuous extraction at pH 7 must therefore be carried out, to remove fraction C completely.
Tenuazonic acid is conveniently stored as the copper salt, whioh is quite stable. The free acid is readily regenerated by shaking the copper salt with CHCl3 and 2N-HCl until all the solid has disappeared, and all the blue-green colour is in the aqueous layer. The CHR13 layer is separated and the aqueous layer further extracted with CHC13. The combined CHC13 extracts are washed with 2N-HCl and water. Evaporation of the solvent leaves the tenuazonic acid as a nearly colourless gum, not crystallizing even on standing for some days at 050 Fraction E. The brown gum remaining after evaporation of the solvent was treated with dry ether (about 50 ml.) and allowed to stand. Nearly colourless crystals separated and were filtered. The filtrate was allowed to evaporate slowly in air, and was further treated with small volumes of ether and filtered. In this way crude mixed altenuic acids were obtained; the yields were variable, but usually ranged from 1 to 4 g./batch of 100 flasks.
Usually the first crop was fairly pure altenuic acid III, and could be recrystallized directly from aq. methanol. Some later crops gave little sign of the presence of altenuic acid III, judged by the FeCl3 test. These were separated by extraction with ether in a Soxhlet apparatus: this removed altenuic acid I, leaving the insoluble altenuic acid II in the thimble. The altenuic acid I could also be extracted with boiling water as described above. The altenuic acid I was recrystallized from water or ethanol, and altenuic acid II from aqueous dioxan.
Mixtures of altenuic acids I and III are difficult to separate by crystallization. However, the presence of these acids in mixtures could be detected by paper chromatography, which also separated them from alternariol and its methyl ether, present as impurities. The system used was butanol equilibrated with citrate or phosphate buffer (M/15) on Whatman no. 3 paper previously sprayed with the same buffer and dried (we are grateful to Mr N. Spencer for suggesting this system). Buffers of various pH values between5 and 6-5 were used, but the most convenient was found to be pH 5-25. Very good separation of altenuic acids I and III was achieved (R. values 0.50 and 0-36 at pH 5-25, citrate buffer), while the phenolic impurities had Rp>0-9.
The spray used was diazotized sulphanilic acid followed by Na2CO3.; the acids showed up as clearly defined brown spots.
These mixtures could also be analysed by using ethyl acetate-citrate buffer (pH 5.25), and this served as a model for separation on a preparative scale (0-5 g.) by means of a Craig countercurrent-distribution apparatus (capacity of tubes: 25 ml. + 25 ml.). Thus in one example a mixture (0-45 g.) was distributed through twenty tubes. One drop from the ethyl acetate layer of each tube was spotted on filter paper and detected with the above spray: this showed that there had been a clear separation into three zones, identified by paper chromatography as altenuic acid III (tubes 1-3), altenuic acid I (tubes 6-13), and alternariol and its methyl ether (tubes 17-20). Acidification and extraction of the first two fractions yielded practically pure altenuic acid III (0-25 g.) and altenuic acid I (0.10 g.).
Isolation of altertenuol. One of the earlier batches was extracted by hand at pH 7 with ether (3 x 1 vol.). After removal of the solvent there remained a solid, which gave a green ferric reaction. Crystallization first from ethanol, then twice from 90% acetic acid, yielded slightly buff-coloured needles, m.p. 283-285°with darkening and sublimation. The substance did not depress the m.p. of altertenuol isolated from strain no. 108, and the colour reaction with FeCl3 was identical.
Isolation and identification of alternariol and alternariol methyl ether. The first batch of 100 flasks of this strain was filtered and concentrated as described above. Inorganic material was filtered off, and the filtrate acidified to Congo red. The brown sludge formed was separated by centrifuging and dried (19.7 g.); the filtrate yielded in the manner already described a mixture of altenuic acids. The deposit (19.7 g.) was treated with dry ether (insoluble residue 3 05 g.), and the ether solution was extracted with aq. 2% NaHCO3. The ether layer was then evaporated, leaving a neutral, partly crystalline gum (1-45 g.). Most of the gum was removed on washing with ether, leaving a pale-brown solid (1.0 g.), which gave the ferric reaction of alternariol.
A sample heated in a high vacuum at 2200 partially sublimed, yielding a colourless sublimate, m.p. 2550 (decomp.); on raising the sublimation temperature to 250°a second colourless sublimate formed, m.p. 340-350°(decomp.). The first sublimate crystallized from ethanol in needles, m.p. 263-264°(decomp.) (uncorr.), undepressed on admixture with the alternariol methyl ether obtained from the mycelium, and giving thetypicalcolourreactionswithFeCl3 and conc. H2S04 (Raistrick et al. 1953). The second sublimate, after crystallization from aq. ethanol, was similarly shown to consist of alternariol. Theratio of alternariol toits methyl ether was approximately 1:4. Isolation of products from culture filtrates ofstrain no. 628 The ferric reaction of this filtrate was similar to that of no. 430, but the final colour was redder. Preliminary tests showed that a substantial amount of tenuazonic acid was present, but the other compounds appeared to be absent.
The culture filtrate was concentrated as already described, then extracted by hand at pH 7 with ether. Evaporation yielded only a small residue, which gave a brown ferric reaction.
The pH was adjusted to 1-2 with HCI, and the solution again extracted by hand with ether until the extract no longer gave the orange ferric colour. Evaporation of the ether yielded a brown gum (11 *2 g.). This was not completely petroleum-soluble, so was subjected to a preliminary purification by dissolving in ether (60 ml.) and adding light Vol. 67 petroleum (b.p. 40-60°, 200 ml.), which precipitated a gum. The solvents were decanted and evaporated. The residue (6-4 g.) was again dissolved in ether and the procedure repeated. After a third treatment, the crude tenuazonic acid (4-6 g.) was converted into the copper salt, which was purified as already described (yield, 4-1 g.); [x]20 1 -117 ± 50 in methanol (c, 0.2); a further quantity (0-4 g.) was obtained as a second crop. The precipitated gums were combined and retreated in the same way, to give a further quantity of copper salt (0-8 g.).

I957
Properties of altenuic acid III. Altenuic acid III crystallizes from aq. methanol or glacial acetic acid in colourless prisms. The m.p. is variable; material from aqueous methanol usually melts at about 1850 with effervescence, followed by resetting, complete by about 1950; a further melt occurs between 2150 and 2350 with effervescence. When crystallized from acetic acid, the substance melts first in the range 198-2020 with effervescence, resetting immediately, and remelting at about 2250 with effervescence. In addition, the acid crystallizes from ethyl acetate and water; the latter is not to be recommended for purification, since altenuic acid III is partly decomposed on boiling with water (Found: C, H,[4][5][4][5][6][7][8]OMe,equiv. by titration, 157. C15Hl408 requires C, H,lOMe, 1C-Me, 4-65%; equiv., titrating as a dibasic acid, 161). Altenuic acid III is readily soluble in ethanol and methanol, sparingly soluble in ether, and even less soluble in CHCI3, benzene or light petroleum. An ethanolic solution gives a pale-wine colour with ethanolic FeCI3, but on the addition of water this deepens to an intense purple. An aqueous solution gives no precipitate with Brady's reagent.

Conversion of altenuic acid I into altenuic acid III.
Altenuic acid I (19 mg.) was dissolved in a slight excess of 2N-NaOH, giving a pale-yellow solution. On acidifying to pH 2 with 2N-HCl a white precipitate formed, which was filtered (5 mg.). Ether extraction of the filtrate gave a further quantity (10 mg.). Unlike the starting material, which gives a pale purple-brown ferric colour in aqueous ethanol, this gave an intense purple coloration. The product crystallized from aq. methanol in prisms, m.p. 175-181°with effervescence, resetting onfurtherheating and remelting at 227-232°with further decomposition. The m.p. was essentially unchanged on admixture with altenuic acid III, but in view of the indefinite nature of this m.p. the product was methylated, to provide further evidence of identity.
The product (7 mg.) was treated with ethereal diazomethane at 00 for 0-5 hr. The solvent was removed and the residue crystallized from methanol to give colourless tablets, m.p. 142-145°, which did not depress the m.p. of the dimethyl derivative of altenuic acid III.
Additional confirmation of the conversion was provided by paper chromatography, with the system described above [butanol-citrate buffer (pH 5.25) on buffered paper]. The RF values were: altenuic acid I, 0-50; the above product, 0-37; altenuic acid III, 0-36.
Conversion of altenuic acid II into altenuic acid III. Altenuic acid II, similarly treated, gave an identical product, m.p. 175-181°with effervescence, resetting on further heating and remelting at 227-234°with further decomposition, and essentially unchanged on admixture with altenuic acid III. The methylation product melted at 142-142-5°, not depressed when mixed with a sample of the dimethyl derivative of altenuic acid III.
Confirmation was again provided by paper chromatography. Altenuic acid II does not give a coloured spot with the diazotized sulphanilic acid-Na2CO3 spray, but after treatment with alkali and reacidification it gives a brown spot, R. 0 35, at pH 5-25.
Altenusin and dehydroaltenwsin Properties of altenusin. Altenusin crystallizes from CHC13 in colourless prisms. After drying at 100°under reduced pressure, it melts at 202-203°with effervescence to a yellow liquid; if not dried, the crystals melt at about 950, resolidify, and remelt at the higher temperature. Altenusin can also be crystallized from benzene or water (Found, after drying at 100°in a high vacuum: C, 62-1; H, 5-2; OMe, 10-4; C-Me, 5-2. C,5H.406 requires C, 62-1; H, 4-9; lOMe, 10-7; 1C-Me, 5-2 %). The substance is readily soluble in ether, ethanol and methanol; it also dissolves in aq. NaHCO3. Its ethanolic solution, treated with ethanolic FeCl3, gives a pale-grey colour, turning to deep brown with excess. If the altenusin is dissolved in aq. ethanol, and aq. FeCl3 is added dropwise, the first drops give a violet colour, quickly fading to leave a colourless solution; with increasing amounts of FeCl3 this reaction is no longer observed, but a permanent intense brown colour appears. During the first stages of this reaction the presence of ferrous ions can be shown by the usual ferricyanide test. Altenusin does not reduce Fehling's solution in the cold, though the solution turns green; on boiling, the usual reduction takes place. However, cold aqueous copper acetate is immediately reduced on addition of an ethanolic solution of altenusin. An aqueous solution gives no precipitatewith Brady's reagentafterseveral hours.
(b) Altenusin was dissolved in a little methanol, cooled in anice bath and treated with excess ofethereal diazomethane. After standing overnight at 0-5°, the ether was evaporated. The gummy residue was dissolved in ether and the solution was filtered, washed with 2N-NaOH, then water, dried and again evaporated to dryness. The pale-brown gum was recrystallized several times from methanol to give colourless prisms, m.p. 116-117°, unchanged on admixture with the derivative above.
Tetramethylaltenusin shows no ferric reaction, is insoluble in cold 2N-NaOH, and gives no precipitate with Brady's reagent.
Properties of dehydroaltenusin. Dehydroaltenusin obtained from strain no. 94 crystallized from ethanol in yellow needles, m.p. 189-190°(decomp.) (Found: C, 62-4, 62-3; H, 4.4, 4-5; OMe, 11-1, 11-1. C15H1206 requires C, 62-5; H, 4-2; lOMe, 10-8 %). Dehydroaltenusin is only slightly soluble in water. Aq. NaHCO3 readily extracts the compound from ethyl acetate solution but dissolves the solid only slowly; the bicarbonate solution is coloured yellow. In aqueous NaOH the colour is yellow-green, soon changing to yellow. An ethanolic solution gives with FeCl3 an intense brown colour, identical with that produced by the action of excess of FeCl3 on altenusin; the colour is essentially unchanged by the addition of water. An ethanolic solution yields a red precipitate with Brady's reagent (see below).
Oxidation of altenusin to dehydroaltenusin with ferric chloride. Altenusin (100 mg.) was dissolved in a minimum volume of ethanol and an equal volume of water was added. Aq. FeCl3 was added dropwise with shaking until the evanescent violet colour was no longer produced, and a slight permanent brown could be seen. By this time a yellow precipitate had formed. After standing this was filtered, washed with water, dried (87 mg.) and recrystallized from methanol, from which it separated in yellow needles, m.p. 189-190°(decomp.), not depressed on admixture with the material obtained from strain no. 94 (Found: C, 62-2; H, 4-4; OMe, 11-1, 11-2, 11-1. Calc. for C15H1206: C, 62-5; H, 4-2; lOMe, 10-8%). The properties of this substance were identical with those of dehydroaltenusin, given above.
Reduction of dehydroaltenusin to altenusin. Dehydroaltenusin was dissolved in the minimum quantity of boiling ethanol and excess of saturated aqueous Na2S204 was added; the yellow colour was immediately discharged. The mixture was cooled and water was added. A colourless solid separated, and was filtered, dried and recrystallized from CHC13. Colourless crystals were obtained, which possessed the properties of altenusin; after drying at 1000 they melted at 198°(decomp.) (uncorr.), rising to 2010 (decomp.) (uncorr.) after admixture with altenusin.
It dissolves in conc. H2SO4 to give a lime-green solution which shows a blue fluorescence in u.v. light; solutions in ethanol are also fluorescent. It is insoluble in aqueous NaHCO8, but gives a yellow solution in aqueous Na2CO,or NaOH, deepening to orange on standing. In ethanolic solution FeCl8 gives a grey-green colour, which becomes a more intense bottle-green on adding water. Altertenuol does not react with Brady's reagent.

Tenuazonic acid and isotenuazonic acid
Properties of tenuazonic acid. Tenuazonic acid hasnot been obtained in crystalline form. When recovered from the pure copper salt it remains as a pale-brown viscous gum, which can be distilled in a high vacuum without essential change in properties or loss of optical activity. The distillate is a faintly straw-coloured gum, b.p. about 1170/0.035 mm.; 5461 -136 ± 50, 132 ± 20 in CHCl3 (c, 0-2, 0-5) (Found: C, 60-6; H, 7-9; N, 7-1%; equivalent by titration, 197. CLOH1503N requires C, 60-9; H, 7-7; N, 7-1%; equivalent (monobasic), 197). The compound is'readily soluble in all the usual organic solvents, including light petroleum, but is sparingly soluble in water. Its aqueous or aqueous ethanolic solution is strongly acidic. Addition of ethanolic FeCl3 to an ethanolic solution of tenuazonic acid produces a brilliant orange-red colour, unaltered by the addition of water. An ethanolic solution treated with excess of aqueous Brady's reagent soon clouds and gives a microcrystalline yellow precipitate. When tenuazonic acid is dissolved in an equivalent of aq. NaOH and treated dropwise with aqueous copper acetate, a green precipitate is formed, soluble in excess of copper acetate, and also soluble in CHC13.
Tenuazonic acid slowly changes on long standing. The optical rotation becomes less negative, and eventually crystallization begins, owing to separation of isotenuazonic acid (see below). It is difficult to separate pure tenuazonic acid from its mixtures with any substantial quantity of the iso-acid, and it is better to convert it immediately into the copper salt, which is stable.
The compound dissolves slowly in aq. NaHCO3 to give a dull yellow solution. A dilute ethanolic solution treated with a drop of aq. NaOH yields a deep-red colour. Addition of FeCl3 to an ethanolic solution produces only a slight deepening of colour.
Semicarbazone of tenuazonic acid. Tenuazonic acid (0 49 g.), hydrated sodium acetate (2-5 g.) and water (2-5 ml.) were warmed slightly to give a clear solution, cooled, and treated with semicarbazide hydrochloride (0-5 g.), which dissolved at once. After some hours a gum separated, which hardened on rubbing; the mixture was then left for some days at 0-5°to complete the separation of the derivative. The brownish solid was then filtered, washed with a minimum amount of ice-cold water and dried. The crude derivative (0.59 g.) was recrystallized from boiling water (7 ml.), cooling slowly with seeding to avoid separation as an oil. After 2-3 hr. a pale-brown first crop was filtered which had been standing at room temperature for 2j years, had darkened in colour, and was largely crystalline. It was dissolved in benzene, and some brown flocculent insoluble matter removed by filtration. The filtrate (15 ml.) was added slowly to light petroleum (b.p. 60-80°, 100 ml.) with shaking. More amorphous solid was precipitated, and was removed by filtration. After evaporation of the solvents, there remained a pale-yellow oil (1-13 g.) which soon began to crystallize, and which was dissolved without residue in light petroleum (b.p. 60-80°, 6 ml.). The solution was set aside to crystallize at 0-50 for 3 days, and then filtered, and the crystals were washed with a minimum volume of icecold light petroleum. The crude isotenuazonic acid (0-63 g.), m.p. 47-55°, was dissolved in light petroleum (b.p. 60-80°, 20 ml.) and passed through a short alumina column (4.5 cm. x 1-5 cm.) to remove impurities: this involved considerable loss, the eluate yielding on evaporation only 0-12 g. of nearly colourless crystalline material. Repeated recrystallizationfrom small volumes of light petroleum gave pure isotenuazonic acid (0.059 g.) as colourless needles,  , 60-9; H, 7-7; N, 7.1%; mol.wt., 197].
isoTenuazonic acid is very similar in chemical properties to its isomer. The two compounds are indistinguishable by the FeCl3 and copper acetate reactions, but Brady's reagent gives a yellow precipitate which is mainly amorphous. isoTenuazonic acid is unstable; even when kept in a desiccator it tends to liquefy slowly.
Copper salt of isotenuazonic acid. Prepared in the same way as copper tenuazonate, the copper salt separates from aq. methanol in green needles, m.p. from 1750; [c]461 I957 SUMMARY 1. The culture filtrates of each of four strains of Alternaria tenuis auct., grown on Czapek-Dox medium, have been shown to contain one or more of five metabolic products or groups of products.
2. One group, altemariol and alternariol methyl ether, has previously been shown to occur in the mycelium of this species; the remaining compounds have not previously been described.
3. A second group, the altenuic acids, consists of three closely related isomeric colourless substances, C15H1408, containing one carbon-methyl and one methoxyl group. Altenuic acid I, m.p. 183-184°, and altenuic acid II, m.p. 245-246o, are readily converted into altenuic acid III, which has a variable melting point and gives a strong purple colour with ferric chloride in aqueous ethanol.
5. Fourthly, altertenuol, C14H1006, buff-coloured needles, m.p. 284-285°, has been isolated in small amounts from two strains. It contains one methoxyl group, but no carbon-methyl group, and gives an intense green ferric colour.
8. The molecular formulae indicate some relationship between all these metabolic products except tenuazonic acid.