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  1. Abstract

    MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of aminomore »acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.

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  2. Abstract The search for more effective and highly selective C–H bond oxidation of accessible hydrocarbons and biomolecules is a greatly attractive research mission. The elucidating of mechanism and controlling factors will, undoubtedly, help to broaden scope of these synthetic protocols, and enable discovery of more efficient, environmentally benign, and highly practical new C–H oxidation reactions. Here, we reveal the stepwise intramolecular S N 2 nucleophilic substitution mechanism with the rate-limiting C–O bond formation step for the Pd(II)-catalyzed C(sp 3 )–H lactonization in aromatic 2,6-dimethylbenzoic acid. We show that for this reaction, the direct C–O reductive elimination from both Pd(II) andmore »Pd(IV) (oxidized by O 2 oxidant) intermediates is unfavorable. Critical factors controlling the outcome of this reaction are the presence of the η 3 -(π-benzylic)–Pd and K + –O(carboxylic) interactions. The controlling factors of the benzylic vs ortho site-selectivity of this reaction are the: (a) difference in the strains of the generated lactone rings; (b) difference in the strengths of the η 3 -(π-benzylic)–Pd and η 2 -(π-phenyl)–Pd interactions, and (c) more pronounced electrostatic interaction between the nucleophilic oxygen and K + cation in the ortho-C–H activation transition state. The presented data indicate the utmost importance of base, substrate, and ligand in the selective C(sp 3 )–H bond lactonization in the presence of C(sp 2 )–H.« less
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