Synthesis of 11-hydroxyl O-methylsterigmatocystin and the role of a cytochrome P-450 in the final step of aflatoxin biosynthesis.

The major skeletal rearrangements (anthraquinone --> xanthone --> coumarin) that occur in the complex biosynthesis of aflatoxin B(1) are mediated by cytochromes P-450. Previous experiments have suggested that two successive monooxygenase reactions are required to convert the xanthone O-methylsterigmatocystin (OMST) to aflatoxin, a process we demonstrate is mediated by a single P-450, OrdA, in Aspergillus parasiticus in accord with findings in A. flavus. The first oxidative cycle is proposed to result in the formation of 11-hydroxy O-methylsterigmatocystin (HOMST), while the second entails aryl ring cleavage, demethylation, dehydration, decarboxylation, and rearrangement to give aflatoxin - a remarkable sequence of transformations. To test this hypothesis, HOMST has been synthesized by an alkylnitrilium variant of the Houben-Hoesch reaction. The troublesome xanthone carbonyl was protected as a butylene to allow further elaboration of the molecule, and then the product xanthone was restored in a uniquely facile peracid deprotection. Methods were devised to construct the sensitive dihydrobisfuran and to maintain the oxidation state of the partially methylated hydroquinone. Expression of ordA in a yeast membrane preparation enabled the intermediacy of HOMST both to be detected in the conversion of OMST to aflatoxin and to be established directly in the biosynthesis of the mycotoxin. Having secured the role of HOMST in aflatoxin formation, the mechanism of the second oxidative cycle of this P-450 is considered.

Synthesis of sterigmatocystin derivatives and their biotransformation to aflatoxins by a blocked mutant of Aspergillus parasiticus.

Seven alkyl and aryl homologues of O-methylsterigmatocystin (OMST) were synthesised and fed in separate experiments to a mutant of Aspergillus parasiticus capable of converting sterigmatocystin (ST) to aflatoxin B1 (AFB1). Their conversion to AFB1 was followed over a time period and it was found that O-propylsterigmatocystin (OPRST) was converted to AFB1 more rapidly than O-ethylsterigmatocystin (OEST) or OMST or ST itself. The aryl derivative O-benzoylsterigmatocystin (OBzST) was converted at the slowest rate. These results show that alkyl and aryl homologues of OMST may be converted to AFB1, suggesting that the methylation of ST is not an absolute requirement for its conversion to AFB1. It seems likely that whatever enzyme(s) are involved in this process exhibit relative specificity. As to whether alkylation of ST is an obligatory step in AFB1 biosynthesis is neither supported nor disproved as the fungal cells used are presumably capable of methylating ST. The fact that the propyl derivative showed fastest conversion is not necessarily significant as this may be due to faster diffusion of the least polar of the derivatives through the cell membrane.

Preparation and antitumor activities of some derivatives of 5-methoxysterigmatocystin.

A series of derivatives of 5-methoxysterigmatocystin (3a,12c-dihydro-8-hydroxy-6,11-dimethoxy-7H-furol[3',2':4,5]furo[2,3-c]xanthen-7-one) has been prepared and evaluated for antitumor activity. The potency of the parent compound has been associated with the intact bisfurano ring system and with the double bond in the terminal furan ring. It has been shown that new substituents can be introduced in the xanthone portion of the molecule and that the antitumor activity is in some cases preserved.