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Efficient synthetic techniques for the diversification of natural products are incremental for drug discovery processes of the pharmaceutical industry because these complex bioactive compounds often require an adjustment of properties. Human liver P450 3A4, key player of the body's detoxification system and decisive factor of a drug's metabolic fate, is renowned for its broad substrate scope including many natural products. In this study, we investigated the synthetic potential of human P450 3A4 for the diversification of natural product classes and isolated the produced metabolites of six selected natural products at a preparative 100‐mg scale. Aided by efficient expression levels in P. pastoris, this whole‐cell biocatalyst was found to be highly effective at the intended job allowing the identification of a total of 31 authentic human metabolites, many of them for the first time. By revealing an unprecedented degree of diversification, this study extends the synthetic repertoire for efficient enzymatic natural product modification in a one‐step fashion and adds a completely new view to an old enzyme traditionally used for inhibition and toxicology studies. [ABSTRACT FROM AUTHOR]

UDP‐glucuronic acid (UDP‐GlcA) is a central precursor in sugar nucleotide biosynthesis and common substrate for C4‐epimerases and decarboxylases releasing UDP‐galacturonic acid (UDP‐GalA) and UDP‐pentose products, respectively. Despite the different reactions catalyzed, the enzymes are believed to share mechanistic analogy rooted in their joint membership to the short‐chain dehydrogenase/reductase (SDR) protein superfamily: Oxidation at the substrate C4 by enzyme‐bound NAD+ initiates the catalytic pathway. Here, we present mechanistic characterization of the C4‐epimerization of UDP‐GlcA, which in comparison with the corresponding decarboxylation has been largely unexplored. The UDP‐GlcA 4‐epimerase from Bacillus cereus functions as a homodimer and contains one NAD+/subunit (kcat = 0.25 ± 0.01 s−1). The epimerization of UDP‐GlcA proceeds via hydride transfer from and to the substrate's C4 while retaining the enzyme‐bound cofactor in its oxidized form (≥ 97%) at steady state and without trace of decarboxylation. The kcat for UDP‐GlcA conversion shows a kinetic isotope effect of 2.0 (±0.1) derived from substrate deuteration at C4. The proposed enzymatic mechanism involves a transient UDP‐4‐keto‐hexose‐uronic acid intermediate whose formation is rate‐limiting overall, and is governed by a conformational step before hydride abstraction from UDP‐GlcA. Precise positioning of the substrate in a kinetically slow binding step may be important for the epimerase to establish stereo‐electronic constraints in which decarboxylation of the labile β‐keto acid species is prevented effectively. Mutagenesis and pH studies implicate the conserved Tyr149 as the catalytic base for substrate oxidation and show its involvement in the substrate positioning step. Collectively, this study suggests that based on overall mechanistic analogy, stereo‐electronic control may be a distinguishing feature of catalysis by SDR‐type epimerases and decarboxylases active on UDP‐GlcA. [ABSTRACT FROM AUTHOR]

The Pd‐catalyzed S‐allylation of thiols with stable allylcarbonate and allylacetate reagents offers several advantages over established reactions for the formation of thioethers. We could demonstrate that Pd/BIPHEPHOS is a catalyst system which allows the transition metal‐catalyzed S‐allylation of thiols with excellent n‐regioselectivity. Mechanistic studies showed that this reaction is reversible under the applied reaction conditions. The excellent functional group tolerance of this transformation was demonstrated with a broad variety of thiol nucleophiles (18 examples) and allyl substrates (9 examples), and could even be applied for the late‐stage diversification of cephalosporins, which might find application in the synthesis of new antibiotics. [ABSTRACT FROM AUTHOR]