BIOCHEMISTRY
The Biosynthesis of Pulvilloric Acid. I. Production, Isolation, and Degradations* S. W. Tanenbaum and S. Nakajima
ABSTRACT : A
study of the environmental conditions needed for maximal laboratory yields of pulvilloric acid, an antibiotic substance produced by Penicillium pulvillorum, led to the estimated production in stationary cultures of quantities over 1.3 g/l. The requisite medium is 5 % glycerol-Czapek-Dox supplemented with 0.1 ml of corn steep liquor/l. Improvements in isolation and purification steps were found which overcame the major difficulty involved in work with this metabolite, i.e., its instability. A complete stepwise degradation procedure, adaptable for radioactive tracer experiments, was developed. This process involved hydrolysis of pulvilloric acid into the known 1-(3-dihydroxyphenyl)-2-heptanol,followed by methylation and dehydration which provided 1-(3,5dimethoxypheny1)-1-heptene. The latter intermediate was
P
ulvilloric acid is a yellow antifungal and antibacterial substance produced by Penicillium pulvillorum (Brian et al., 1957). Its molecular structure (I) has been determined by degradative (McOmie et al., 1963, 1966) and synthetic (Bullimore et al., 1967) studies. Both in biogenetic and structural terms, pulvilloric acid closely resembles two other secondary metabolites. These are citrinin, whose biogenesis has been worked out by several groups (Birch et al., 1958; Schwenk et at., 1958; Rodig et al., 1966) and has been shown to involve the acetate-polymalonate single carbon-transfer pathways; and ascochitine, more recently characterized by Iwai and Mishima (1965). This paper reports strain and fermentation improvements which have lead to consistent, high yield production of pulvilloric acid; and describes a facile isolation method for obtaining the analytically pure antibiotic. As a preliminary to radioactive tracer experiments on the elucidation of the biosynthesis of pulvilloric acid, a carbon-by-carbon degradative procedure, with satisfactory yields of intermediates at each stage, has been developed. Experimental Procedures and Results Fermentations. It was earlier reported (Nakajima and Tanenbaum, 1968) that strain selection by sectoring of P. pulvillorum (ACC 1124) as originally received, led to isolates
* From the Department of Microbiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032. Received June 6, 1969. This investigation was supported by a grant from the Public Health Service (AI-06801). Preliminary accounts of this work have been presented at the 3rd International Fermentation Symposium, Rutgers, N. J., Aug 1968; and a t the 156th National Meeting of the American Chemical Society, Atlantic City, N. J., Sept 1968.
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cleaved, either by use of ozone or better with permanganate, into 3,5-dimethoxybenzoic and hexanoic acids. The aromatic moiety was converted into styphnic acid cia 3,5-dimethoxy2,6-dinitrobenzoic acid, 4,6-dimethoxy-1,3-dinitrobenzene, and 2,4-dimethoxy-l,3,5-trinitrobenzene. It was further degraded by the bromopicrin method. The hexanoic acid was degraded by successive stepwise Schmidt reactions. The terminal two carbons in the aliphatic side chain of pulvilloric acid could also be directly liberated as acetic acid by the Kuhn-Roth oxidation. Attempts to synthesize carrier quantities of 1-(3,5-dimethoxypheny1)-2heptanol from hexanal and 3,5-dimethoxybenzylmagnesium bromide led only to the isolation of bis-l,2-(3,5-dimethoxypheny1)ethane.
which exhibited diminished formation of intractable, violetcolored side products. The replacement of glucose by glycerol in the standard Czapek-Dox medium (Figure 1) caused a marked increase in product yield, which is analogous to earlier findings with Raulin-Thom medium (Brian et al., 1957). The addition of 0.1 ml of corn steep liquor (Corn Products Co. Steepwater E801)/1. further enhanced the production of pulvilloric acid. In 500-ml erlenmeyer flasks, yields of 1.3 gfl. of pulvilloric acid were routinely attained. As seen from these data, maximal production of metabolite was noted during 14-17-days growth. Using 2.8-1. fernbach flasks with 500 ml Czapek-Dox medium modified as given above, estimated yields (see legend to Figure 1) of 1.5 g/l. of the metabolite were frequently found. The alternative synthetic medium described previously (Nakajima and Tanenbaum 1968), favored accumulation of glycerides rather than pulvilloric acid formation. Isolation and Purification. After incubation for 15 days at 26O, the culture filtrates were strongly acidified by careful addition of concentrated HCl(200 ml/l,). This step was found helpful in preventing emulsification which interferes with the separation of organic and aqueous layers on subsequent extraction. The acidified beer was then extracted twice with ether (200 rnlil.), and the combined organic layer was filtered through Whatman No. 1 paper to remove small amounts of emulsion. The ethereal extract was washed with water, dried over Na2S04, and was then evaporated to dryness in cacuo at room temperature. The crude residual, brown-yellow powder was in a Illinirna1 Of and was subjected to chromatography on anhydrous MgS04. Reagent grade diethyl ether was used for development. ~~l~~~~ of 2 40 cm dimensions were used to resolve the process can be amounts Of crude scaled up with little difficulty. Optimal yields of highly purified
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product accrued when the chromatographic column was developed in dim light. The violet-colored impurities, presumably decomposition products of pulvilloric acid, remained a t the upper portion of the chromatograms, while pulvilloric acid readily progressed through the column as a bright yellow band. The eluate was immediately concentrated under a steam of nitrogen. The precipitated pulvilloric acid was then treated in ether with a small amount of decolorizing charcoal, and was again subjected to the chromatographic step. Upon addition of a small amount of hexane to the concentrated eluate, crystallization of pure, bright yellow needles of pulvilloric acid commenced. The product had the following characteristics:' mp 76.5-77.5' (softens at 71S0), 255 mp (log e 4.02),and 313 mp (log E 3.51); XzBc:ohexane215 (log E 4.07), 227 (4.00),263 (3.62),317 (4.16),and 387 mp (3.37). Its methanol adduct melted a t 88-89'. Chemical Degradations (Figure 2). HYDROLYSIS OF PULVILLORIC ACID (I). The procedure of McOmie et a f . (1966), with some modifications for tracer techniques, was followed. In a 100-ml three-necked flask provided with a nitrogen inlet tube, a dropping funnel, and a reflux condenser connected with baryta traps, was placed 1.6 g of NaOH in 16 ml of C0,-free water. Pulvilloric acid (268 mg) was then added, the temperature was slowly raised to 50', and was maintained at this point for 1 hr. The reaction mixture was then refluxed for 4 hr. After cooling, 15 ml of 30% H2S04was cautiously added. The resultant BaC03 was collected and was washed with water and acetone. After being dried in L;acuo over P206, the yield was 57 mg (31 %). The acidified reaction was steam distilled. The distillate, which contained formic acid, was neutralized with NaOH. and was concentrated in cacuo to 10 ml. The p H was adjusted to 5 with HCI, and the solution was refluxed for 10 min under a nitrogen stream to remove Cor. Pirie's solution (250 ml) (Pirie, 1946) was then slowly added, which resulted in the formation of 81 mg (53%) of BaC03. In another modification, the formate was converted into its p-formaminoazobenzene derivative (Kinoshita and Nakajima, 1960). This derivative was purified by silica gel chromatography, and was recrystallized from chloroform (mp 162'). The formate oxidation mixture was filtered while still hot, which removed resinous by-products. The filtrate was decolorized with charcoal, and upon cooling there was deposited colorless flakes of pure 1-(3,5-dihydroxyphenyl)-2-heptanol (11), mp 144'. The yield was 154 mg (60%). 1-(3,5-DIMETHOXYPHENYL)-2-HEPTANOL (III). The detailed preparation of this compound is reported elsewhere (Barrett et af., 1969). In essence, the methylation of I1 was carried out with methyl iodide in acetone in the presence of anhydrous KICOP.The dimethoxy derivative (111) (bp 205-210" (10mm)) was formed in 92% yield. 1 -(3,5-DIMETHOXYPHENYL)-l-HEPTENE (v). A. Dehydration with KHSOI. Potassium hydrogen sulfate (5 g) was melted at 220-230' under a nitrogen atmosphere, and 1.8 g of 1-(3,5-dimethoxyphenyl)-2-heptanol(111) was slowly added in the course of several hours. After cooling, the fused melt was taken up in water and was extracted with ether. The extract was washed with water, dried, and was distilled
1 There are minor differences in these data from those which are recorded by McOmie et al. (1966).
'.c
t
Time, days
1: Chronologic analysis of pulvilloric acid production in stationary cultures at 26". Erlenmeyer flasks (500 ml), each containing 200 ml of the appropriate medium were used. Amounts of pulvilloric acid (each point averages duplicate determinations) were estimated by acidification of aliquots of the beer with HCl, ether extraction, evaporation to a residue, and measurement of the OD of the alcohol-soluble portion at 255 mp. Standard CzapekCzapek-Dox-glycerol); Dox (A-A); Czapek-Dox-glycerol (e. corn steep medium ( G O ) ; synthetic medium of Nakajima and Tanenbaum (1968; A-A). FIGURE
in vacuo t o give a first colorless fraction (bp 120°,(0.7 mm), 800 mg).This was followed by a small (50 mg) higher boiling fraction (bp 180-250' (0.7mm)). Anaf. Calcd for C1SHz202: C? 76.88; H, 9.46. Found: major fraction, C, 76.89;H , 9.50;minor fraction, C, 76.15; H, 9.33. The peak positions for ultraviolet light absorption of both components were identical A(?": 259 mp and 293 mp). These data suggest that both cis and trans isomers of 1-(3,5-dimethoxypheny1)-1-heptene were formed during the course of the elimination reaction. B. Chugaev Reaction. 1 -(3,5-Dimethoxyphenyl)-2-heptanol was converted either into its methyl xanthate (IVa) or amyl xanthate (IVb) derivative by the procedure of Sjoberg et a/. (1962). The purified xanthates were decomposed by heating at 220' (oil bath). The resultant 1-(3,5-dimethoxyphenyl)-lheptene (V) was purified as outlined above. Yields of V were 86 from the methyl and 96 cia the amyl xanthate, respectively. CLEAVAGE OF 1-(3,5-DIMETHOXYPHENYL)-l-HEPTENE.1-(3,5Dimethoxypheny1)-1-heptene (1.5 g) was dissolved in 80 ml of acetone. Powdered K M n 0 4 (5.4 g) was added with cooling during the course of 30 min. The reaction was stirred overnight at room temperature and was made alkaline with dilute KOH.
ISOLATION A N D DEGRADATION OF
PULVILLORICACID
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BIOCHEMISTRY
C5HllCOOH
-
1
I NaN3 HG04
2 KMn04 3.Repeat l a 2
P
2,3.4,5,6
5 c02 I
=a E b
R=CSSCH~ R=CSSC~HII
+
CH3NH2
X FIGURE 2 :
Stepwise degradation procedure for obtaining the individual carbon atoms of pulvilloric acid.
The precipitated MnOy was filtered, washed with water and then with NaHC03, and the combined filtrates were evaporated to dryness. The residue was taken up in dilute H2S04 and was steam distilled. The neutralized distillate was concentrated, giving the crude fatty acid sodium salts. These were treated with H2S04,and the liberated fatty acids were extracted into 50 ml of chloroform.* The resultant fatty acid mixture when assayed by gas-liquid partition chromatogr a p h ~was ~ shown to contain hexanoic (64%), valeric (24%), butyric (8%), propionic (1 Z), and acetic acids (3%). The fatty acids were separated by passage of the chloroform through a glycine-buffered silica gel column (5.5 X 27 cm) which contained cresol red (Nakajima and Tanenbaum, 1969). The yield of pure hexanoate which was obtained after this chromatographic purification on silica gel was 140 mg (16%). Treatment of the warmed residual reaction mixture with a 2 This CHCla solution should not be concentrated, since a large proportion of hexanoic acid will be lost. 3 Gas-liquid chromatography was performed with an F & M Model 402 gas chromatograph equipped with a Disc Instruments 228A chart integrator. Column: 6 poly(diethyleneglyco1 succinate) on 8Cb100 mesh Diatoport S . It was found (Nakajima and Tanenbaum, 1969) that the free fatty acids, in chloroform solution, could be directly analyzed when the injection port, column, and flame detector block temperatures were kept at 135, 110, and 150", respectively, Comparable results were found on analysis of the fatty acids as methyl esters, by the protocols previously described (Nakajima and Tanenbaum, 1968).
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small amount of charcoal, filtration, and then chilling, provided 3,5-dimethoxybenzoic acid (VI; 560 mg, 66 %). An analytical sample made by repeated recrystallization (mp 184') possessed an infrared spectrum identical with that of the authentic material. 3,5-DIMETHOXY-2,6-DINITROBENZOICACID (VII). 3,5-Dimethoxybenzoic acid (10 g) was carefully added in small portions to 40 ml of fuming nitric acid (90%) which had been precooled to -40' in a Dry Ice-acetone bath. After an initial 6 g of (VI) had been caused to react, another 40 ml of nitric acid was added, followed by the remainder of the starting material. The mixture was then stirred at room temperature for 15 hr. Dilution with 300 ml of ice-water gave the crude product. This was collected, dissolved in NaHC03, and filtered. Acidification deposited crystals of VII. It was crystallized from methanol as yellow cubes, mp 261-265"; yield, 8.0 g ( 5 4 z ) . Anal. Calcd for CsHsNzOs: C, 40.11; H, 3.00; N, 9.80. Found: C, 39.72; H , 2.96; N, 10.29. Spectral studies showed CHCll A,, 832, 1598, 3057 (aromatic); 1050, 2822 (OCH3); 1337, 1447,1711 (COOH); and 1550(N02)cm-l. 4,6-DIMETHOXY-I ,3-DINITROBENZENE (VIII). 3,5-Dimethoxy2,6-dinitrobenzoic acid (1 gm) was heated at 265" (bath temperature). Vigorous and complete decarboxylation took place within 5 min. The crystalline mass was washed with NaHC03, then water, and was dried. Crystallization from ethanol-ethyl acetate gave 775 mg (9373 of orange prisms, mp 155.5'. Dreyer et a/. (1964), who previously synthesized
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VI11 by nitration of 1,3-dimethoxybenzene, reported the same melting point. 2,4-DIMETHOXY-I ,3,5-I KINITKOBENZCNE (1x). A PreViOUSly cooled mixture of 1.5 ml of HaS04 and 1.0 ml of fuming nitric acid (9073 was added to 300 mg of 4,6-dimethoxy-1,3dinitrobenzene. The resultant suspension was left at room temperature for 2 hr, during which solution occurred, followed by deposition of fine crystals. After further dilution with icewater, the precipitated material was collected to give 320 mg (92 %) of crude product. Recrystallization from methanol afforded yellow prisms, mp 126" (lit. (Dreyer ef a/., 1964) mp 126'). STYPHNIC ACID (X). DEMETHYLATION OF IX. A suspension 2,4-dimethoxy-1,3,5-trinitrobenzene (212 mg) was refluxed for 4 hr with 10 ml of 4 8 x HBr. The starting material initially dissolved, but was followed by precipitation of the product. After dilution with water, (X) was collected by filtration, yield, 169 mg (8873. It was crystallized from dilute alcohol, mp 177". Further identification was made by comparison of its infrared spectrum with that of an authentic sample. This demethylation could also be effected with concentrated HC1 in 44 yield. Standard Degradatice Procedures. Brompicrin reactions with styphnic acid were carried out essentially as described by Werbin and Holoway (1956). Adherence to a reaction time of 40 min in ice-water and then another 40 min at room temperature gave brompicrin free of CBr4 or CHBr3 (cf. also Birch er a/., 1962). Stepwise Schmidt degradations followed the method of Phares (1951). Kuhn-Roth oxidation of pulvilloric acid was performed using the protocol of Awasthy et a/. (1967), which includes M n S 0 4 in the reaction medium. Purification of the mixed fatty acids resultant from Schmidt and Kuhn-Roth oxidations were carried out by the procedure indicated earlier. a BIS-l,2-(3,5-DIMETHOXYPHENYL)ETHANE.The starting material, 3,5-dimethoxybenzyl chloride, was prepared from 3,5-dimethoxybenzoic acid by reduction with LiAIH, in tetrahydrofuran (Wenkert et ul., 1964) followed by chlorination with SOClr (Adams et a/., 1948). To 7.2 g of Mg turnings covered with 150 ml of absolute ether was added 5 g of the benzyl chloride in 30 ml of ether, followed by a crystal of iodine. The remainder of the acid chloride (total, 50 g in 300 ml of ether) was run into the reaction slowly, and heat was applied to keep the mixture under reflux for 1 hr. The Grignard reagent was then slowly reacted with 38 g of hexanal in 100 ml of ether, and the reaction was maintained under reflux for a n additional 2 hr. Upon cooling, 300 ml of water was cautiously added for decomposition of the reaction complex. Following the addition of dilute HnS04,the products were extracted into ether. The organic phase was washed with N a n S 0 3and water and was dried over Na2S04.Removal of solvent gave a yellow oil, which was triturated with 500 ml of petroleum ether (bp 30-60'), and was then kept in the cold. The product (6.2 g) crystallized from methanol as leaflets, mp 105.5". Infrared analysis 840, 1600, 3000 (aromatic), 1460, 2850, 2940 (CH?), 1060, and 1200 (aromatic ether) cm-*) demonstrated that the compound lacked a hydroxyl group, but contained instead an ether linkage. These spectral data suggested that the compound was XI. This supposition was confirmed on elemental analysis. Anal. Calcd for CI8HTYOI:C, 71.50; H, 7.33. Found: C, 71.43; H, 7.67.
I S O L A T I O N A N D
Discussion By a combination of strain improvement, medium modifications, and changes in the isolation procedure, the yield of pulvilloric acid from stationary cultures of P. pulriilorwn was increased approximately twofold over that which WAS reported by the original investigators (Brian et ai., 1957). Of particular relevence in obtaining pure pulvilloric acid is adherence to details of the fermentation work-up methodology. Isolation attempts based on earlier procedures, which included chloroform extraction of beers and solvent evaporation at atmospheric pressure (Brian et a/., 1957), together with crystallization from toluene in the presence of ethanol (McOmie et a/., 1966), only confirmed prior observations on the accumulation of considerable quantities of brown-yellow to violet-colored resinous material. In order to prevent such losses, which are presumably due to rearrangements and decomposition reactions of crude pulvilloric acid, the MgS04 chromatographic separation described here was substituted. This step was best carried out in subdued light using ether for development, and the solvent was removed at reduced pressure under a nitrogen stream. When these precautions were followed, the metabolite was routinely obtained in good yield as bright yellow needles of apparent homogeneity. The degradation procedure for pulvilloric acid (Figure 2) which embodies individual stages of relative experimental ease and high yield, was developed as a prelude to a radioactive tracer examination of the biosynthesis of this compound. Initially in the study of these proposed degradation reactions, a n attempt was made to synthesize the required intermediate 1-(3,5-dimethoxyphenyl)-2-heptanol (111) from 3,5-dimethoxybenzyl chloride in a Grignard reaction with hexanal. However, instead of 111, the hitherto unreported side-product from this reaction, bis-1,2-(3,5-dimethoxypheny1)ethane (XI), was obtained. This approach was then abandoned in favor of accumulating I11 cia the known alkaline rearrangement of I, which had now become readily available due to the foregoing fermentation and isolation modifications. Furthermore, while the dehydration of 1-(3,5-dimethoxyphenyl)-2-heptanol (111) to 1-(3,5-dimethoxyphenyl)-l-heptene (V) could be effected in moderate yield simply by heating with K H S 0 4 , the yield of olefin was increased to almost quantitative by employing the two-step Chugaev reaction on the amyl xanthate of V. Similarly, the important cleavage reaction of 1-(3,5-dimethoxyphenyl)-l-heptene (V) into 3,5-dimethoxybenzoic (VI) and hexanoic acids, first carried out in 7 z yield by ozonolysis, was later improved by substituting instead of permanganate oxidation. The remainder of the degradative pathway then followed fairly standard isotopic technics. The application of this series of reactions in biosynthetic experiments on pulvilloric acid formation is given in the accompanying paper (Tanenbaum and Nakajima, 1969). References Adams, R., MacKenzie, Jr., S., and Loewe, S. (1948), J. Am. Chem. SOC.70,664. Awasthy, A. K., Belcher, R., and Macdonald, A. M. G. (1967), J. Chem. SOC.,C , 799. Barrett, G. C., McOmie, J. F. W., Nakajima, S., and Tanenbaum, S. W. (1969),J. Chem. Soc., C, 1068.
D ~ , G R A D A T I O NO F
PULVILLORIC A C I D
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BIOCHEMISTRY
Birch, A. J., Fitton, P., Pride, E., Ryan, A. J., Smith, H., and Whalley, W. B. (1958), J. Chem. Soc., 4576. Birch, A. J., Moye, C. J., Rickards, R. W., and Vanek, Z. (1962), J. Chem. Soc., 3586. Brian, P. W., Curtis, P. J., Hemming, H. G., and Norris, G. L. F. (1957), Trans. Brit. Mycol. SOC.,40, 369. Bullimore, B. K., McQmie, J. F. W., and Turner, A. B., Galbraith, M. N., and Whalley, W. B., (1967), J. Chem. SOC.,C, 1289. Dreyer, D. L., Tabata, S., and Horowitz, R. M. (1964), Tetrahedron20,2977. Iwai, I., and Mishima, H., (1965), Chem. Znd. (London), 186. Kinoshita, K., and Nakajima, S. (1960), Chem. Pharm. Bull. (Tokyo) 8,56. McQmie, J. F. W., Turner, A. B., and Tute, M. S. (1966), J. Chem. Soc., C, 1608. McQmie, J. F. W., Tute, M. S., Turner, A. B., and Bullimore,
B. K. (1963), Chem. Znd. (London), 1689. Nakajima, S., and Tanenbaum, S. W. (1968), Arch. Biochem. Biophys. 127, 150. Nakajima, S., and Tanenbaum, S. W. (1969), J . Chroinatog. 43,444. Phares, E. F. (1951), Arch. Biochem. Biop1ij.s. 33, 173. Pirie, N. W. (1946), Biochem. J. 40, 100. Rodig, 0. R., Ellis, L. C., and Glover, I. T. (1966), Biochemistry 5, 2458. Schwenk, E., Alexander, G. J., Gold, A. M., and Stevens, D. F. (1958), J . Biol. Chem. 233, 1211. Sjoberg, B., Cram, D. J., Wolf, L., and Djerassi, C. (1962), Acfa Chem. Scand. 16,1079. Tanenbaum, S. W., and Nakajima, S. (1969), Biochemistrj8 , 4626. Wenkert, E., Loeser, E. M., Mahapatra, S. N., Schenker, F., and Wilson, E. M. (1964), J. Org. Chem. 29,435. Werbin, H., and Holoway, C. (1956). J . Bid. Chem. 223, 651.
The Biosynthesis of Pulvilloric Acid. 11. Studies on Incorporation of Radioactive Precursors” S. W. Tanenbaum and S. Nakajima
: The biosynthesis of pulvilloric acid, an unstable secondary metabolite produced by Penicillium pulvillorum, was studied with the use of 14C-labeled small precursor molecules. Radioactive pulvilloric acid obtained from acetate-l-14C supplementation was subjected to a complete series of degradations. Of the fifteen carbon atoms in the molecule, nine were assayed individually for their specific radioactivities. The results revealed that the acetate-polymalonate pattern, though possibly not involving a single C14 polyketide chain, was operative in its biogenesis. Following growth experiments in the presence of H’COOH and partial degradation of the resultant antibiotic, the carboxyl ABSTRACT
P
ulvilloric acid (I) is a metabolite accumulated by Penicillium pulvillorum (Brian et al., 1957) which is closely related in its structure to ascochitine (Ia; Iwai and Mishima, 1965) and to citrinin (11), also secondary metabolites of higher fungi. Of biosynthetic interest is the fact that pulvilloric acid contains a n-pentyl side chain at a carbonyl-derived point of
* From the Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032. Received June 6, 1969. Brief accounts of this work were presented at the 3rd International Fermentation Symposium, Rutgers, N. J., Aug 1968, and at the 156th National Meeting of the American Chemical Society, Atlantic City, N. J., Sept 1968. This investigation was supported by a grant (AI-06801) from the U. S . Public Health Service.
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N A K A J I M A
function of pulvilloric acid was demonstrated to have arisen from the C1-transfer pool. These findings parallel the known pathway of biosynthesis of citrinin, a metabolite closely related in structure. Ethyl h e ~ a n o a t e - l - ~was ~ c fed to the growing mold to test the possible role of hexanoyl coenzyme A or other C,~4‘-phosphopantetheineintermediate as a “starter” in pulvilloric acid formation. However, the antibiotic which was isolated in this experiment exhibited an isotopic distribution pattern which reflected its prior degradation to the level of acetate-l-IC. Alternative pathways for the biosynthesis of pulvilloric acid, and their biogenetic relationships to structurally similar polyacetate metabolites and to the formation of fatty acids, are discussed.
juncture with its benzopyran ring system; it thus presents biogenetic features which may also relate it to many lichen depsides and depsidones (Asahina and Shibata, 1954) which embody the olivetolcarboxylic acid fragment (111). In light of the knowledge that the biosynthesis of citrinin (Birch et al., 1958; Schwenck el al., 1958; Rodig et al., 1966) has been shown to involve the acetate-polymalonate condensation route, followed by donation of extracyclic methyl groups as well as the carboxyl function from C1-transfer systems, a grossly similar pathway for pulvilloric acid formation was anticipated. From the work of Curtis el d . (1968) with mutant strains of Penicillium citrinum and their accumulation products, it would appear furthermore that C-acyl-o-orsellinic acids might be involved in the biosynthesis
