| The biosynthesis of lovastatin (1) by Aspergillus terreus was examined using Penicillium aurantiogriseum which produces compactin (5), a 6-desmethyl analogue of 1, and mutated fungal strains prepared by Professor Hutchinson (University of Wisconsin). [2,3-13C2]-3-Acetoacetic acid, N-acetylcysteamine thioester (28a) and [2,3- 13C2]-3-hydroxybutyric acid, N-acetylcysteamine thioester (29a) are not incorporated into 5.; Aspergillus nidulans lovB, a strain in which the lovastatin nonaketide synthase (LNKS, lovB) gene from A. terreus has been overexpressed, produces 4-hydroxy-6-[(1E,3 E,5E)-1-methylhepta-1,3,5-trien-1-yl]-2-pyrone ( 41) and 4-hydroxy-6-[(1E,3E,5 E,7E)-3-methylnona-1,3,5,7-tetraen-1-yl]-2-pyrone (42). 13C-Labeled sodium acetate is incorporated into 41 and 42 at a rate of 70% rate.; A. nidulans lovB+lovC, a strain in which both lovB and lovC genes have been overexpressed, produces 4a,5-dihydromonacolin L (11) both in vivo and in vitro. 13C Labeled sodium acetate is incorporated into 11 with an efficiency of 20%, but neither 29a nor [2,11-13C2]-(2 E,8E,10E)-(R)-6-methyldodecatri-2,8,10-enoic acid, N-acetylcysteamine thioester (16a) are transformed into 11 using intact cells or cell free systems.; A. terreus lovC, a strain in which the lovC gene has been inactivated, produces 41 and 42 , but not 1, nor 11. This strain allows successful incorporations of monacolin J into 1, 6-desmethylmonacolin J into 5, and 11 into 1, but not of ML-236C into 5.; LNKS was purified and tested for Diels-Alderase activity. LNKS catalyzes the Diels-Alder reaction of 16 with the correct stereochemistry required for the biosynthesis of 1. LNKS does not catalyze the Diels-Alder cyclizations of [3-carbomethoxy-(E)-2-propen-1-oxy]-( E,E)-2,4-hexadien-1-oxy] diphenylsilane (59) or cis-(E,E)-2,4-hexadien-1-yl ( E)-3-carbomethoxy-2-propen-1-yl cyclohexane-1,2-dicarboxylate ( 60).; Model studies on potential enzyme-catalyzed Diels-Alder reactions were undertaken. Compounds 59 and 60 remain unchanged when reacted with alcohol dehydrogenase, subtilisin Carlsberg, or carbonic anhydrase [4-Hydroxy-(E)-2-buten-1-oxy]-(E,E)-2,4-hexadien-1-oxy1 dipbenylsilane 78 is not cyclized by alcohol dehydrogenase. 2-(4-Penten-1-yl)phenol (82), 2-(5-hexen-1-yl)phenol (83), 4-(pent-4-enyl)phenol (92), 4-(5-hexen-1-yl)phenol(93), and 4-(6-hepten-1-yl)phenol (94) were synthesized and reacted with tyrosinase. Compounds 82 and 83 are not transformed by this enzyme, whereas 92, 93, and 94 are oxidized to ortho -quinones which polymerized before an intramolecular Diels-Alder reaction can occur. The reaction of 92 with tyrosinase in presence of ethyl vinyl ether affords the intermolecular Diels-Alder adduct exo-7-ethoxy-6-(5-hexen-1-yl)bicyclo[2.2.2]-2,3-dioxo-octa-5-ene. Ethyl 7-(4--methoxyphenyl)-2E-hexenoate, 6-ethoxy-1-(4-metboxyphenyl)-5-hexene, 6-(4-methoxyphenyl)-1-hexyne, and 1-(3,4-dimethoxyphenyl)-5E,6-octadiene were prepared, but deprotection of the methyl ethers to give the corresponding phenols proved difficult. |
