We report the identification and characterization of the caz biosynthetic cluster from C. globosum and the characterization of a highly-reducing polyketide synthase (PKS) that acts in both a sequential and convergent manner with a nonreducing PKS to form the chaetomugilin and chaetoviridin azaphilones. Genetic inactivation studies verified the involvement of individual caz genes in the biosynthesis of the azaphilones. Through in vitro reconstitution, we demonstrated the in vitro synthesis of chaetoviridin A 3 from the pyrano-quinone intermediate cazisochromene 8 using the highly-reducing PKS and an acyltransferase.
The echinocandins are a small group of fungal N-acylated cyclic hexapeptides that are fungicidal for candida strains and fungistatic for aspergilli by targeting cell wall 1,3-β-glucan synthases. The side chains of all six amino acid building blocks have hydroxyl groups, including the nonproteinogenic 4R,5R-dihydroxy-Orn1, 4R-OH-Pro3, 3S, 4S-dihydroxy-homoTyr4 and 3S-OH-4S-Me-Pro6. The echinocandin (ecd) gene cluster contains two predicted nonheme mononuclear iron oxygenase genes (ecdG,K) and one encoding a P450 type heme protein (ecdH). Deletion of the ecdH gene in the producing Emericella rugulosa generates an echinocandin scaffold (echinocandin D) lacking both hydroxyl groups on Orn1. Correspondingly, the ΔecdG strain failed to hydroxylate C3 of the homoTyr residue, and purified EcdG hydroxylated free L-homoTyr at C3. The ΔecdK strain failed to generate mature echinocandin unless supplemented with either 4R-Me-Pro or 3S-OH-4S-Me-Pro, indicating blockage of a step upstream of Me-Pro formation. Purified EcdK is a Leu 5-hydroxylase, acting iteratively at C5 to yield γ-Me-Glu-γ-semialdehyde in equilibrium with the cyclic imine product. Evaluation of deshydroxyechinocandin scaffolds in in vitro anticandidal assays revealed up to 3-fold loss of potency for the ΔecdG scaffolds, but a 3-fold gain of potency for the ΔecdH scaffold, in line with prior results on deoxyechinocandin homologs.
The communesins are a prominent class of indole alkaloids isolated from Penicillium species. Due to their daunting structural framework and potential as pharmaceuticals, communesins have inspired numerous total synthesis efforts. However, the genetic and biochemical basis of communesin biosynthesis has remained unexplored. Here, we report the identification and characterization of the communesin (cns) biosynthetic gene cluster from Penicillium expansum. We confirmed communesin is biosynthesized from the coupling of tryptamine and aurantioclavine, two building blocks derived from L-tryptophan. The post-modification steps were mapped by targeted-gene deletion experiments and structural elucidation of intermediates and new analogues. Our studies set the stage for biochemical characterization of the communesin biosynthesis towards understanding how nature generates remarkable structural complexity from simple precursors.
Dirhodium(II) caprolactamate exhibits optimal efficiency for the production of the tert-butylperoxy radical, which is a selective reagent for hydrogen atom abstraction. These oxidation reactions occur with aqueous tert-butyl hydroperoxide (TBHP) without rapid hydrolysis of the caprolactamate ligands on dirhodium. Allylic oxidations of enones yield the corresponding enedione in moderate to high yields, and applications include allylic oxidations of steroidal enones. Although methylene oxidation to a ketone is more effective, methyl oxidation to a carboxylic acid can also be achieved. The superior efficiency of dirhodium(II) caprolactamate as a catalyst for allylic oxidations by TBHP (mol % catalyst, % conversion) is described in comparative studies with other metal catalysts that are also reported to be effective for allylic oxidations. That different catalysts produce essentially the same mixture of products with the same relative yields suggests that the catalyst is not involved in product forming steps. Mechanistic implications arising from studies of allylic oxidation with enones provide new insights into factors that control product formation. A previously undisclosed disproportionation pathway, catalyzed by the tert-butoxy radical, of mixed peroxides for the formation of ketone products via allylic oxidation has been uncovered.
Our data suggest that in essentially all clinical parameters, there is no difference between free flap type used for soft tissue coverage of the lower extremity. Patients undergoing reconstruction with a fasciocutaneous flap may return to weight bearing earlier--although they are more likely to require elective flap revisions. These results imply essentially equivalent outcomes regardless of flap type or operative indication, in contrast with some of the biases in the orthopedic community. The particular flap chosen for any reconstruction should remain solely at the discretion of the plastic surgeon.
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