In vitro biosynthesis, isolation, and identification of predominant metabolites of 2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one (RVX-208).

The structures of the two predominant metabolites (M4 and M5) of RVX-208, observed both in in vitro human and animal liver microsomal incubations, as well as in plasma from animal in vivo studies, were determined. A panel of biocatalytic systems was tested to identify biocatalysts suitable for milligram scale production of metabolite M4 from RVX-208. Rabbit liver S9 fraction was selected as the most suitable system, primarily based on pragmatic metrics such as catalyst cost and estimated yield of M4 (∼55%). Glucuronidation of RVX-208 catalyzed by rabbit liver S9 fraction was optimized to produce M4 in amounts sufficient for structural characterization. Structural studies using LC/MS/MS analysis and (1)H NMR spectroscopy showed the formation of a glycosidic bond between the primary hydroxyl group of RVX-208 and glucuronic acid. NMR results suggested that the glycosidic bond has the ß-anomeric configuration. A synthetic sample of M4 confirmed the proposed structure. Metabolite M5, hypothesized to be the carboxylate of RVX-208, was prepared using human liver microsomes, purified by HPLC, and characterized by LC/MS/MS and (1)H NMR. The structure was confirmed by comparison to a synthetic sample. Both samples confirmed M5 as a product of oxidation of primary hydroxyl group of RVX-208 to carboxylic acid.

Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone.

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.

Extractive biotransformation for production of metabolites of poorly soluble compounds: synthesis of 32-hydroxy-rifalazil.

A novel reaction system was developed for the production of metabolites of poorly water-soluble parent compounds using mammalian liver microsomes. The system includes the selection and use of an appropriate hydrophobic polymeric resin as a reservoir for the hydrophobic parent compounds and its metabolites. The utility of the extractive biotransformation approach was shown for the production of a low-yielding, synthetically challenging 32-hydroxylated metabolite of the antibiotic rifalazil using mouse liver microsomes. To address the low solubility and reactivity of rifalazil in the predominantly aqueous microsomal catalytic system, a variety of strategies were tested for the enhanced delivery of hydrophobic substrates, including the addition of mild detergents, polyvinylpyrrolidone, glycerol, bovine serum albumin, and hydrophobic polymeric resins. The latter strategy was identified as the most suitable for the production of 32-hydroxy-rifalazil, resulting in up to 13-fold enhancement of the volumetric productivity compared with the standard aqueous system operating at the solubility limit of rifalazil. The production process was optimized for a wide range of reaction parameters; the most important for improving volumetric productivity included the type and amount of the polymeric resin, cofactor recycling system, concentrations of the biocatalyst and rifalazil, reaction temperature, and agitation rate. The optimized extractive biotransformation system was used to synthesize 32-hydroxy-rifalazil on a multimilligram scale.

Reversible derivatization to enhance enzymatic synthesis: chemoenzymatic synthesis of doxorubicin-14-O-esters.

An efficient three-step, chemoenzymatic synthesis of unprotected doxorubicin-14-O-esters from doxorubicin hydrochloride salt is described. The key step is a lipase-catalyzed regioselective transesterification/esterification using commercially available acyl donors and doxorubicin reversibly derivatized with N-alloc to improve substrate loadings. The overall yield is ca. 60% and chromatographic purification is not required, thereby making the process more amenable to scale-up.

14-Hydroxylation of opiates: catalytic direct autoxidation of codeinone to 14-hydroxycodeinone.

Codeinone (3) was efficiently and directly converted to 14-hydroxycodeinone (1) by catalytic air oxidation in aqueous solution. A number of simple manganese and copper salts were identified to be effective catalysts, including MnSO4, KMnO4, and CuSO4. An appropriate reducing agent, such as sodium thiosulfate, is required in the reaction mixture presumably for the reduction of a detrimental peroxide intermediate. This discovery allows the more abundant codeine to be employed as the starting material for the synthesis of 14-hydroxylated opiate drugs without recourse to a thebaine-like intermediate. These discoveries were inspired from our study of microbial transformation of codeine to 14-hydroxycodeine by Mycobacterium neoaurum, where we found the actual 14-hydroxylation step is a chemical reaction rather than an enzymatic reaction, as previously believed.

Combinatorial biocatalysis.

The published applications of combinatorial biocatalysis have continued to expand at a growing rate. This is exemplified by the variety of enzyme catalysts and whole-cell catalysts used for the creation of libraries through a wide range of biocatalytic reactions, including acylation, glycosylation, halogenation, oxidation and reduction. These biocatalytic methods add the capability to perform unique chemistries or selective reactions with complex or labile reagents when integrated with classical combinatorial synthesis methods. Thus, applications towards the production of libraries de novo, the expansion of chemically derived combinatorial libraries, and the generation of novel combinatorial reagents for library synthesis can be achieved. Theoretically, these results illustrate what is already evident from nature: that complex, biologically active, structurally diverse compound libraries can be generated through the application of biocatalysis alone or in combination with classical organic synthesis approaches.