Marko D. Mihovilovic

Discovery and resupply of pharmacologically active plant-derived natural products: A review.

Medicinal plants have historically proven their value as a source of molecules with therapeutic potential, and nowadays still represent an important pool for the identification of novel drug leads. In the past decades, pharmaceutical industry focused mainly on libraries of synthetic compounds as drug discovery source. They are comparably easy to produce and resupply, and demonstrate good compatibility with established high throughput screening (HTS) platforms. However, at the same time there has been a declining trend in the number of new drugs reaching the market, raising renewed scientific interest in drug discovery from natural sources, despite of its known challenges. In this survey, a brief outline of historical development is provided together with a comprehensive overview of used approaches and recent developments relevant to plant-derived natural product drug discovery. Associated challenges and major strengths of natural product-based drug discovery are critically discussed. A snapshot of the advanced plant-derived natural products that are currently in actively recruiting clinical trials is also presented. Importantly, the transition of a natural compound from a “screening hit” through a “drug lead” to a “marketed drug” is associated with increasingly challenging demands for compound amount, which often cannot be met by re-isolation from the respective plant sources. In this regard, existing alternatives for resupply are also discussed, including different biotechnology approaches and total organic synthesis. While the intrinsic complexity of natural product-based drug discovery necessitates highly integrated interdisciplinary approaches, the reviewed scientific developments, recent technological advances, and research trends clearly indicate that natural products will be among the most important sources of new drugs also in the future.

Monooxygenase-Mediated Baeyer−Villiger Oxidations

Enzyme-mediated Baeyer−Villiger oxidations offer a “green chemistry” approach for the production of chiral lactones. Several organisms have been found to catalyze this reaction in the course of their metabolic pathways. A number of flavin-dependent monooxygenases have been characterized, and acceptance of a multitude of non-natural substrates has been found. Such biocatalysts are used in synthetic chemistry either as isolated enzymes in combination with appropriate cofactor recycling systems or as living whole cells, in native or recombinant form, for the production of valuable intermediates. This review gives an overview of the most widely utilized enzymes and the corresponding substrate profiles, together with applications in natural product and bioactive compound synthesis. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Recent Developments in the Application of Baeyer–Villiger Monooxygenases as Biocatalysts

Baeyer–Villiger monooxygenases (BVMOs) represent a specific class of monooxygenases that are capable of catalyzing a variety of oxidation reactions, including Baeyer–Villiger oxidations. The recently elucidated BVMO crystal structures have provided a more detailed insight into the complex mechanism of these flavin‐containing enzymes. Biocatalytic studies on a number of newly discovered BVMOs have shown that they are very potent oxidative biocatalysts. In addition to catalyzing the regio‐ and enantioselective Baeyer–Villiger oxidations of a wide range of carbonylic compounds, epoxidations, and enantioselective sulfoxidations have also been shown to be part of their catalytic repertoire. This review provides an overview on the recent developments in BVMO‐mediated biocatalytic processes, identification of the catalytic role of these enzymes in metabolic routes and prodrug activation, as well as the efforts in developing effective biocatalytic methodologies to apply BVMOs for the synthesis of high added value compounds.

Direct Functionalization of (Un)protected Tetrahydroisoquinoline and Isochroman under Iron and Copper Catalysis: Two Metals, Two Mechanisms

A highly facile, straightforward synthesis of 1-(3-indolyl)-tetrahydroisoquinolines was developed using either simple copper or iron catalysts. N-protected and unprotected tetrahydroisoquinolines (THIQ) could be used as starting materials. Extension of the substrate scope of the pronucleophile from indoles to pyrroles and electron-rich arenes was realized. Additionally, methoxyphenylation is not limited to THIQ but can be carried out on isochroman as well, again employing iron and copper catalysis.

Self-Sufficient Baeyer–Villiger Monooxygenases: Effective Coenzyme Regeneration for Biooxygenation by Fusion Engineering†

Over the past few years, industrial interest in biocatalysts that perform selective oxidative reactions has increased significantly. Baeyer–Villiger monooxygenases (BVMOs) have been identified as a highly versatile class of enzymes for the efficient catalysis of chemo-, regio-, and/or enantioselective oxygenation reactions. Although the most prominent transformation catalyzed by these biocatalysts is a chiral variant of the classical Baeyer–Villiger reaction, the oxygenation of heteroatoms and epoxidation reactions have also been reported. Stoichiometric amounts of O2 and NADPH are required for these reactions. A complication for the largescale application of these reactions is the high cost of the reduced nicotinamide coenzyme. To overcome this problem, several electrochemical and photochemical approaches have been explored. However, the efficiency of these approaches is typically poor. Furthermore, it has been shown that BVMOs require NADP for stability and enantioselective catalysis. An efficient and commonly used method for coenzyme regeneration employs whole cells, especially in combination with the recombinant expression of the required biocatalysts. This strategy has been implemented in BVMOmediated biotransformations with wild-type strains and has proved particularly successful with recombinant overexpression systems. The approach avoids laborious enzyme purification steps and exploits the coenzyme regeneration capacity of the host. Although whole cells have been shown to be effective catalysts for Baeyer–Villiger oxidation, they also exhibit limitations, such as cellular toxicity, enzyme inhibition by the substrate/product, degradation of the product, and poor oxygen-transfer rates. Coenzyme regeneration by using isolated enzymes has also been studied extensively in the past few years. Well-known examples of such NADPH-regenerating enzymes are alcohol dehydrogenase and formate dehydrogenase. A phosphite dehydrogenase (PTDH) was also identified as an effective enzyme for coenzyme regeneration. The favorable thermodynamic equilibrium constant makes the oxidation of phosphite a nearly irreversible process. The exquisite selectivity of PTDH for phosphite also precludes any side reactions, such as those that can occur, for example, when an alcohol dehydrogenase is used. These characteristics make PTDH an ideal candidate for use as a coenzyme regenerating enzyme (CRE) in combination with BVMOs or other NAD(P)H-dependent enzymes. Herein, we report a novel approach to the combination of the catalytic activity of a redox biocatalyst with concomitant coenzyme recycling in a single fusion protein (Scheme 1). During the last decade, a number of fusion protein tags have been developed. These tags are used intensely in life-sciencerelated research and commercial activities. Although the fusion of proteins is a widely applied strategy in, for example, enzyme purification (e.g. the use of glutathione S transferase (GST) tags) and the subcellular visualization of target proteins (e.g. with a green fluorescent protein (GFP) tag), this concept is hardly ever encountered in the context of synthetic applications. Only a few isolated examples in the literature provide evidence that the fusion of separate enzymes can result in improved biocatalytic properties. We report herein on the engineering of a number of representative BVMOs that are linked covalently to soluble NADPH-regenerating phosphite dehydrogenase. This construct enables the use of phosphite as a cheap and sacrificial electron donor with whole cells, cell extracts, and purified enzyme. It was our particular goal to design a self-sufficient two-in-one redox biocatalyst that does not require an additional catalytic entity for coenzyme recycling. As model

Efficient Biooxidations Catalyzed by a New Generation of Self-Sufficient Baeyer―Villiger Monooxygenases

Over the last decades industrial interest in oxidative biocatalysis has increased significantly. One of the most prominent enzyme families that catalyze a variety of different oxidations are Baeyer–Villiger monooxygenases (BVMOs). These biocatalysts are part of an exclusive family of flavin-dependent enzymes that catalyze the biotransformation of aldehydes and (a)cyclic ketones to their corresponding esters and lactones. [1] Additionally, these enzymes are also known for their capability to oxidize heteroatoms (sulphur, nitrogen, boron) and perform epoxidation reactions. [2] Type I BVMOs utilize flavin adenine dinucleotide (FAD) as cofactor and NADPH as electron donor in order to activate molecular oxygen (O2) and generate a reactive C4a-peroxyflavin intermediate. This enzyme intermediate acts similarly to an organic peracid and reacts with the organic substrate and this results in formation of the oxygenated product. [3] BVMOs have been shown to carry out these oxidative reactions in a highly regio-, stereo- and enantioselective manner; this indicates that these enzymes are interesting candidates for various biocatalytic applications. [4] An obstacle of using BVMOs in a cost efficient way is the requirement of stoichiometric amounts of expensive NADPH coenzyme. Several coenzyme regeneration methods have been explored in the recent years. [5] The most efficient approach is based on the regeneration of NADPH by using a two-enzyme system (either as isolated enzyme or in whole cells). [6]

Halogen dance reactions—A review

Halogen Dance (HD) reactions are a useful tool for synthetic chemists as they enable access to positions in aromatic and heteroaromatic systems for subsequent functionalization which are often difficult to address by other methods, hence, allowing entry to versatile scaffolds. While the method can be extremely useful, this transformation is often neglected upon designing synthetic sequences. This may be largely attributed to the lack of comprehensive reference works covering the general principles and outlining the versatility and limitations of the technique. The following review tries to present HD reactions in a clear and concise manner in order to convince more chemists of its advantages. It covers the field of HD reactions from their first observation in 1951 until the present. The important contributions leading to the elucidation of the mechanism are briefly outlined followed by a detailed mechanistic section and a discussion of factors which influence HD reactions. Finally, an overview of HD reactions on various carbocyclic and heterocyclic ring systems and its applications in the synthesis of complex compounds is given.

Tandem Catalysis: From Alkynoic Acids and Aryl Iodides to 1,2,3-Triazoles in One Pot

A tandem catalysis protocol based on decarboxylative coupling of alkynoic acids and 1,3-dipolar cycloaddition of azides enables a highly efficient synthesis of a variety of functionalized 1,2,3-triazoles. The three-step, one-pot method avoids usage of gaseous or highly volatile terminal alkynes, reduces handling of potentially unstable and explosive azides to a minimum, and furnishes target structures in excellent yields and a very good purity without the need for additional purification.

ASYMMETRIC OXIDATIONS AT SULFUR CATALYZED BY ENGINEERED STRAINS THAT OVEREXPRESS CYCLOHEXANONE MONOOXYGENASE

Recombinant strains of bakers yeast (Saccharomycescerevisiae) and Escherichiacoli expressing cyclohexanone monooxygenase from Acinetobacter sp. NCIB 9871 have been used as whole-cell biocatalysts for oxidations of several sulfides, dithianes and dithiolanes to the corresponding sulfoxides. The enantio- and diastereoselectivities of these reactions compare favorably with oxidations catalyzed by the purified monooxygenase or the parent microorganism (a class II pathogen). The facility of handling yeast reactions makes these biotransformations an attractive alternative route to optically pure sulfoxides.

An Enzymatic Toolbox for Cascade Reactions: A Showcase for an In Vivo Redox Sequence in Asymmetric Synthesis

Single-step transformations of non-natural substrates by enzymes have been successfully established in the last decades as highly valuable techniques for the synthesis of chiral building blocks. Owing to their properties, biocatalysts can be employed as catalytic tools for the facile transformation of functional groups in organic synthesis. By exploiting the manifoldness of enzymes and their different catalytic activities, it is possible to design new artificial biosynthetic pathways on the basis of the “retrosynthetic approach” that is commonly applied in chemical synthesis and that was only very recently proposed as a novel concept for biocatalysis. This design principle is used in the strategic planning of organic syntheses by transforming a target molecule into simpler precursors in which molecular complexity is reduced by manipulation of functional groups. The concept of multistep one-pot reactions has caught the attention of synthetic chemists in recent years. To address the increasing demands by society to further improve the sustainability of chemical processes, one-pot cascades of reaction sequences can substantially decrease the amount of chemicals used for each reaction and subsequent down-stream processing by concomitantly optimizing the energy requirements and operation expenses. As pointed out in a recent review, such cascades not only improve processes by saving time and reducing waste, but they also offer advantages if unstable or toxic intermediates are involved, as these do not accumulate. Nature uses the design principle in a highly successfully manner, as all metabolic pathways are interconnected and conducted within the “single-vessel” environment of a cell. The concerted interaction of numerous enzymes within the cell allows exceptionally high yields in multistep biosynthetic pathways. 5] However, this approach is largely limited to alreadyexisting metabolic reaction sequences (e.g. , 1,3-propane diol and 1,4-butane diol) and does not necessarily deliver the high structural diversity of compounds utilized by the chemical industry. The major difference between biocatalysis and classical fermentation is the extension of the enzymatic transformations to non-natural substrates. Consequently, the extension of singlestep biotransformations, which are particularly powerful in asymmetric synthesis, is a logical development. 8] In this context, we recently reported the successful conversion of unsaturated fatty acids into medium-chain alcohols, w-hydroxy acids, and dicarboxylic acids through the combination of appropriate enzymes in a cascade reaction by employing a combined in vivo/in vitro strategy towards nonchiral products. Very recently, Schrewe et al. presented an interesting three-step synthetic approach including two enzymes for the production of terminal alkylamino functionalization in a recombinant E. coli strain. Notably, however, only a single substrate was investigated, but even so, the versatility of the presented enzyme was highlighted. Another approach was published very recently by the group of Li who applied a twostrain-mixed-culture strategy for the synthesis of d-lactones. Both studies showed the potential of redox cascades in living organisms and at the same time pointed out their limitations. A simple transfer from a single-step biotransformation (including broad substrate acceptance and high selectivity) to enzyme cascades still remains a challenge. The prime novelty and major aim of the present study was to combine the efficiency of biosynthetic redox pathways, the modularity of synthesis by simple functional group transformations, and the substrate promiscuity of enzymes. Designing and evaluating the feasibility of a multienzyme-catalyzed cascade process in living microbial cells enabled the creation of an artificial “mini”-metabolic pathway connected to primary metabolism through redox-cofactor regeneration. This approach was applied in an in vivo environment and concomitantly provided access to diverse chemical entities through divergent reaction pathways. Figure 1 illustrates the straightforward concept of this work. In lieu of looking at different specific target compounds, for which one synthetic step is performed biocatalytically, we reduced the complexity of the molecule to the functional groups of the compound regardless of the residual structure. From a synthetic point of view, oxidation of a simple allylic alcohol starting material to the corresponding a,b-unsaturated ketone [a] C. Peters, J. Muschiol, Dr. M. Kadow, S. Saß, Prof. Dr. U. T. Bornscheuer Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis Greifswald University Felix-Hausdorff-Str. 4, 17487 Greifswald (Germany) Fax: (+ 49) 3834-86-794367 E-mail : uwe.bornscheuer@uni-greifswald.de [b] N. Oberleitner, T. Bayer, P. Schaaf, Dr. N. Iqbal, Dr. F. Rudroff, Prof. Dr. M. D. Mihovilovic Institute of Applied Chemistry Technical University Vienna Getreidemarkt 9/163-OC, 1060 Vienna (Austria) E-mail : marko.mihovilovic@tuwien.ac.at florian.rudroff@tuwien.ac.at [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cctc.201300604.