Peniphenone and Penilactone Formation in Penicillium crustosum via 1,4-Michael Additions of ortho-Quinone Methide from Hydroxyclavatol to γ-Butyrolactones from Crustosic Acid

Abstract

Penilactones A and B consist of a γ-butyrolactone and two clavatol moieties. We identified two separate gene clusters for the biosynthesis of these key building blocks in Penicillium crustosum. Gene deletion, feeding experiments, and biochemical investigations proved that a nonreducing PKS ClaF is responsible for the formation of clavatol and the PKS-NRPS hybrid TraA is involved in the formation of crustosic acid, which undergoes decarboxylation and isomerization to the predominant terrestric acid. Both acids are proposed to be converted to γ-butyrolactones with involvement of a cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol by a nonheme FeII/2-oxoglutarate-dependent oxygenase ClaD and its spontaneous dehydration to an ortho-quinone methide initiate the two nonenzymatic 1,4-Michael addition steps. Spontaneous addition of the methide to the γ-butyrolactones led to peniphenone D and penilactone D, which undergo again stereospecific attacking by methide to give penilactones A/B.

Synthetic Utility of One-Pot Chemoenzymatic Reaction Sequences

“…For example, ortho-quinone methides (o-QMs) are high-energy intermediates which have been implicated as biosynthetic intermediates in numerous natural product pathways. 27 o-QMs, such as 47, are derived from ortho-phenolic compounds and typically react with nucleophiles or dienophiles in 1,4-additions or cycloaddition reactions, respectively. These reactive intermediates have been used extensively in the total synthesis of natural products and can be accessed through a variety of approaches.…”

“…36,37 Efforts such as these are critical for improving access to these compounds to enable biological studies and the determination of structureactivity relationships. [25][26][27] As the field of biocatalysis continues to expand and play a greater role in synthetic chemistry, it is reasonable to expect that the development of innovative one-pot chemoenzymatic processes will likewise see continued growth. In particular, the combination of biocatalytic and transitionmetal-catalyzed transformations is representative of this new frontier in biocatalysis.…”

Oxepinamide F biosynthesis involves enzymatic d-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration

“…2a ). To prove its function, opaA in A. ustus was replaced with a hygromycin B resistance cassette by using a split marker gene replacement protocol 14 . The generated mutants were verified by PCR (Supplementary Fig.…”

Biocatalyst-Initiated Ortho-Quinone Methide Formation and Derivatization

“…We anticipated that following benzylic C-H bond oxidation, an o-QM intermediate (see demonstrated to occur naturally in the biosynthesis of peniphenones and penilactones in the fungus P. crustosum, highlighting the biomimetic nature of our approach. 34 Furthermore, we propose that our chemoenzymatic methodology for the generation of o-QMs can be adopted broadly for use with other enzymes capable of mediating benzylic C-H hydroxylation on ortho-phenolic compounds.…”