- The in vitro conversion of norsolorinic acid to aflatoxin B1. An improved method of cell-free enzyme preparation and stabilization
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The biosynthesis of the environmental carcinogen aflatoxin B1 (13) is initiated by the formation of a C6-primer by a dedicated yeast-like fatty acid synthase. Homologation of this starter unit by a polyketide synthase gives the anthraquinone norsolorinic acid (2). Approximately 15 chemical steps follow from this first stable intermediate to the mycotoxin (13) itself. A new protocol of cell-free enzyme preparation has been developed from the fungus Aspergillus parasiticus which carries out all of these transformations for the first time. The key experimental step involves rapid concentration and efficient dialysis by membrane filtration to remove primary and secondary metabolites, cofactors, and small biomolecules (MW 1 (13) has been investigated, cofactor requirements defined for each step, and a time-course run in which only versicolorin A (9) and sterigmatocystin (11) were observed to accumulate. The post-bisfuran skeletal rearrangement of versicolorin A (9) to demethylsterigmatocystin (10) was studied in O-methylsterigmatocystin (12) in the presence of D20 and/or d7-glucose or stereospecifically labeled NADPD. Unexpectedly high extents of proton exchange were found in the A ring during this transformation, including at a site of formal reduction. A tentative mechanism is discussed to account for this multi enzyme process.
- Watanabe, Coran M. H.,Townsend, Craig A.
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- Evidence for the Probable Final Steps in Aflatoxin Biosynthesis
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The final steps in the biosynthesis of the potent environmental carcinogen aflatoxin B1 (8) are believed to involve the oxidative cleavage and rearrangement of O-methylsterigmatocystin (7) with loss of a C1-unit.The means by which this overall transformation occurs is not known and has been addressed using cell-free conversions of samples of radiolabeled 7 that were obtained by the incorporation of either - or acetate.The proportion of radioisotope detected in aflatoxin B1 relative to that of the C1-unit liberated (formaldehyde, formic acid, or carbon dioxide) was tested. Carbon dioxide alone was isolated in the proper stoichiometry to limit the possible mechanisms that can be acting at the conclusion of this biosynthetic pathway.
- Chatterjee, Moneesh,Townsend, Craig A.
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- Palladium catalyzed kinetic and dynamic kinetic asymmetric transformations of γ-acyloxybutenolides. Enantioselective total synthesis of (+)-aflatoxin B1 and B2a
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The reaction of γ-tert-butoxycarbonyloxy-2-butenolide with phenol nucleophiles in the presence of a Pd(0) complex with chiral ligands may be performed under conditions that favor either a kinetic resolution or a kinetic asymmetric transformation (KAT) or dynamic kinetic asymmetric transformation (DYKAT). Performing the reaction at high concentration (0.5 M) in the presence of a carbonate base favors the former, i.e., KAT; whereas, running the reaction at 0.1M in the presence of tetra-n-butylammonium chloride favors the DYKAT process. Syntheses of aflatoxin B1 and B2a employs the DYKAT to introduce the stereochemistry. Starting with Pechmann condensation of the monomethyl ether of phloroglucinol, the requisite phenol nucleophile is constructed in two steps. The DYKAT proceeds with > 95% ee. A reductive Heck cyclization followed by a lanthanide catalyzed intramolecular acylation completes the synthesis of the pentacyclic nucleus in 3 steps. Reduction of the lactone provides aflatoxin B2a and its dehydration product B1. This synthetic strategy creates an asymmetric synthesis of the former in only 7 steps and the latter in 9 steps. Thus, the ultimate synthetic sequence involves 3 + 5 → 39 → 40 → 42 → 43 → 46 → 47 → 48 (aflatoxin B2a) → 49 (aflatoxin B1.
- Trost, Barry M.,Toste, F. Dean
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p. 3090 - 3100
(2007/10/03)
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