biosynthesis of natural products14]. In this reaction a hydroxy group has to be removed from C-6 and an additional one in- corporated at C-4. Thus besides emodin (l), the natural an- thraquinones chrysophanol (2), islandicin (3), and catenarin (4), whose hydroxy group arrangements resemble fully or partly that of a half-molecule of (5) (see box), are also of in- terest as intermediates in biosynthesis.

OH 0 OH OH 0 OH

OH 0 OH

COzCH3

Table 1. Competitive incorporation of the radioactive anthraquinones ( l ) - ( 4 ) in secalonic acid D (S) by Penrcillium oxulicum [a].

Expt. Anthraquinone Spec. a/b incorp. [b] WI

1.03 a) [U-"'C]emodin ( I ) 0.432 b) [U-'Hlemodin ( I ) 0.418

1

~~

a) [U-'4C]chrysophanol (2) 3.42 2 3.56 b) [U-'Hlemodin (I) 0.96

~~

0.301 a) [U-'"C]islandicin (3) 0.163 b) [U-'Hlemodin ( I ) 0.541

3

0.022 a) [U-''C]catenarin (4) 0.008 4 b) [U-'HH]emodin (I) 0.37

[a] ATCC 10476. [b] Specific radioactivity of the isolated secalonic acid D (5) divided by that of the fed anthraquinone and by the number of anthraquinone molecules which can be incorporated in (5) x 100.

For the competitive incorporation, pairs composed of bio- synthetically prepared uniformly labeled [U-3H]emodin (1) and one of the [U-I4C]anthraquinones (1) to (4)151 were fed to the mold Penicilliurn onalicum[61. After isolation and purifica- tion to constant radioactivity, the 3H/'4C ratio in (5) was de- termined in a scintillation counter (Table 1).

As experiment 1 shows, [U-3H]emodin loses only 3% or less of its tritium radioactivity (predominantly located in the methyl group) during conversion into (5) by the mold. It is therefore suitable as reference compound for competitive in- corporation reactions. From the incorporation values (ratio a/b) referred to emodin ( I ) of the other experiments it signif- icantly follows, irrespective of deviations of the enzyme ac- tivity of various components, that chrysophanol (2) (Experi- ment 2) is incorporated into (5) 3.6 times better than (1) and 11.8 times better than (3) (Experiment 3) . In contrast, (4) can be ruled out as a biosynthetic prec~rsorl'~.

From these findings it can be concluded that emodin (1) and islandicin (3) are converted into the ergochromes, e.g.

(5), via chrysophanol (2). The hydroxy group on C-4 is thus incorporated after oxidative ring opening which precedes the dimerizatiod3.*1. The preferred suitability of (2) for prepara- tively useful biomimetic ring-opening reactions is already well known'''.

Received: March 20. 1980 [Z 466a [El German version: Angew. Chem. 92, 483 (1980)

CAS Registry numbers: (I), 518-82-1; (2), 481-74-3; (3), 476-56-2 (4). 476-46-0; (5), 35287-69-5

111 B. Frunck. G. Fels. G. Ufer, Angew. Chem 89,677 (1977): Angew Chem. Int

[2] B. Frunck, H Flusch, Fortschr. Chem. Org. Naturst. 30. 151 (1973). [3] B. Frunck in P. S. Sreynt The Biosynthesis of Mycotoxins. Academic Press,

New York, in press. [4] B. Frunck, Angew. Chem. 91, 453 (1979): Angew Chem. Int. Ed. Engl. 18,

429 (1979). 151 [U-'4C]islandicin (3) and [U-'4C]catenarin (4) were obtained directly from

12-'4C]acetate with Penrcillium islundrcum (ATCC 10 127) and Helmmthospo- rium cutenurium (CBS 191.29). respectively. [U-'H]- and [U-"'C]emodin ( I ) and [U-'4Cjchrysophanol (2) were obtained from [2-ZH,]acetate and [2- ''C]acetate respectively with Penicillium islundicum (CBS 338.48) and Tulu- romyces worrmunnii (ATCC 10517) by reductive and alkaline cleavage of the initially formed bisanthraquinonyls skyrin and rugulosin (ATCC. American Type Culture Collection; CBS: Centralbureau voor Schimmelcultures, Baarn, Holland).

Ed. Engl. 16, 652 (1977).

[6] P. S. Sreyn, Tetrahedron 26. 5 1 (1970) [7] Since none of the anthraquinones (I) to (4) have previously been found in

cultures of Penicillium oxulicum the incorporation values might not be essen- tially determined by different pool sizes.

[8] I . Kurobune. L. C. Vining. Tetrahedron Lett. 1978, 1379. [9] B. Frunck, B. Berger-Lohr, Angew. Chem. 87.845 (1975): Angew. Chem. Int.

Ed Engl. 14, 818 (1975).

Citreoviridins from Aspergiffus terreus[**l By Burchard Franck and Henning-Peter Gehrkenl'] Dedicated to Professor Rolf Huisgen on the occasion of his 60th birthday

The potent neurotoxic mycotoxin citreoviridin ( la ) is re- sponsible for the epidemic-like occurrence of cardiac beri- beri''] in East Asia. Its acute toxicity, comparable to that of the aflatoxins, is caused by inhibition of the mitochondria1 ATP synthetases['I. ( la) was previously found in moldy rice and in Penicillium citreoviride and related Penicillium spe- c i e ~ ~ ~ ] . We isolated citreoviridin ( la) and further previously unknown derivatives of similar structure from the mold As- pergillus terreus, one of the commonest organisms occurring in soil, on various types of grain and sugar-containing nu- trients and which is used biotechn~logically~~~. It deserves special mention that the occurrence of citreoviridin, which belongs to the most dangerous mycotoxins, is not restricted to East Asian mold fungi, and that the citreoviridin produc- tion by strains of Aspergillus terreus is extremely high-up to 2% of the dried mycelium. This made possible a thorough biogenetic analysis of the mycotoxin.

On closer examination of Aspergillus terreus for active sub- stances, we discovered in the case of 5 of 13 strains['] brilliant yellow metabolites showing typical polyene UV/VIS spectra. The main metabolite (deep-yellow needles, m. p. 109- 110 "C, from MeOH, A,,, = 237,287,295, 388 nm) proved to be identical to citreoviridin (la)[61; in addition we found

['I Prof. Dr. B. Franck, Dr. H.-P. Gehrken Organisch-chemisches Institut der Universitat Orleansring 23, D4400 Munster (Germany)

["I Metabolites of Fungi, Part 31.-Part 30: B. Frunck, G. Bringmunn. G. Flohr. Angew. Chem. 92. 483 (1980); Angew. Chem. Int. Ed. Engl. 19. 460 (1980).

Angew. Chem. In!. Ed. Eng!. 19 (1980) No. 6 0 Verlug Chemie, GmbH, 6940 Weinherm, I980 OS70-0833/80/0606-461 $ 02.50/0 46 1

small amounts of five very similar metabolites. We therefore give the citreoviridin (la) the suffix "A" and the other com- ponents in order of their R, values (1.44, 1.35, 1.24,0.72,0.62; ethyl acetate on TLC strips SI F-Riedel de Haen) referred to (la) = 1.00 as B, C, D, E, and F. The components C and D could be assigned the structures (lb) and (lc) from spectros- copic data. Fragments formed in the mass spectrometer by cleavage of the C-5/C-6 bond indicated that (la), (lb), and (lc) differ only by methyl groups on C-2 and C-4. Accord- ingly, only the signals of C-2 and C-4 showed significant dif- ferences in the I3C-NMR spectra ([D6]acetone).

( l a ) , R' = H, R2 = CH,; ( I h ) , R' = CH3, R 2 = H; ( l r ) , R', R 2 = CH3

OCH? 0

0 ACH3 0 COZH Ho

2, (3) x, 0 : labeling with 11 -13C]-

and [2-'3C] ace ta te , r e s p .

Table 1. "C-NMR data and "C-enrichment of the isotopomeric citreoviridins A (la), which were obtained after feeding [l-"C]- and [2-'3C]acetate on Aspergillus terreus (CBS 503.65) (measured in [Dolacetone, values of the enrichment in mul- tiples of the natural abundance [7]).

C atom S and "C-enrichment multiplicity (la) from (la) from

[ 1 - "C]acetate [2-"C]acetate

c-1 c -2 c -3 C-4 c - 5 C-6 C-7 [a] C-8 [b] C-9 [a] C-10 [b] C-11 [a] c-12 C-13 [a] c-14 C-15 C-16 C-17 c-18 c-19 c-20 c-21 c-22 OCH,

163.5 s 88.9 d

171.2 s 108.3 s 155.0 s 119.6 d 136.3 d 127.0 d 139.4 d 131.5 d 142.2 d 132.9 s 144.5 d 84.8 s 86.5 d 80.8 s 78.5 d 13.0 q 8.94 q

21.8 q 19.3 q 13.5 q 56.8 q

10.32

9.44

9.58

-

-

- 19.4

14.9

17.8

16.4

13.6

18.0

-

-

-

-

-

- - - - - -

-

26 2

17.0

21.4

23.7

21.2

18.9

17.4

18.8

24.6

-

-

-

-

-

-

-

-

- - - - -

[a, b] These assignments can be interchanged

The structure of citreoviridin A (la) suggests a biosynthe- sis from nine acetate moieties via acetyl- and malonylcoen- zyme A and the Ci8 polyketide (3), and its C-methylation at four positions (C-4, C-12, C-14, C-16). This is consistent with previous incorporation with acetate and me- thionine, in which, however, only few positions of incorpora- tion could be determined by chemical degradation because of labeling with 14C. Application of [1-'3C]- and [2-13C]ace- tate (90% 13C-enrichment; 200 mg/100 ml nutrient) to Asper-

gillus terreus (CBS 503.65)" gave isotopomeric citreoviridins A (la). Their unusually high incorporation of 17.6 and 25.7%, re~pectivelyr~], enabled accurate determination of I3C- positions by I3C-NMR spectra (Table 1). It follows from the spectra that all odd C-atoms ( x ) of polyketide chain (3) of (la) originate from the carboxy groups of the acetate build- ing block and the even C-atoms (.) of (3) from the methyl groups. The C-atoms of the methoxy and of the C-methyl groups, like the unlabeled C-atoms of the C18 chain, showed no significant signals consistent with their origin from the C, pool of the organism.

As an advanced intermediate of the citreoviridin biosyn- thesis from acetate moieties, attention was directed to oleic acid. It contains as many C-atoms as the polyketide (3) and is a key building blockl81 for the biosynthesis of many poly- dehydrocarboxylic acids1'1. The radioactivity of citreoviridin (la) isolated after the feeding of Aspergillus terreus with [I- 14 Cloleic acid corresponded to a small incorporation of only 0.00093%. KMn0,-oxidation of this citreoviridin (la) af- forded a cumalic acid derivative (2), which contained only 36% of the radioactivity to be expected on direct incorpora- tion. The low incorporation of the [l-C'4]oleic acid might therefore be best explained in terms of a degradation to I4C- acetate.

Procedure

Isolation of (la): 200 surface cultures of Aspergillus terreus (CBS 503.65) on 350 ml batches of Czapek-Dox nutrient("1 in 1 1 Erlenmayer flasks, which were grown for 21 d at 26 "C, gave 750 g of freeze-dried mycelium. This was washed conse- cutively, five times with 3.5 1 petroleum ether (126 g residue) and acetone (65.2 g residue). The acetone residue was taken up in ethyl acetate, the insoluble material removed by filtra- tion and the filtrate chromatographed several times with ethyl acetate on columns of silica gel (Merck, particle size 0.063-0.2 mm). The residue obtained on evaporation of the combined eluates was recrystallized from methanol; yield 15.1 g (2.01% of the dry mycelium) (la), m. p. 108-1 10 "C.

Received: March 20, 1980 [Z 466 b IE] German version: Angew. Chem. 92, 484 (1980)

CAS Registry number: (la), 25425-12-1

[I] Y. Ueno in I . F. H. Purchase: Mycotoxins. Elsevier. New York 1974, p. 283.

121 P. E Linnefl. A . D. Milchell, M. D. Osselron, L. J. Mulheirn, R. B. Beechey, Biochem. J . 170, 503 (1 978).

[3] a) D. W. Nugel, P. S Steyn, D. B. Scotf, Phytochemistry 11. 627 (1972); b) D. W. Nagel, P. S. Steyn, N. P. Ferreira, Phytochemistry 11, 3215 (1972).

141 K. B. Roper, D. I . Fennell: The Genus Aspergillus. Williams and Wilkins, Baltimore 1965.

151 The citreoviridin-forming strains of Aspergillus terreus have the designa- tions: CBS (Centraalbureau voor Schimmelcultures. Baarn. Holland) 503.65, 594.65. 130.55; ATCC (American Type Culture Collection) 16.792; CMI Commonwealth Mycological Institute 61.457. Of these, the first men- tioned shows the highest hitherto known formation of citreoviridin (la).

[6] N. Sakube, T. Golo, Y. Hirafa, Tetrahedron Lett. 27, 1825 (1964). 171 The incorporation was determined mass spectrometrically. For calculation

of the "C enrichment in the labeled positions of the citreoviridin (la) ob- tained after feeding [1-'3C]- and [2-"C]acetate. the intensities of their I3C- NMR signals are divided by the corresponding intensities of unlabeled (la). The reduced intensities I, thus obtained are multiplied by the quotients A,/ I, (A,,, =mass spectrometrically determined, average "C enrichment at the labeled positions in multiples of the natural "C abundance).

[XI B. Frunck, Angew. Chem. 91, 453 (1979); Angew. Chem. Int. Ed. Engl 18, 429 (1 979).

191 F Bohlmann, Fortschr. Chem. Org. Naturst. 25, 1 (1967); E. D. Korn, J. Biol. Chem. 239, 396 (1964).

[lo] 0. W. Thiele: Lipide, Isoprenoide mil Steroiden. Thieme, Stuttgart 1979, p. 58.

f l t ] As described by S . Gatenbeck, Acta Chem. Scand. 23, 3493 (1969).

462 0 Verlag Chemie, GmbH, 6940 Wernheim, 1980 0570-0833/80/0606-462 $ 02.50/0 Angew. Chem. Inl. Ed. Engl. 19 (1980) No. 6